Underground Tank Technology Update

16

(d) risk assessment

25 Yes
9 Site-specific
1 Optional
1 No response

10 No
4 Within RBCA
1 Planning to

(e) monitoring
48 Yes
1 No response

1 Site-specific

5. Is RNA an acceptable cleanup remedy for the following

contaminants?
(a) gasoline/BTEX compounds
46 Yes
4 Yes site-specific
1 No
(b) diesel fuels/PAH compounds
40 Yes
9 Yes site-specific
2 No
(c) MTBE
22 Site-specific
13 Not regulated
5 Yes
3 Yes possible w/acceptable risk
3 Probably no
3 No NA policy
2 No acceptable risk data available

Vol. 12, No. 3

May/June 1998

7. What evidence does your state require to demonstrate

RNAs effectiveness?
(a) shrinking or stable plume
47 Yes
1 Yes not always

Department of Engineering Professional Development The College of Engineering University of WisconsinMadison

2 No formal method/policy
1 Preferred models OK
(b) decreasing contaminant concentrations
44 Yes
3 No
2 No formal method/policy
1 Yes not always
1 Preferred models OK
(c) geochemical indicators
20 Yes
20 No
11 Site-specific
8. Does your state have groundwater regulations for MTBE
as of January 1998?

24 Yes
24 No

3 Site-specific

9. Of the 24 states without current MTBE regulations,

does your state anticipate adopting MTBE groundwater
regulations in the future?

9
8
1
6

(a) specify a minimum number of monitoring points?

28 No
15 Yes
8 Site-specific

(c) require a minimum duration of monitoring?

17 1-2 yrs
17 Site-specific
15 to <GW standards/goals
2 >2 yrs

Underground Tank
Technology Update

(d) specify a monitoring frequency (typical)?

38 1 to 4 events per year
13 Site-specific

6. For RNA, does your state:

(b) specify the number of points?

32 Site-specific
19 Other

May/June 1998

No change
Adopt regulations in 1998
Adopt regulations in 1999
Adopt regulation pending EPA issuing final
health advisory/MCL

UTTU thanks Mike Martinson, Delta Environmental Consultants,

Inc., for contributing this article. Mike can be reached at
612-697-5165 or mikema@deltaenv.com.
The next issue of UTTU will contain the specific data that Mike
Martinson obtained during the course of this survey.

Underground Tank Technology Update is

published bimonthly by the University of
WisconsinMadison, Department of
Engineering Professional Development.
UTTU supplies useful information to
federal, state, and local officials working
with groundwater technology and to other
interested technical specialists. For new
subscriptions or address corrections,
use the form on inside back page.
UTTU is funded by the U.S. EPA under
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to the University of WisconsinMadison,
which is responsible for its preparation.
Mention of trade names or commercial
products does not constitute endorsement
or recommendation for use.
Comments and suggestions are welcome
and may be directed to John T. Quigley,
Project Director, 432 N. Lake St., Madison,
WI 53706. Tel 608/265-2083.
If you have a problem locating a reference
cited in UTTU, please contact Pat Dutt
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or call her at 607/257-6801.
Advisory Board
Gilberto Alvarez, Environmental Engineer
OUST, U.S. EPA, Region 5, Chicago, Illinois
Bruce Bauman, Research Program
CoordinatorSoil and Groundwater, API
Washington D.C.

Article summaries
Discussion of bioremediation terms ............................... 2
This is the first of three articles summarizing bioremediation
issues originally debated in November 1997 by members of the
BioGroup on the Internet.

Engineered microbe effectiveness, bioaugmentation

in lab vs. field, and transgenic microorganisms ............ 5
In this article researchers give practical information on
bioaugmentation.

Acceptable hydrocarbon concentrations in soil ............ 7

This article gives technical and philosophical perspectives on what
is the acceptable concentration of hydrocarbons left in soil?

History of bioremediation references ............................ 11

For those interested in the history of bioremediation, a list is
provided.

1998 national RNA survey .............................................. 14

This article summarizes the states RNA practices.

Robert Hitzig, Geologist

OUST, U.S. EPA, Washington D.C.

Underground Tank Technology Update

Engineering Professional Development

432 North Lake Street
Madison, Wisconsin 53706

George Mickelson, Environmental Engineer

Wisconsin Department of Natural Resources
Madison, Wisconsin
Nonprofit
Organization
U.S. Postage
PAID
Madison, WI
Permit No. 658

Mark D. Millsop, Hydrogeologist

GME Consultants, Crosby, Minnesota
Phil OLeary, Professor
Department of Engineering Professional
Development, UWMadison
Gerald W. Phillips
U.S. EPA, Region 5, Chicago, Illinois
Matt Small, Hydrogeologist
U.S. EPA, Region 9
San Francisco, California
Staff
John T. Quigley
Pat Dutt Komor
Darrell Petska
Debbie Benell
Susan Kummer/Artifax

Project Director
Geologist/Writer
Copy Editor
Program Assistant
Graphics

EPAs internet chat room at www.epa.gov/swerust1/flags.htm

consists of EPA, New Mexico, Alaska, Arizona, Montana,
Minnesota and California sites. The Minnesota site, for instance,
lists documents relating to land treatment, composting and release
investigations in karst areas. For a list of state contacts, see the
site http://www.epa.gov/swerust1/states/statcon1.htm.
UTTUs home page, http://epdwww.engr.wisc.edu/uttu/, contains
an alphabetical and topical list of every article that has appeared
in UTTU since 1987.

For information on The Environment: The Cleanup and

Re-use of Brownfields, a workshop presented in Seattle,
Washington, May 11-12, send an e-mail to dwert@aipt.org
or see the web site http://aipt.org/environment.html.

Underground Tank Technology Update

Discussion of
bioremediation terms
A member of the GZA GeoEnvironmental Inc., BioGroup
was approached by a vendor selling treated microbes. The
member wanted information on microaerophillic bacteria
and degradation/transformation pathways. Here are the
responses the member received.

Microaerophillic bacteria, from Whittaker, 1997. Used in

bioaugmentation, these guys purportedly need just the
minutest amounts of oxygen (no specifications provided)
and can biodegrade just about anything. One vendor claims
that they are neither aerobic nor anaerobic (rather,
microaerophillic) and do not require any nutrient addition. I
am not familiar with the term microaerophillic (though I can
make the obvious assumptions based on the name itself); in
the absence of any technical information provided by the
vendor, I wondered if anyone out there could enlighten me.
Degradation/transformation pathways. I have been told from
unreliable sources that during bioremediation, one should
expect an increase in the abundance of low molecular
weight alkanes/aromatic compounds (including BTEX)
because they are produced from the transformation of
larger molecules, although this will pass as these compounds are themselves degraded. I expressed deep
scepticism about this, since from my understanding, aerobic
biotransformation proceeds via carboxylic acid/catechol
formation, not the discrete alkane/aromatic compounds.
Questions: Can biotransformation really result in an
increase in lower-molecular-weight compounds (in whatever
form) to the extent that BTEX (for example) concentrations
would go up? Or more likely, can the actual lowermolecular-weight intermediates be produced in such
abundance that they could be mistaken for alkane/aromatic
compounds during analysis?
Microaerophillic bacteria, from Schaffner, 1997. It doesnt
matter whether they are microaerophillic, or winged pixies,
for that matter. The primary rate-limiting factor for biodegradation of contaminants such as petroleum hydrocarbons
(PHC) is the electron acceptor. If the superbugs are viable
using a lower electron acceptor concentration, I believe that
means theyll require a longer time to degrade targeted
compounds. The bottom line, as always, remains reaction
stoichiometry, i.e., you must meet the electron acceptor
demand exerted by the PHCs.
Degradation/transformation pathways. I believe you are
correct regarding PHC metabolites, i.e., functional groups
are not merely stripped from compounds. Regarding
increased BTEX concentrations, however, it is possible
that stimulation may induce biosurfactant production which
may temporarily mobilize sorbed constituents. I have not
observed this myself, but I recall that this mechanism has
been evoked in some of the early literature to explain
increases in contaminant concentrations following
biostimulation.

May/June 1998

Microaerophillic bacteria, from Barden, 1997. It sounds like

another case of overselling things. Microaerophillic refers
to conditions of very low dissolved oxygen (generally less
than 1 mg/L) where dissolved oxygen is not completely
absent, hence the description of the condition as not
anaerobic, but not fully aerobic. These microbes are capable
of various degradation pathways but primarily can utilize
organic substrates such as BTEX. They are not superbugs
and have essentially the same nutrient requirements as
other microbes. Very likely they are using nitrate as a
terminal electron acceptor (nitrate reduction). If your vendor
cant provide you with specific factual technical information
supporting their product, I would tell them to take a hike.
Degradation/transformation pathways. The biodegradation/
biotransformation does not produce BTEX compounds
(complete aromatic rings). In some cases, degradation of
straight- or branched-chain alkanes could potentially
produce a slight increase in low-molecular-weight alkanes,
but youd be unlikely ever to see it. It is possible that some
degradation intermediates (organic acids) from anaerobic
degradation could show up in the same range as BTEX in
a GC scan. The lower-molecular-weight hydrocarbons are
generally more biodegradable and dont tend to accumulate
in the system.

May/June 1998

states did not widely accept using RNA for MTBE

contamination:
only 5 states permit an RNA remedy for MTBE
25 states consider RNA of MTBE to be a
site-specific decision
24 states have MTBE regulations
3 states require that MTBE cleanup requirements
be site-specific
24 states currently have no MTBE regulations
9 states expect to adopt MTBE regulations in
1998-99
6 states anticipate adopting regulations soon after
the U.S. EPA issues its final health risk advisory
and/or establishes an MTBE maximum contaminant
level (MCL)
The following section lists the questions posed to each of
the 51 state contacts and their responses.
1. Does your state allow the use of natural attenuation (often
termed remediation by natural attenuation, or RNA) as a
groundwater cleanup remedy for UST sites with petroleum
hydrocarbon contamination:
(a) in combination with other treatment technologies or
methods?
49 Yes
1 Yes site-specific
1 Yes post-remediation

Microaerophillic bacteria, from LaMountain, 1997. By

definition, these bacteria are oxygen-requiring organisms
that grow only at reduced oxygen concentrations, less than
one atmosphere. These organisms are supposedly aerobes
that do not have all of the protective enzymes to ward off
oxygen toxicity, so they can only grow at low levels of O2.
Degradation/transformation pathways. I think the vendors
are pushing organisms that will use nitrate as an electron
acceptor and use O2 only for the oxygenase enzymes. Ron
Olsen has performed some research on these. These
organisms would still require nutrient addition, depending on
the groundwater characteristics, e.g. nitrate or phosphates.
Olsen has said that if you add O2, then you will select for
other organisms that like O2 better, and these organisms will
never get a chance to operate. You are correct to assume
that the intermediates would be more polar than the original
hydrocarbons. You should see fatty acids and catechols or
carboxylic acids as degradation products. I think your source
of information was thinking lower molecular weight in
general, e.g. big multiple rings chopped into smaller ring
units, and not thinking about their substitution, e.g. having
hydroxyls or carboxylic acid groups on them.
I have dealt with many vendors of bioremediation techniques
or organisms, and many do not understand their products.
This is because they are salespeople. I usually ask them to
get their companys microbiologist to call me, so I can get
the straight story about the product.
If this is a major remediation project or a major expense for
your company, I would certainly ask for much more background than they have apparently given to you. Do not buyin to anything without a good deal of technical information
that a trusted microbiologist says is reliable; there are too
many people trying to make a quick buck in this business.

Underground Tank Technology Update

(c) Is a contingency plan required prior to RNA

implementation?
34 No
13 Yes
2 Yes afterwards
2 Site-specific
2. Pertaining to the specific use of natural attenuation or RNA,
has your state established:
(a) statutes
46 No
1 Planning to

4 Yes

(b) regulations
39 No
1 Planning to

11 Yes

(c) guidance
17 Yes
20 No
14 Considering/Developing
3. What cleanup goals are available for closure of groundwater
contamination in your state?
(a) Generic levels
44 Yes
1 Planning to
(b) Site-specific levels
46 Yes

6 No

5 No

4. To implement natural attenuation (or RNA, or monitoring

only), does your state require:
(a) site characterization
51 Yes
(b) source/hot spot control
43 Yes
2 Preferred
1 No

(b) as a sole remedy?

38 Yes
3 Yes site-specific
1 Yes not always
1 Yes as monitoring only
1 No site-specific possible
7 No

15

3 Site-specific
2 Yes not always

(c) free-phase/free-product removal

51 Yes to either minimal measurable levels or
to extent of technical feasibility

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14

Underground Tank Technology Update

1998 national RNA survey

by Mike Martinson
The May/June 1997 UTTU gave results from a national
survey of states on the use of remediation by natural
attenuation (RNA). The survey, using written questionnaires
or phone calls, gathered information from UST programs.
By late January 1997, all 50 states, the District of Columbia
and Guam had responded to the survey.
In January 1998, in cooperation with UTTU, Mike Martinson
of Delta Environmental Consultants, Inc. completed a similar
telephone survey. The original respondents from the UTTU
survey (if available) were asked basically the same questions to facilitate comparison with the UTTU 1997 survey. In
addition, questions pertaining to upcoming RNA and methyl
tert-butyl ether (MTBE) issues were posed.
Both surveys contained questions that could be construed
as somewhat ambiguous. The 1998 Delta survey, however,
was undertaken completely over the phone so as to assist in
clarifying questions. Authors of each survey acknowledge
that responses were subject to human bias: that is, even
regulators from the same state might not give the same
answer to the same question. In addition, the 1998 survey
queried other staff experts as suggested by the initial
regulatory contacts.
The telephone poller began the survey by specifying that the
topic was RNAs use in state UST programs, specifically for
groundwater petroleum hydrocarbon cleanup; thus,
responses were focused on the major chemical constituents
(i.e., BTEX, PAHs, and MTBE) of gasoline and diesel, rather
than on the rarer UST chlorinated compounds. In addition,
the focus of the 1998 survey was on groundwater contamination; very little information was obtained concerning soil
contamination.
The respondents were familiar with the definition of natural
attenuation: biological, physical and chemical transformations that can make contaminants less mobile and toxic, as
well as reduce their mass, volume and concentration. RNA
is regarded as a passive remediation technique whereby
natural processes are used to clean up a site; in contrast,
active remediation involves an engineered intervention that
expedites or enhances natural processes.
Comparison of results from both surveys indicate the
following:
all states continued in their previous assessment that
RNA was acceptable in combination with other remedies
to clean up petroleum-contaminated sites
the use of RNA as a sole groundwater cleanup remedy
increased from 1997 results (to about 75%), with only 7
states (<14%) in 1998 specifying that RNA was not
allowed as a sole remedy

May/June 1998

the 1997 survey indicated that most states had or

planned to establish statutes, regulations or guidance on
RNA; the 1998 survey further clarified this data:
4 states had statutes specific for RNA
1 state would add statutes by 1999
11 states had RNA-specific regulations
1 state planned to have new regulations for RNA
17 states currently had RNA-specific guidance
14 states planned to develop or consider guidance
the 1998 requirements for implementing RNA on a
groundwater remediation project were even more
consistent than those reported in 1997:
site characterization was mandatory for all states
all states favored free-phase removal to either
minimal measurement levels or until technical
infeasibility was demonstrated
the majority of the states required or preferred
groundwater monitoring (49 states) and source control
(45 states)
as stated in the 1997 survey, a stable or shrinking
groundwater plume extent (47 states) was considered
the most important (or primary) line of evidence to
demonstrate RNA effectiveness; decreasing concentration trends, according to 44 states, were also a primary
line of evidence
Additional RNA trends emerged from the 1998 survey:
groundwater RNA (including monitoring-only policies)
was considered an acceptable or site-specific remedy
alternative for most states gasoline/BTEX-impacted
sites (50 states) and diesel fuel/PAH-impacted sites
(49 states)
only 13 states required a contingency plan prior to RNA
implementation; 34 states did not require such a plan
risk assessment requirements to implement RNA were
approximately the same in 1997 (24 states) compared to
1998 (25 states); in addition to the states requiring risk
assessment, 9 states required site-specific
consideration for risk assessment in 1998, compared
to 4 states in 1997
groundwater cleanup goals for petroleum hydrocarbon
contamination were set at generic levels for 44 states
and as site-specific levels for 46 states
states increased their use of required and site-specific
use of geochemical indicators (i.e., secondary lines of
evidence) to support plume extent and concentration
trend data; geochemical indicators were required by
19 states in 1997; 20 states in 1998 had requirements
while 11 additional states were evaluating site-specific
cleanup requirements for secondary lines of evidence

May/June 1998

Underground Tank Technology Update

Microaerophillic bacteria, from Zang, 1997. I have seen some

of these products under different names. They may contain
nutrients like nitrate, which can be used as the electron
acceptor, for instance.
Degradation/transformation pathways. I really cannot see
how a lesser degradable PAH (higher molecular weight) can
result in an increase in BTEX. For one thing, when the
aromatic rings are broken, the molecule would be destabilized. The thing would essentially fall apart. More importantly, the resulting BTEX would be more biodegradable than
the parents.
Having said all that, I have even seen BTEX generating out
of thermal desorption processes. I have also seen a graduate
of UBC tell me that he saw increased BTEX through slurry
phase biotreatment. I think we should go back to the laboratory. If a sample has high hydrocarbons other than BTEX,
they will mask the BTEX because in order to perform GC
analyses, the laboratory will have to dilute the sample to a
certain degree. Thus, the BTEX may become non-detectable
in the diluted extract. In the treated sample, when background hydrocarbons are reduced to a lower level, BTEX
stands out.

Microaerophillic bacteria, from Gerlach, 1997. By the way,

cant we try to define the terms aerobic, anaerobic, anoxic,
microaerophillic . . . so that we can use them consistently
throughout our discussions? From a microbiological standpoint, you can always group bacteria into either aerobic or
anaerobic species; however, many bacteria have the
metabolic capability to grow aerobically or anaerobically,
depending on the conditions they discover; they are called
facultative aerobes, or facultative anaerobes.
In my opinion (I am an engineer) and the opinion of many
microbiologists:
aerobic means that oxygen (dissolved oxygen)
is available
anaerobic means that no (dissolved) oxygen is available.
For metabolism (biodegradation) to occur, other electron
acceptors must be available. Electron acceptors in
anaerobic environments can be nitrate, sulfate, iron,
manganese, carbon dioxide, or organics (fermentation);
and there may be a few more that are of little relevance
with respect to bioremediation
Saying no oxygen, however, has an inherent problem: Is
no oxygen absolutely no oxygen, or is oxygen just below
the detection limit of whatever method is being used? People
started using the term microaerophillic, or anoxic, for
environments where only little oxygen is available, usually
less than 0.2 mg/L, which is coincidentally the detection limit
of many oxygen tests! In my opinion anoxic has mostly
been used for environments where no, or little, oxygen is
available and nitrate is the dominant electron acceptor.
Anoxic is most widely being used in wastewater treatment.
For biodegradation to occur, electron acceptors must be
available. Thus, bacteria in microaerophillic environments
have to use other electron acceptors to biodegrade contaminants efficiently. Id be really careful if somebody tells me that

his bugs do not need any nutrient-addition while others do,

something the vendor might not explicitly state, but suggest.
Nutrients are required for bacterial growth and for efficient
bioremediation. If the required nutrients are not available in
the system (bioreactor, aquifer), they must be supplied.
There is no direct relationship between nutrient supply
(usually N, P) and electron acceptor; however, there might
be electron acceptors that can also serve as a nutrient, e.g.
nitrate. What your sources might be meaning to say is that
the relative abundance of BTEX and other aromatics is
increasing. That indicates that easily degradable compounds such as straight-chain aliphatic hydrocarbons are
being degraded more readily than aromatics or branched
aliphatics. Thus, the overall TPH concentration can decrease, but the relative abundance of persistent compounds
can increase . . . unless there is a mobilization mechanism,
such as biosurfactant production, that increases the
concentration of dissolved contaminants. I do not have an
idea why low-molecular-weight alkanes should increase.
Alkanes are usually degraded via carboxylic acids; these
concentrations can increase but are usually of little
regulatory concern. However, as mentioned above, the
relative abundance of branched alkanes can increase,
e.g., pristane and tristane in a diesel contamination.

Terminology consensus
From LaMountain, 1997. I agree that a consensus on
terminology would be great. I have found the following to be
a general consensus among anaerobic microbiologists, and
consequently this is what I teach to my students:
aerobicwhen O2 is present
microaerophilliclimiting O2 (less than 1 mg/L);
this describes organisms, rather than environmental
conditions; the organisms have specific requirements
of low O2
anoxicnitrate is the main electron acceptor
anaerobicno O2, no nitrate, usually sulfate or
CO2 as electron acceptor
fermentativeno electron acceptor available, characterized by production of low-molecular-weight acids and
solvents; this is unusual in engineering reactors or soils
but common in industry
I havent heard of any opinions or new names for ironreducing conditions; I suppose they would have to go in
with anoxic.

Descriptions of aerobic, anoxic-anaerobic and

fermentative organisms
Aerobic, from Pelmont, 1997. Aerotolerant organisms
should be mentioned, for instance, Lactobacilli. They are
anaerobes with no respiratory chain and no use of O2.
However, they still do fermentation in the presence of
some O2, i.e. in aerobic conditions. Other anaerobic bugs
such as some sulfate-reducing species have been shown
to stand low levels of O2. This is very important in the
environment since they can thrive at the limit between
aerobic conditions and oxygen-depleted media in water,
mats and even biofilms.

Underground Tank Technology Update

Anoxic-anaerobic, from Pelmont, 1997. Nitrate is just one

electron acceptor. Here we call denitrification anaerobic
respiration, using nitrate as an acceptor. This is dissimilatory reduction of nitrate, producing typically N2O and N2.
Assimilation of nitrate is reduction to ammonia. The
distinction between these two modes, however, is not
always clear. Nitrate-using respiration shifts easily to other
acceptors when nitrate is depleted: dimethylsulfoxide,
trimethylamine oxide, fumarate, and others. E. coli does so,
with a hierarchy of acceptors under control at the transcription level. I do not see the usefulness of this distinction
between anoxic and anaerobic. Other respirations involve
the reduction of Fe(III), Mn(II). Methanogenesis has been
shown to be a respiratory process producing energy with
CO2 or acetate as an acceptor.

May/June 1998

than in the gas phase, an oxic system can become anoxic

quite quickly, where there is lots of water. The next most
efficient electron acceptor commonly found in the
environment is nitrate, which has a redox equilibrium (Eh)
of 421 mV. Nitrate, nitrite and nitrous oxide are utilized, in
lieu of dioxygen, as oxygen acceptors by many common
aerobic soil bacteria, e.g. Pseudomonas, Bacillus,
Alcaligenes. Denitrifying bacteria may thus be considered
as facultative aerobes. They simply switch over to using
the next best electron acceptor when O2 is not available.
Ferric iron (Eh= 357, pH 7.0) is the next best electron
acceptor. Several books give an erroneous value of 770 mV,
which is the value at pH 0. Many, but not all, denitrifying
bacteria can use Fe+3 as an electron acceptor.

May/June 1998

Underground Tank Technology Update

Oppenheimer, C.H., Miget, R.J. and H.I. Kator, Ecological

Relationships Between Marine Microorganisms and
Hydrocarbons in the O.E.I. Study Area, Louisiana, Rice
University Studies, Vol. 65, p. 287-325, 1979.

Stotzky, G., Influence of Clay Minerals on Microorganisms:

III Effect of Particle Size, Cation Exchange Capacity and
Surface Area on Bacteria, Canadian Journal of Microvvbiology, Vol. 12, p. 1235-1246, 1966.

Oppenheimer, C.H. and F.K Hiebert, Microbially Enhanced

Oil Production Field Tests in Texas, Proceedings of the
Symposium on Applications of Microorganisms to
Petroleum Technology, USDOE/NIPPER, Bartlesville,
Oklahoma, 1988.

Tausson, W.O., Bacterial Oxidation of Crude Oils,

Neftyanoe Khoz, Vol. 14, p. 220-230, 1928.

Oppenheimer, C.H. and F.K. Hiebert, Microbiological

Techniques for Paraffin Reduction in Producing Oil Wells,
Department of Energy Final Report, DOE/BC/14014-9
(DE89000741), 67 p., 1989.
Pasteur, L., Ann.Chim.Physics., Vol. 58, 323 p., 1860.

Fermentative. We here use a biochemical definition of

fermentation. Fermentative processes produce ATP at the
substrate level. Respiration is an energetic process building
directly a membrane potential, ATP being made with H+ or
Na+ translocating ATP syntheses. This is usually a clear-cut
definition, as proposed in the past by Slater . . . According
to this definition, acetic acid production from ethanol is not a
fermentation, just a respiration going to CO2 and H2O if not
stopped in time. Fermentation is a word commonly used in
industry, whatever the biochemical mechanisms are.

Obligate anaerobes (respiratory), from Focht, 1997.

These are the domain of the sulfidogens (sulfate reducers,
Eh =-190mV), which may be thought of as having a very
truncated respiratory system. This is a tough way to make a
living: note the small energy difference, about 200 mV, in
oxidizing H2 this way as opposed to coupling H2 oxidation
with O2, 1230mV. But if table scraps are all that remain, then
these organisms prevail under these meager conditions. The
sulfide producers and the methanogens use molecular H2 as
their energy source. H2 is generated by the other group of
anaerobes immediately below.

Pashley, R.M., McGuiggan, P.M., Ninham, B.W. and D.F.

Evans, Attractive Forces Between Uncharged Hydrophobic
Surfaces: Direct Measurements in Aqueous Solution,
Science, Vol. 229, p.1088-1089, 1985.

Since fermentation is often a lower energy-yielding process,

it is usually characterized by the making of large amounts
of organic by-products. Fermentation cannot be carried in
oligotrophic conditions. But organic compounds are not the
sole by-products: CO2 and H2 are also abundant sometimes, as in the case of some obligate anaerobes as
Clostridia doing efficient fermentation with the production
of a lot of gas. A bug like Clostridium thermoaceticum,
however, is a true acetogenic species, producing acetic
acid according to a mechanism that is interpreted as a
CO2-using respiration, according to the above definition.

Obligate anaerobes (fermentative), from Focht, 1997. So far,

we have been talking about inorganic electron acceptors
(respiration). Fermentation, by Pasteurs definition, is the use
of organic electron acceptors. The production of lactic acid
(e.g. yogurt production) from glucose by the lactic acid
bacteria (which are microaerophiles) results actually from the
reduction of pyruvate to give a stoichiometric balance of
protons and electrons in lieu of an inorganic electron
acceptor. The bacteria of the genus Clostridium actually
produce H2 gas during the production of protons and
electrons.

Science News Letter, Nov. p. 297, 1942.

Shabtai, Y. and D.L. Gutnik, Exocellular Esterase and
Emulsan Release from the Cell Surface of Acinetobacter
Calcoaceticus, Journal of Bacteriology, Vol. 161, p.11761181, 1985.
Sharpley, J.M., Elementary Petroleum Microbiology, Gulf
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Shennan, J.L. and I. Vance, Microbial Enhanced Oil Recovery
Techniques and Offshore Oil Production, in Microbial
Problems in the Offshore Oil Industry, p. 73, Ed. E.C. Hill,
et.al., Wiley and Sons, Chinchester, 1987.
Sieburth. J.Mc.N., Microbial Seascapes, University Park Press,
Baltimore, 248 p., 1975.
Sieburth, J.Mc.N., Sea Microbes, Oxford University Press,
491 p., 1979.
Simanov, A.I., Nazarov, M.I., Gruzinov, N.V., Afanas-Eva,
M.A. and V.P. Andryukov, Meteorologiya i Gidrologiya,
Vol. 3, p. 64-72, 1984.
Smith, P.V., Preliminary Note of Origin of Petroleum, Bulletin
of American Association of Petroleum Geologists, Vol. 36,
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Sondheimer, E. and J.B. Simeone, Chemical Ecology,
Academic Press, N.Y., 336 p., 1970.
Stephenson, M., Bacterial Metabolism, Longmans, Green
and Co., London, 398 p., 1949.
Stone, R.W., White, A.G.C. and M.R. Frenske, Journal of
Bacteriology, Vol. 39, p. 91, 1940.

Terms applied to bacterial physiology

and thermodynamics
The words describing aeration have unfortunately become
confusing due to incorrect usage, even among microbiologists. Lets consider these terms as they pertain to bacterial
physiology and thermodynamics.

Aerobic, from Focht, 1997. Aerobic refers to life with air,

ergo, molecular oxygen. We fall into this category. O2 is the
most efficient electron acceptor known because of the high
redox potential (816 mV, pH 7.0) for the O2/H2O couple at a
1:1 molar ratio, referred to as the Eh. The Eh for the H2/2H+
is 414 mV, pH 7.0). Rather than argue about how little
oxygen it takes to be anaerobic or anoxic (without molecular
oxygen), we should view oxidation/reduction reactions from
the perspective of the Nernst equation; that is the basis of
electro-chemistry. Energy can be evaluated and compared
by use of electro-chemical potentials.
Anoxic, from Focht, 1997. Consider what happens in the
environment as a result of respiration. Because diffusion of
molecular oxygen in water is about 100,000 times slower

Facultative anaerobes, from Focht, 1997. Microorganisms,

including yeasts and other eucaryotes as well as bacteria,
that can grow aerobically by respiration or anaerobically by
fermentation, fit this term as defined by Pasteur. Unfortunately, too many textbooks have corrupted this term by
incorrectly calling denitrifying bacteria facultative anaerobes.
Denitrifying bacteria are not fermentative: they are strictly
respiratory and are unable to use organic substrates as
electron acceptors. Hence, they (and iron and manganesereducing bacteria) should correctly be referred to as facultative anaerobes, i.e. they are obligate respiratory bacteria.
Microaerophiles, from Focht, 1997. These bacteria are
basically fermentative anaerobes. They are different,
however, from obligate anaerobes because they are able to
tolerate small quantities of oxygen during growth. How
small? That depends on the environment. Microaerophiles
have an incomplete cytochrome system, such that when
protons react with O2, hydrogen peroxide is produced. Even
more toxic are super oxide radicals (O2-) that are generated
during respiration. All aerobic organisms have superoxide
dismutase (SUD), which converts super oxide radicals to

13

Perfiliev, B.V. and D.R. Gabe, Capillary Methods of Investigating Microorganisms, Translated from Russian, University
of Toronto Press, J.M. Shewan, Editor, 1969.
Perry, J.J., Microbial Cooxidations Involving Hydrocarbons,
Microbiological Reviews, Vol. 43, p. 59-72, 1979.
Pinta, M., Detection and Determination of Trace Elements,
Translated from French, Ann Arbor-Humphrey Science
Publisher, Ann Arbor MI, 588 p., 1970.

Teal, J.M. and R.W. Howarth, Oil Spill Studies: A Review of

Ecological Effects, Environmental Management, Vol. 8,
p. 27-44, 1984.
Thimann, K.V., The Life of Bacteria, Macmillan, N.Y., 909 p.,
1964.
Twenhofel, W.H., Treatise on Sedimentation, Dover Publications, New York, Two Volumes, 926 p., 1961.
Vandermeulen, J.H., Some Conclusions Regarding Long-Term
Biological Effects of Some Major Oil Spills, Phil. Trans. R.
Society of London, Vol. 297, p. 335-351, 1982.
Van der Linden, A.C. and G.J.E. Thijsse, The Mechanisms of
Microbial Oxidations of Petroleum Hydrocarbons, Adv.
Enz., Vol. 27, 1965.
Ward, C.H., Bender M.E. and D.J. Reish, The Offshore
Ecology Investigation, Rice University Studies, Vol. 65,
Nos. 3&4, 589 p., 1979.
Warner, J.S., Determination of Aliphatic and Aromatic
Hydrocarbons in Marine Organisms, Analytical Chemistry,
Vol. 48, p. 576-583, 1976.
Wentworth, C.K., A Scale of Grade and Class Terms for
Classifying Sediments, Journal of Geology, Vol. 30,
p. 377-391, 1922.
Yen, T.F., University of Southern California, A State of the
Art Review on MEOR, NSF Grant OIR-8405134, 1988.
Young, L.Y. and C.E. Cerniglia, Eds., Microbial Transformation
and Degradation of Toxic Organic
Chemicals, Wiley-Liss Inc., New York, 1995.
ZoBell, C.E., The Effect of Solid Surfaces Upon Bacterial
Activity, Journal of Bacteriology, Vol. 46, p.39-56, 1943.
ZoBell, C.E., Marine Microbiology, Chronica Botanica,
Waltham, MA., 240 p., 1946.
ZoBell, C.E., U.S. Patent #2,413,278, Described a process by
which bacteria release oil from geological formations, 1946.
ZoBell, C.E., Studies on Redox Potential of Marine Sediments, Bulletin of American Association of Petroleum
Geologists, Vol. 30, p. 477-513, 1946.
ZoBell, C.E., Assimilation of Hydrocarbons by Microorganisms, Advance Enzym., Vol. 10, p. 443-486, 1950.
ZoBell, C.E., Bacterial Activities and the Origin of Oil, World
Oil, Vol. 130, p. 128, 1950.
ZoBell, C.E., U.S. Patent # 2,742,398, described a process to
reduce paraffin in oil wells, 1956.
UTTU graciously thanks Dr. Carl Oppenheimer,
carlo@mail.utexas.edu., for sending us his reference list.

12

Underground Tank Technology Update

Haas, H.F., Yantzi, M.F. and L.D. Bushnell, Microbial Utilization of Hydrocarbons, Trans. Kansas, Academic Science,
No. 44, p. 39-45, 1941.
Hildebrand, H.H. and G. Gunter, Deposition of Petroleum Tars
and Asphalts, Beaches of the Northern Gulf of Mexico,
UTMSI Report, University of Texas, Port Aransas, Texas,
88 p., 1955.
Hitzman, D.O., Petroleum Microbiology and the History of its
Role in Enhanced Oil Recovery, in E.C. Donaldson and
J.B. Clark (Eds.), Proceedings, 1st International Conference
on Microbial Enhanced Oil Recovery, May 16-21, 1982,
Afton, Oklahoma, p.162-218, 1983.
Horvath, R.S., Microbial co-Metabolism and the Degradation of
Organic Compounds in Nature, Bacteriological Reviews,
No. 36, p. 146-155, 1972.
Hunt, John M., Petroleum Geochemistry and Geology, W.H.
Freeman and Company, San Francisco, 617 p., 1979.
Hutner, S.H., Nutrition of Protists, in This is Life, Johnson and
Steere, Editors, Holt Reinhart and Winston, New York,
1962.
James, A.M., The Electrochemistry of the Bacterial Surface,
Proceedings of Biophysics and Biophysical Chemicals,
No. 8, p. 98-144, 1957.
Johnson, F.H., Goodale, W.T. and J. Turkevich, The Bacterial
Oxidation of Hydrocarbons, Journal Cell. Comp. Physiol.,
Vol. 19, No. 163-172, 1942.
Kator, H.I., Utilization of Crude Oil Hydrocarbons by Mixed
Cultures of Marine Bacteria, Unpublished PhD Thesis, the
Florida State University, Department of Oceanography,
1972.
Kator, H.I., Oppenheimer, C.H. and R.J. Miget, 1971, Microbial
Degradation of a Louisiana Crude Oil in Closed Flasks and
Under Simulated Field Conditions, Proceedings of the Joint
Conference on Prevention and Control of Oil Spills,
American Petroleum Institute, EPA, and U.S.C.G.,
p. 287-296, 1971.
King, J.W. and D.A. Stevens, Proceedings of the First International MEOR Workshop, Department of Energy, April 1-3,
1986, Bartlesville, Oklahoma, NTIS DOE/BC/10852-1,
(DE87001216), 373 p., 1987.
Kinghorn, R.R.F., An Introduction to the Physics and Chemistry
of Petroleum, Wiley and Sons, New York, 420 p., 1983.
Krumbein, W.E., Editor, Microbial Geochemistry, Blackwell
Science Publications, 330 p., 1983.
Kuznetsov, S.I., Ivanov, M.V. and N.N. Lyalikova, Introduction
to Geological Microbiology, Trans. 1963, McGraw-Hill,
New York, 1962.
La Riviere, J.W.M., The Production of Surface Active Compounds by Microorganisms and its Possible Significance
in Oil Recovery II, Antonie v. Leeuwenh, Journal of
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Lederberg, J. and E.M. Lederberg, Journal Bacteriology,
63:399, 1952.
Lee, C.C. and W.K. Craig, Water Soluble Hydrocarbons
from Crude Oil, Bulletin Environmental Contaminated
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Leeuwenhoek, A. van, 1694, Ondervindingen en
Beschouwingen der Onsigtbar Geschapene Waarheden,
2nd Ed., p 45., Delft, in Dobell, 1932.

May/June 1998

McKenna, E.J. and R.E. Kallio, The Biology of Hydrocarbons,

Annual Reviews of Microbiology, Vol. 19, p.183, 1965.
Marr, E.K., The Bacterial Oxidation of Benzene, Dissertation,
Pennsylvania State College, 1959.
Margulis, L., Early Life, Jones and Bartlett, Boston. 260 p.,
1984.
Mason, S.F., Chemical Evolution, Origin of the Elements,
Molecules and Living Systems, Clarendon Press, Oxford,
317 p., 1992.
Master, M. and C.H. Oppenheimer, On the Solution of Quartz
and Precipitation of Dolomite in Sea Water During
Photosynthesis and Respiration, Zeit. F. Allgemeine
Mikrob, Vol. 5, p.48-51, 1965.
Meinschein, W.G., Origin of Petroleum, Bulletin of the
American Association of Petroleum Geologists, Vol. 43,
p. 925, 1959.
Meinschein, W.G., Biological Markers and n-Alkanes as
Geological Agents, in Organic Geochemistry of
Contemporaneous and Ancient Sediments, Ed. Meinschein,
Great Lakes Section Society of Economic Paleontologists
and Mineralogists, Bloomington, Indiana, Chapter 2,
p. 29, 1983.
Miget, R.J., Oppenheimer, C.H., Kator, H.I. and P.A. LaRock,
Microbial Degradation of Normal Paraffin Hydrocarbons
in Crude Oil, Proceedings for the Joint Conference on
Prevention and Control of Oil Spills API-FWPCA,
December, p. 327-331, 1969.
Montgomery, C.W., Fundamentals of Geology, W.C. Brown,
357 p., 1989.
National Academy of Sciences, Productivity of World
Ecosystems, NAS, Washington D.C., 166 p., 1975.
Oppenheimer, C.H., Thesis, University of California at
Los Angeles, Effect of High Pressure on Marine
Microorganisms, 1951.
Oppenheimer, C.H. and L. Kornicker, Effect of Microbial
Production of Hydrogen Sulfide and Carbon Dioxide on the
pH of Recent Sediments, Publication of the Institute of
Marine Science, The University of Texas, Vol. 5, p. 5-15,
1958.
Oppenheimer, C.H., Bacterial Production of Hydrocarbon-like
Materials, Zeitschrift fur Allgemeine Mikrobiologie, Vol. 5,
No. 4, p. 284-307, 1965.
Oppenheimer C.H., Eh and pH of Marine Sediments, in
Encyclopedia of Earth Sciences, Reinhold Publications
Company, New York, 1966.
Oppenheimer, C.H., Testimony Presented to the Oversight
Hearings on the Effect to the United States on the Blowout
of the Pemex IXTOC Oil Well, September 11, 1979.
Oppenheimer, C.H., Personal Paper, Collection of Papers
Prepared for the Bureau of Land Management, Offshore
Oil Lease Hearings, Louisiana, Mississippi, Florida, New
York, 1980.
Oppenheimer, C.H., Oil Ecology, Chapter 1, Marine Environmental Pollution,1, Hydrocarbons, R. Geyer, Editor, p. 2135, Elsevier Oceanography Series, Amsterdam, 1980.
Oppenheimer, C.H. and W. Drost-Hansen, A Relationship
Between Multiple Temperature Optima for Biological
Systems and the Properties of Water, Journal of
Bacteriology, Vol. 80, p.21-24, 1960.

May/June 1998

Underground Tank Technology Update

hydrogen peroxide. We and all other aerobes also have

catalase, which converts H2O2 to O2 and H2O.
Microaerophiles have SUD, but not catalase, while obligate
anaerobes have neither.
Clinical bacteriologists readily observe that it is quite easy to
grow hemolytic streptococci on blood agar plates, but not on
other agar plates. So guess where microaerophiles are
most commonly found, and what is so unique about blood:
the mammalian mouth and the lining of other body cavities,
which contain lots of catalase. Not surprisingly, most
microaerophiles that reside at low oxygen concentrations
are pathogens (hemolytic streptococci, diplococci) while
others that reside in anaerobic environments (lactic acid
bacteria) are not pathogenic. Based on what we know
about the ecology of soil bacteria, the significance of
microaerophiles is obscure and not likely to be important vis
a vis biodegradation because other bacteria would be better
competitors under microaerophillic or anaerobic conditions.

Additional information
For additional information on definitions of anaerobes or
associated metabolic processes, consult the issues of The
American Society of Microbiology News published in the
early 1980s. A number of definitions were proposed and
debated in the Letters to the Editor (Liss, 1997).

Acknowledgments
UTTU thanks the following for their contributions to this
article: Dr. Martin Whittaker, Ph.D., Golder Associates Ltd.,
905-567-4444, mwhittaker@golder.com; Dr. Debbie Roberts
LaMountain, Department of Civil and Environmental
Engineering, University of Houston, 713-743-4281,
djroberts@uh.edu; Mike Barden, Geoscience Resources
LTD, Albuquerque, New Mexico, 505-821-5508,
mike-barden@ibm.net; Robin Gerlach, Center for Biofilm
Engineering, Montana State University, Bozeman, Montana,
406-994-4770, robing@erc.montana.edu; Dr. Steven Liss,
Ryerson Polytechnic University, Toronto, Ontario, 416-9795000, sliss@acs.ryerson.ca; Dr. Allan Zhang, OConnor
Associates Environmental, Inc., Langely, British Columbia,
604-513-1005, allan-zhang@oconnor-associates.com;
Dr. Jean Pelmont, Universite Joseph Fourier, Grenoble,
France, 33-0-476-51-48-05, jean.pelmont@ujf-grenoble.fr;
Dr. Dennis Focht, University of California, 909-787-3446,
focht@citrus.ucr.edu. The views of these individuals are not
necessarily those of their organizations.
UTTU also thanks Richard Schaffner, P.G., technical
specialist, GZA GeoEnvironmental Inc., moderator of the
Bioremediation Discussion Group. For administrative
information on the BioGroup, please visit the BioGroup
home page (http://biogroup.gzea.com); for additional
information, send a message to rschaffner@gzea.com.

Engineered microbe effectiveness,

bioaugmentation in lab vs. field
and transgenic microorganisms
This article is based on a November 1997 discussion within
the BioGroup. The exchange began when a member
wanted to verify a claim that a catalyst that contained
facultative anaerobes and microaerophiles would draw
oxygen down into the substrate and would complete
petroleum hydrocarbon degradation in 60 days. The
member wanted to know if there were any methods to
demonstrate the superbugs effectiveness.

Assessing microbe effectiveness

From Rie, 1997. Several methods are available to check
the effectiveness of the additions. First, a word of caution:
I have yet to find a refereed, peer-reviewed article in any
publication that shows any more than a brief (30-day or
less) acceleration of natural biodegradation when microbes
are added. This is in comparison to equal treatment of a
control plot without microbe addition. Normally, some
combination of moisture, nutrient, and/or air addition is
enough to stimulate existing microbes. See, for instance,
the numerous Air Force studies.
As for assuring the effectiveness of the native or foreign
(indigenous or added) microbes, several methods exist.
Probably the best cheap method is a simple respiration test,
measuring oxygen and carbon dioxide in test wells that are
already in place. Water samples could be checked for
dissolved oxygen and pH, which will decrease with increasing carbon dioxide levels.
A second method would be a relatively inexpensive lab
count of hydrocarbon-degrading microbes, using core
samples, carefully taken during soil sampling for hydrocarbons. Such testing would have to be carefully planned,
but it could yield total heterotrophic plate counts as well
as hydrocarbon degraders for a cost of less than $2,000
$3,000. We recently developed a method for specifically
measuring hydrocarbon degraders, which, when combined
with TPH data, can give an excellent picture of what is
going on. To complete the picture, TPH measurements
can be taken for the same series of samples. Several
laboratories, including ours, provide the biological testing
and planning of sample taking at nominal fees.

Potential biodegradation: the lab vs. the field

From Oppenheimer, 1997. A quantitative jump exists
between the ideal lab and the field. Believe me, Ive taught
geo and marine microbiology for 40 years and for the past
7 years Ive been attempting to be a businessman. The
bottom line for the multitude of small projects is to get
results without an expensive procedure.

Underground Tank Technology Update

We screen all sites by a simple method. Homogenize

thoroughly a 500 g soil samplewater is easyand run
a baseline analysis on a composite of at least five grab
samples. Separate the soil into two 200 g samples. Place
the samples in shallow glass trays to a depth of one inch.
One is the control. To the second add your product. Moisten
the two samples to a mud consistency. Place on the bench
and allow to dry, stirring once a day. If working with high
volatiles, use a closed sealed glass pan. There will still be
sufficient oxygen because only two atoms of oxygen are
required to oxidize one hydrocarbon molecule. The open
trays will dry in about seven days. Residual water in the
covered trays will require adjustment.
Mix thoroughly all samples and take a composite of at least
five small samples from each tray. Mix the composites for
analysis. The results will give you information on potential
biodegradation rate, and you dont have to bill your customer
some outlandish fee. If the test is negative, go back to the
lab bench or change microorganisms. Old sites that have no
history of contamination are always suspect. The basic
problem is to select the right bacteria and understand the
environmental conditions to keep the living system viable.

From Wrenn, 1997. Although this method can probably tell

you if bioaugmentation will not work at a particular site
(assuming an adequate number of replicates are used), it
almost certainly will not give you enough information to
determine that it will. Two processes are important in
determining whether bioaugmentation will be effective:
bacterial transport and survival. The extensive and frequent
mixing that Dr. Oppenheimer uses simulates landfarming
reasonably well, but most other bioremediation processes
(especially in-situ processes) are much less well mixed.
Transport of bacteria from an injection well into a contaminated formation is difficult, because bacteria are sticky
particles that will be filtered out of suspension by the soil
particles. Also, if some type of inducer (for a desirable
enzyme), or solubility-enhancing compound (to improve the
bioavailability of the contaminants) is included in the product
being tested, these materials will remain in close proximity to
the added bacteria in the laboratory microcosms; however,
they probably will not move through the subsurface at the
same rate as the bacteria. This will obviously change the
way the microbial product performs in the field relative to
the laboratory. Furthermore, there is no reason to assume
that survival of an introduced microbial population in a
well-mixed and aerated laboratory microcosm is an indicator
of how well those organisms will survive in the subsurface,
where the concentration gradients of oxygen and hydrocarbons can be steep.
Bioaugmentation should always be evaluated in the field. It
should work in the laboratory. If it doesnt, the microorganisms in the product are poorly matched to the target contaminants or there is a problem with their viability. Success
in the lab, however, doesnt indicate that the product will
work in the field. The factors that determine success in the
field cannot be evaluated in simple laboratory microcosms.
Field studies must be carefully designed to distinguish
between the effects of bioaugmentation and those due to

May/June 1998

pumping water that contains oxygen, nutrients, and/or

surfactants.
One minor correction on the subject of laboratory treatability
studies: more than two atoms of oxygen are required to
oxidize most hydrocarbon molecules. Two moles of O2 (i.e.,
four oxygen atoms) are required to completely oxidize one
mole of methane, the simplest hydrocarbon. In general,
between 1.2 and 1.5 moles of O2 are required per carbon
atom to completely oxidize the hydrocarbons of interest in
bioremediation (e.g., BETX and above). Most target hydrocarbons have at least six carbon atoms and will require at
least 7.5 molecules of O2 for complete oxidation of each
molecule. The rule of thumb is 3 mg O2 per mg of hydrocarbon, which is equivalent to 11 ml of air per mg hydrocarbon.
Obviously, the volume of air required to completely mineralize the hydrocarbons in 200 g of soil depends on the degree
of contamination. If the soil has 5,000 ppm of degradable
hydrocarbons, 11 liters of air are required to provide sufficient oxygen for complete biodegradation. Most treatability
studies wont be conducted long enough to achieve that
degree of mineralization, but it would be foolish to draw
conclusions based on less than 10% mineralization, and
even that minimal level of treatment requires more than 1
liter of air. Also, the biodegradation rate will become oxygen
limited long before the O2 in the headspace is completely
exhausted. So, it isnt valid to assume that enough O2 will be
available, unless you do a few quick calculations on the size
of your system relative to the amount of hydrocarbon
degradation that you want to observe.

Questions raised concerning transgenic bugs

Another member wanted specific information on a genetically modified microorganism with hydrocarbon-degrading
capabilities that was patented in 1981. He also wanted to
know if transgenic bacteria were used for ex-situ bioremediation purposes. Finally, he wanted to know what risks
transgenic microorganisms presented to the environment.

From Glass, 1997. I have a great deal of experience with

U.S. regulation of genetically engineered microbes, including
nine years in the agricultural biotech industry when field tests
were first conducted in the mid to late 1980s. My company
conducted early field tests of genetically engineered
rhizobia for nitrogen fixation. I have been involved with the
bioremediation industry since 1990, and I have been a
proponent of using engineered microbes for bioremediation,
when their use made sense technically and economically.
Most individuals believe that transgenic microbes pose no
additional risk and that the regulatory process to gain
approval for their use is manageable and achievable. Many
bioremedial professionals, however, are apprehensive about
using engineered microbes because of possible adverse
public or governmental reaction; the simple fact is, for most
currently utilized applications of bioremediation, engineered
bugs are just not needed.
With respect to the microorganism that A.M. Chakrabarty
patented in 1981, I believe these microbes were never used.
There is generally no reason to improve hydrocarbondegrading bacteria through genetic engineeringnaturally

May/June 1998

Underground Tank Technology Update

History of bioremediation
references
The following references offer a glimpse of bioremediation
history.
Alexander, M., Introduction to Soil Microbiology, Wiley, N.Y.,
472 p., 1961.
American Petroleum Institute, Federal Water Pollution Control
Administration, Proceedings to the Joint Conference on
Prevention and Control of Oil Spills, 345 p., 1969.
American Petroleum Institute, Annual Oil Spill Conference,
Washington D.C., 1969 to present.
Atlas, R.M., Microbial Degradation of Petroleum Hydrocarbons: an Environmental Perspective, Microbiological
Reviews, No. 45, p. 180-209, 1981.
Azoulay, E. and J.C. Senez, Degradation bacterienne des
hydrocarbures parafinniques II. Determination des produits
intermediares par la methode des adaptations
simultanees, Ann. d. Inst. Pasteur, No. 8, p.868-879, 1960.
Baas Becking, L. G. M., Kaplan, I. R. and D. Moore, Limits of
the Natural Environment in Terms of pH and Oxidation
Reduction Potentials, Journal of Geology, No. 68, p. 243284, 1960.
Baas Becking, L. G. M., Geology and Microbiology, Contr.
Marine Microbiology, New Zealand, New Zealand Oceanographic Institute, Memoir 3, p. 48-64, 1959.
Beerstecher, E., Petroleum Microbiology, Elsevier Press,
375 p., 1954.
Blank, M., Ed., Chemistry of Biological Systems, Advanced
Experimental Medicine and Biology, Vol. 7, Plenum Press,
New York, 1970.

11

Clerk, R.B., Marine Pollution, Oxford, Clarendon Press,

168 p., 1992.
Colwell, R.R. and J.D. Walker, Ecological Aspects of Microbial
Degradation of Petroleum in the Marine Environment,
Critical Reviews in Microbiology, No. 5, p. 423-445, 1977.
Conservation Foundation, State of the Environment, An
Assessment Mid-Decade, Conservation Foundation,
Washington D.C., 586 p., 1984.
Corredor, J.E., Morell, J.M. and C.D. Castillo, Persistence of
Spilled Crude Oil in a Tropical Intertidal Environment,
Marine Pollution Bulletin, No. 21, p. 358-388, 1990.
Council on Environmental Quality, Environmental Quality 9th
Annual Report, Washington D.C., 599 p., 1978.
Davis, J.B., Petroleum Microbiology, Elsevier Publications,
Amsterdam, 604 p., 1967.
DeRosa, M., Gambacorta A. and A. Gliozzi, Structure,
Biosynthesis and Physicochemical Properties of
Archaebacterial Lipids, Microbiology Review, No. 50,
p. 70-80, 1986.
Dobell, C., Antonie van Leeuwenhoek and his Little Animals,
London Staples Press, 1932.
Driver, J.I., The Geochemistry of Natural Waters, Prentice Hall,
Englewood Cliffs, New Jersey, 437 p., 1988.
Drucker, H. and R.E. Wildung, Biological Implications of
Metals in the Environment, Proceedings Symposium,
Technical Information Center, Energy Research and
Development Administration, 682 p., 1975.
Ehrlich, H.L., Geomicrobiology, Marcell Dekker, New York,
393 p., 1981.
Englemann, T.W., Bacterium photometricum, Ein Beitrag
zurverglichenden Physiologie des Licht und Farbensinnes,
Pflugers Arch., No. 30, p. 95, 1883.
Fenchel, T. and T.H. Blackburn, Bacteria and Mineral Cycling,
Academic Press, London, 225 p., 1979.

Blumer, M. and J. Sass, Oil Pollution Persistence and

Degradation of Spilled Fuel Oil, Science, No. 176,
p. 1120-1122, 1972.

Ford, B.J., Microbe Power, Stein and Day, New York, 181 p.,
1976.

Bowan, H.J.M., Trace Elements in Biochemistry, Academic

Press, New York, 241 p., 1966.

Garrels, R.M., Mineral Equilibria, New York, Harper Brothers,

254 p., 1960.

Boylan, D.B. and B.W. Tripp, 1971, Determination of Hydrocarbons in Sea Water: Extracts of Crude Oil and Crude Oil
Fractions, Nature, No. 230, p. 44-47, 1971.

Gabler, R.E., Sager, R.E., Brazier, S.M. and D.L. Wise,

Essentials of Physical Geography, Saunders College
Publication, Philadelphia, 550 p., 1987.

Brock, T.D., Smith, D.W. and M.T. Madigan, Biology of

Microorganisms, Prentice Hall, Englewood Cliffs, New
Jersey, 847 p., 1984.

Gibson, D.T., Cardini, G.E., Maseles, F.C. and R.E. Kallio,

Incorporation of Oxygen-18 into Benzene by Pseudomonas Putida, Biochemistry, Vol. 9, p. 1631, 1970.

Brown, S.O., Microbial Oxidation of Crude Oils and Water

Soluble Fraction of Crude Oils as Shown by the B.O.D.
Method, Texas A&M Research Foundation, Project 9, p. 2,
1950.

Gliozzi, A., Rolandi, R., DeRosa, M., Gambacorta, A. and B.

Nicolaus, Membrane Models of Archaebacteria, in
Transport in Biomembranes, Edited by R. Anolini, Raven
Press, New York, 1982.

Bushnell, L.D. and H. F. Haas, The Utilization of Certain

Hydrocarbons by Microorganisms, Journal of Bacteriology,
No. 41, p. 653-673, 1941.

Gortner, R.A. and W. Gortner, Outlines of Biochemistry, John

Wiley, New York, 1078 p., 1949.

Calvin, M., Chemical Evolution, Oxford University Press,

278 p., 1969.
Campbell, R., Microbial Ecology, Blackwell Science Publication, Oxford, 191 p., 1983.
Carey, F.A., Organic Chemistry, McGraw-Hill, New York,
1219 p., 1989.

Gundlach, E.R., Boehm, P.D., Atlas, R.M., Ward, D.M. and

D.A. Wolfe, The fate of AMOCO CADIZ Oil, Science,
No. 221, p. 122-129, 1980.
Gunkel, W., Gassmann, G., Oppenheimer, C.H. and I. Dundas,
Preliminary Results of Baseline Studies of Hydrocarbons
and Bacteria in the North Sea, 1975, 1976 and 1977,
Spanish Conference on Hydrocarbon Pollution, Santiago de
Compostela, 38 p., April 1977.

10

Underground Tank Technology Update

Please note that even a tier 3 based on speciated TPH is

protective of human health and the environment. All RBCA
programs that I am familiar with, including the ASTM
RBCA, are based on the EPA Risk Assessment Guidance
for Superfund (RAGS). Toxicity factors are typically taken
from the integrated risk information system. Yes, there is
conservatism built into the toxicological data, and yes,
there is some redundancy in conservatism with pathway
analysis. In the end, however, we have a system that is
protective of human health and the environment without
burdening the private and public sector with unreasonable
remediation costs.

From Ketcheson, 1997. Im from Canada, and I thought the

majority of cleanup guidelines were based on toxicological
evaluation using a generic, yet conservative set of exposure conditions. The established value actually represents
a no-effects limit for the most sensitive receptor, which has
then been reduced by some factor of safety to establish the
guideline value. I understand that the value represents an
upper concentration limit that will be tolerated in the
environment. Thus any decision above the limit represents
an unsatisfactory condition in the environment that conceptually needs to be mitigated.
I would be concerned if contaminant levels were close to
this value because the sampling is suggesting levels close
to an unsatisfactory limit. Given the heterogeneity at most
sites, I have no confidence that there would not be TPH
levels above the sampled value in close proximity to the
sampling location. This is where geostatistics may prove
useful.

Testing contaminated soil

From Smith, 1997. Regarding TPH cleanup standards, in
many places where large quantities of fuel have spilled into
the subsurface, there are large subsurface fuel pockets that
are difficult to detect with soil sampling and conventional
soil gas sampling. Ive been on several sites where soil and
soil gas have tested clean, but high methane levels and low
oxygen levels indicated TPH pockets somewhere. At one
Air Force base, these pockets supported a soil vapor
extraction system for several years without TPH ever
being detected in soil samples, and TPH being detected
in soil gas only after several hours of vapor extraction. Yet
methane concentrations were high, and low oxygen
concentrations were found in the original samples of
undisturbed soil gas.
Most investigations dont look for methane or measure
oxygen concentrations in the soil gas. In the San Francisco
Bay area some environmentalists and citizens are beginning to insist that these measurements be made before
they will agree to no action. The methane at Naval Air
Station in Alameda, California, constitutes 60 percent of the
soil gas under parking lots. This constitutes a very real
explosive hazard if it leaks into utility trenches or buildings.

May/June 1998

Yet methane is not regulated by federal or state toxics laws,

nor accounted for in quantitative risk assessments. Quantitative risk assessments have a very narrow scope, and
qualitative risks such as transformation into more toxic
products and explosive hazards should be taken into
account. In general, if oxygen is present in the subsurface
where hydrocarbons are found, some Bay Area citizens are
more comfortable with higher TPH cleanup levels.

Original inquiry?
From Morgan, 1997. Some time ago an individual asked a
question regarding permissible or legal limits of hydrocarbon contamination in soil. Our discussion group has
proceeded to give that individual all sorts of information
about what is wrong (or at least inconsistent) with permissible levels of contamination, but little to answer his or her
basic question. The original posting seemed to be asking a
general question and hoping, I think, to receive responses
from various geographic regions regarding cleanup criteria
used in their area. If my premise is correct, I suggest that
the people scan the ASTM document, DS64-Cleanup
Criteria for Contaminated Soil and Groundwater, edited by
A. Buonicore. The document is a summary of criteria used
by various states as well as other countries; it should not,
however, be viewed as the definitive or final document on
this subject. I offer it here only to assist the original poster in
getting an overview of the topic.

Acknowledgments
UTTU thanks the following for their comments:
Austin Cooley, Brown and Caldwell, Houston, Texas,
acooley@brwncald.com; Dr. Dennis Focht, 909-787-3446,
focht@citrus.ucr.edu; University of California, Riverside;
Jim Willits, BioActive Remediations Technologies, Inc., New
Jersey, willits@bioactive.com, http://www.bioactive.com;
Mike Miller, Camp Dresser & McKee Inc., Cambridge,
Massachusetts, millerme@cdm.com; Dr. Carl Oppenheimer,
carlo@mail.utexas.edu; Tony Morgan, hydrogeologist,
LGI, Inc., 909-390-2833, quatinvest@earthlink.net;
William Smith, chair of the Sierra Clubs East Bay Military
Conversion Task Force, Fremont, California, 510-490-3008,
WJASmith@AOL.com; David Ketcheson, dketches@
niagara.com; Mark Rothstein, Peco Energy Co., 215-8414868, mrothstein@legal.peco.com. Comments expressed
by these individuals are not necessarily those of their
affiliated organizations.
UTTU also graciously thanks Richard Schaffner, P.G.,
technical specialist, GZA GeoEnvironmental Inc., moderator
of the Bioremediation Discussion Group. For information on
the BioGroup, please visit the BioGroup home page, http://
biogroup.gzea.com) or send an electronic message to
rschaffner@gzea.com.

May/June 1998

Underground Tank Technology Update

occurring bugs work just fine (particularly indigenous

microbes)and theres no need to incur the expense of any
extensive research program to improve them. To the extent
that genetic engineering will be useful in bioremediation, it
will be for recalcitrant compounds, or for compounds where
natural degradation pathways dont exist.
To the best of my knowledge, transgenic bacteria have not
been used at all in commercial bioremediation, although
some academic groups, notably Gary Sayres at the
University of Tennessee, have obtained government
approval for outdoor testing of engineered microbes in
bioremediation experiments. Under current EPA regulations,
a company could use engineered microbes in a bioreactor
without needing a permit for field trials (experiments), but
EPA approval would be needed for commercial use. There
may have been some experimental, but unlikely commercial,
uses of engineered microbes in reactors.
You asked about the special environmental risks of using
transgenic microorganisms tailored for bioremediation: how
do strains derived from a contaminated site differ from those
derived from a lab culture (with adapted degradation
capability)? This question is probably too complicated to
answer in a brief e-mail. There are likely no unique risks
from transgenic microbes, but there are several risks one
should assess before the large-scale introduction of ANY
non-indigenous microbe, including the ability to survive and
disseminate beyond the plot, and any adverse effects on
nontarget organisms. With respect to the use of
bioaugmentation with natural bugs vs. engineered bugs:
lab cultures often cannot compete with indigenous populations, and most studies do not show any lasting effect of
introduced populations (e.g., in increasing rates of degradation). Thus, introduced populations dont persist long enough
to do any good, and they probably dont last long enough to
do any harm either.

From LaMountain, 1997. Another BioGroup member

expressed similar views in that shed heard that the new
organism could neither survive nor compete in the world
outside the lab. I have never heard that transgenic bacteria
have actually been used to successfully treat anything. In
most cases, if you talk to the authors who published the
wonderful paper about the creation of a strain that could
degrade new compounds, they will tell you that it did not
survive the competitive environment of a treatment process.
Although Gary Sayre has shown that some genetically
altered organisms can survive in the environment for at least
short periods of time, in most cases the organisms released
do not survive long. The major changes in their genome are
in degradative pathways, so it is unlikely that this would
affect their pathogenicity. The largest risk involves wasting a
lot of money.
Organisms that derive their degradative properties naturally through exposure to the compound or a similar
compound are much more stable and usually the best
competitors for that substrate. If they are soil organisms,
and not kept in the lab too long, they will thrive back in the

soil where they came from. If you keep the organisms

happy in the lab too long, they will not be as effective when
put back into the soil.

Acknowledgments
UTTU thanks the following for their comments: Dr. John
Rie, CBRS, Inc., Meriden, Connecticut, 203-237-1382,
cbrs@snet.net; Dr. Carl Oppenheimer, www.obio.com,
carlo@mail.utexas.edu; Dr. David J. Glass, D. Glass
Associates, Inc., Needham, Massachusetts, 617-726-5474,
DGlassAssc@aol.com; Dr. Debbie Roberts LaMountain,
Department of Civil and Environmental Engineering,
University of Houston, 713-743-4281, djroberts@uh.edu;
and Brian Wrenn, Rochester, New York, 715-787-0502,
bawrenn@rpa.net. The comments expressed by these
individuals are not necessarily those of their organizations.
UTTU graciously thanks Richard Schaffner, P.G., technical
specialist, GZA GeoEnvironmental Inc., moderator of the
Bioremediation Discussion Group. Please visit the
BioGroup home page (http://biogroup.gzea.com) for
information or send a message to rschaffner@gzea.com.

Acceptable hydrocarbon
concentrations in soils
A member of the BioGroup posed the following question:
For bioremediation of hydrocarbons in soils, what final
concentration is considered as acceptable? The comments
received from the BioGroup follow.

From Willits, 1997. To accurately respond to the question of

acceptable limits for soils, you need to know the potential
for impact to groundwater and subsequent food chain
consumption. In general, levels in the United States range
between 100 and 1,000 ppm for soils and virtually nondetect for groundwater. Different states have different levels
for standards.
From Cooley, 1997. I must take issue with this response, or
at least part of it. Soil standards for total petroleum hydrocarbons (TPH) have, in the past, been typically set to
arbitrary base-10 numbers (e.g. 100, 1,000 or 10,000 ppm).
Some states are moving toward using risk-based standards
for TPH. These standards usually are based on both
protection of groundwater from cross-media contamination
and consideration of dermal exposure, soil ingestion,
inhalation of volatiles or particulates, or a combination of
these exposure routes. For example, the Texas risk-based
standards take soil ingestion and inhalation of particulates
into account in establishing near-surface soil standards and
potential for leaching to groundwater for all impacted soil.

Underground Tank Technology Update

Because TPH is not a chemical but a mixture, risk analysis

is conducted using surrogate compounds. The TPH is
speciated through GC/MS methods and a surrogate is used
for each carbon range. For example, C9-C12 aromatics are
treated as naphthalene. Alternatively, the TPH Criteria
Working Group has developed transport and fate properties
(e.g. Koc) for these carbon ranges through laboratory
testing. Surrogates are also used for toxicological data.
In our experience, the resulting risk-based standards for soil
vary from approximately 500 mg/kg to concentrations that
exceed soil saturation. Typically they fall in the 1,000-5,000
mg/kg range. The composition of TPH varies significantly
from site to site, depending primarily upon release origin
and TPH degradation. Furthermore, soil properties, such as
fraction of organic carbon used in calculating risk-based
standards, vary from site to site.
Regarding TPH in groundwater, there is no human healthbased justification for requiring non-detect as a cleanup
standard. Even if you treat the TPH mass as consisting
entirely of pyrene (a very conservative assumption), the
resulting risk-based standard for human ingestion in a
residential setting is approximately 1 mg/L. However,
ecological risk issues, such as endangerment of critical
habitats, or aesthetic concerns, such as taste or odor, may
result in more stringent cleanup standards.

From Focht, 1997. I note that scientists have found concentrations that are quite variable. We have found that diesel
fuel-contaminated soil gives an excellent fit to a fractile lognormal distribution plot (27 samples). My question therefore
has less to do with science, but rather with law.
In California the legal limit for TPH-contaminated soil is 100
ppm. But what does this mean? A log-normal mean, or all
samples being less than 100 ppm? I have found no one to
give an answer. The few attorneys who do understand
statistics have little appreciation for the meaning of a lognormal distribution. The general rule of thumb among soil
scientists is that contaminantsincluding natural ones such
as nitrateare log-normally distributed, while most other
intrinsic soil properties (organic matter, texture, mineral
constituents) are normally distributed. Even this is a
questionable generality if one samples soil located on
terminal moraines.
In lieu of recognizing spatial variability of contaminants, I
maintain that we are chasing a rainbow, with no pot of gold
nearbyexcept to snake oil peddlers and their remedies.

From Miller, 1997. Concentrations are quite variable.

But many books, articles, and guidance documents are
available on the subject of environmental statistics. Perhaps
most important are the guidance documents published by
the USEPA. In the absence of any specific state guidelines
on the subject, these documents will provide the recommended rules. The following are some
of the most applicable:
U.S. EPA, Office of Policy, Planning and Evaluation,
Methods for Evaluating the Attainment of Cleanup
Standards, Volume 1: Soils and Solid Media, Statistical
Policy Branch, Washington D.C., 1989

May/June 1998

U.S. EPA, Office of Solid Waste, Statistical Analysis

of Ground-Water Monitoring Data at RCRA Facilities,
Interim Final Guidance, Washington D.C., 1989
U.S. EPA, Office of Solid Waste, Statistical Analysis
of Ground-Water Monitoring Data at RCRA Facilities,
Addendum to Interim Final Guidance, Washington D.C.,
1992
U.S. EPA, Office of Emergency and Remedial
Response, Guidance for Data Usability in Risk
Assessment (Part A), Washington D.C., 1992
U.S. EPA, Office of Policy, Planning and Evaluation,
Methods for Evaluating the Attainment of Cleanup
Standards. Volume 3: Reference-Based Standards for
Soils and Solid Media, Environmental Statistics and
Information Division, Washington D.C., 1994
A very useful textbook on the subject is R.O. Gilberts
Statistical Methods for Environmental Pollution Monitoring,
Van Nostrand Reinhold, New York, 1987. The concentrations of foreign chemicals (xenobiotics) in the soil and
groundwater frequently do follow a log-normal distribution.
Many exceptions exist, however, so it is crucial to determine
the distribution of your data for each measured parameter.
Such tools as probability plotting, Lilliefors normality tests,
and Shapiro-Wilk normality tests can be used here. Once
the distribution has been determined for each chemical,
the appropriate measure of the datas central tendency
(e.g., mean, geometric mean, median) can be calculated.
You will find that the usual regulator-accepted value is an
upper confidence limit for that calculated central tendency.
Note that in your calculations you must use only data from
the contaminated area in your calculationsno averaging
in values from any clean background areas.
On the subject of environmental law: many state regulations
discuss very elementary statistical methods that are
acceptable for evaluating environmental data. (I am not
familiar with California laws.) In the absence of any such
guidance, it is usually acceptable to use the EPA procedures (see references above), but get approval from your
regulators. This may be heresy, but you can read and
interpret the state environmental laws yourselfyou dont
necessarily need a lawyer. I am an environmental chemist,
and I have been interpreting for years the relevant environmental laws of the federal government and of Massachusetts, New Jersey, New York, Pennsylvania, Ohio, and
Illinois. A little patience is required to plow through the
verbiage to find the applicable portions of the law.

How to classify pyrene and its relationship

to RBCA
From Willits, 1997. Maybe pyrene is lumped in with TPH in
Texas, but here in New Jersey, pyrene is grouped with
PAHs with an entirely different standard. In fact, I think that
one of the few things we use TPH analysis for is diesel
contamination. On another note, RBCA certainly seems to
have enabled a lot less actual action and cleanup.
From Cooley, 1997. Pyrene, when detected, is treated as a
separate PAH constituent in Texas and other states that I

May/June 1998

Underground Tank Technology Update

have worked in. From a risk standpoint, pyrene can be used

as a conservative surrogate for an entire TPH mass. In
other words, when calculating risk, treating 1,757 mg/kg
TPA as 1,757 mg/kg pyrene is conservative. Therefore, a
RBCA where TPH is treated as pyrene is protective of
human health and the environment.
I wish there were enough money in the world to remediate
every spill to background concentrations, but there isnt.
Therefore, we have to make intelligent choices on where
and how to spend remediation dollars. The best available
tool is RBCA, which is designed to achieve cleanups
protective of human health and the environment.

From Rothstein, 1997. Concerning what final hydrocarbon

concentration is considered as acceptable?: this is a
loaded question! It depends what compounds are in the
hydrocarbon. Benzo(a)pyrene is only allowed to be in
residential soil to 2.5 ppm while naphthalene is allowed to
3,000 ppm in Pennsylvania. If the oil is fairly innocuous,
such as mineral oil or food grade oil, then any concentration
that does not permit free product to cause a sheen when it
rains would be acceptable. This concentration (when no
sheening will occur during rain) would be the residual
concentration, which for mineral oil ranges from 12,400 ppm
in sand to approximately 50,000 ppm.
Pennsylvania has risk-based standards for a large number
of compounds. The list is contained in the Land Recycling
Act, also known as Act 2. This act has soil- and waterbased numbers. The soil numbers are different for residential and commerical sites; they also differ with proximity to
the aquifer. The big problem with Act 2 is that it doesnt
address oils, such as transformer mineral oil, that contain
some of the compounds listed but all of which are below
PQLs in the oil itself. In this case we had the state of
Pennsylvania agree that we would simply reduce the oil
concentration to below residual saturation so that there
would be no sheen. Another problem with Act 2 is that
virtually all of the methods require EPA GC/MS techniques
such as 8270B, which are very expensive.

From Willits, 1997. Mr. Cooley appears to believe that

certain cleanup criteria are unrealistic when balanced
against costs and risks. I believe that the main reason
we are at this juncture in accepting lowered standards
for environmental quality is that we have propounded a
massive failure in accomplishing cleanups over the past
20 years. It seems that both greed on the part of the
environmental industry as a whole and failure to exercise
proper caution in evaluating and applying technologies has
brought us to this sorry point. Our clients are exhausted
and fed up with spending millions of dollars for systems to
clean up soils and groundwater that are still not cleaned to
acceptable standards. No amount of statistical analysis is
adequate to prevent accidental contact or seepage to some
higher risk location by an unidentified preferential pathway.
Yet what we are doing has had such a high percentage of
failure: pump-and-treat systems, vapor extraction systems,
peroxide treatments, etc.
From Oppenheimer, 1997. We are a company who has
pushed inexpensive applied bioremediation with near 100%

success in cleanup. We are continuously in competition

with large funded companies that have the capability of
selling inefficient systems that can never reach criteria.
Now they are pushing for criteria reduction through risk
assessment or just plain politics.
Our governments are responsible for the health of the
population. It is inexcusable for any government unit to
reduce criteria when potential health hazards are present,
especially in groundwater. Bioremediation, involving proper
use of selected natural microbial populations, is a natural,
sensible way of removing pollutants from soil, air, and
water. We have proven it cost-effective for all natural
hydrocarbons and many chlorinated hydrocarbons in-situ.
It is currently estimated that in the U.S. we use approximately 35% of the GNP for pollution abatement.
A large company in Japan has spent $5-10 million on
research to produce microorganisms that degrade TCE,
and they will spend more on developing mass production.
I have been producing TCE organisms in mass amounts for
two years and have used them successfully in groundwater
cleanup. If the Japanese company (who had been contacted by our Japanese partner) had spent their millions on
the application of our proven microorganisms, they could
have cleaned up a great amount of TCE pollution generated by industry in Japan. Sometimes I think our scientists
are more interested in salaries than cleaning up our
polluted environment. This also applies to certain government agencies. Bioremediation using applied microorganisms and appropriate application technology can and will
turn a pollutant into a resource.

From Cooley, 1997. Certainly using a default-conservative

pyrene standard for evaluating risks due to TPH needs to
be done within the framework of a risk-based corrective
action (RBCA). For example, if you are following the ASTM
RBCA standard, generic non-site-specific tier 1 risk-based
screening levels (RBSLs) would be established for pyrene
for the various media and exposure pathways. If the TPH
values are less than the tier 1 RBSLs, no further action
would be required for TPH. If the TPH values exceeded the
tier 1 pyrene RBSLs, then you would have several options:
1. Speciate the TPH and develop tier 1 RBSLs for the
TPH, then compare the TPH concentrations to the
TPH RBSLs
2. Develop tier 2 site-specific target levels (SSTLs) for
pyrene; compare TPH concentrations to pyrene SSTLs
3. Speciate TPH and develop tier 2 SSTLs for TPH;
compare TPH concentrations to TPH SSTLs
4. Perform a tier 3 evaluation using numerical methods
(i.e. modeling); develop tier 3 SSTLs and compare
TPH concentrations to tier 3 SSTLs for TPH; tier 3
SSTLs could be based on pyrene or speciated TPH
The concept behind RBCA is to do sufficient analysis on
which to base decisions. For example, if you have a small
TPH release and you can live with cleaning up to tier 1
RBSLs for pyrene, then why spend the money performing a
detailed risk assessment? On the other hand, if you have a
very large release, it is worthwhile to develop tier 3 SSTLs
based on TPH speciation.

Underground Tank Technology Update

Because TPH is not a chemical but a mixture, risk analysis

is conducted using surrogate compounds. The TPH is
speciated through GC/MS methods and a surrogate is used
for each carbon range. For example, C9-C12 aromatics are
treated as naphthalene. Alternatively, the TPH Criteria
Working Group has developed transport and fate properties
(e.g. Koc) for these carbon ranges through laboratory
testing. Surrogates are also used for toxicological data.
In our experience, the resulting risk-based standards for soil
vary from approximately 500 mg/kg to concentrations that
exceed soil saturation. Typically they fall in the 1,000-5,000
mg/kg range. The composition of TPH varies significantly
from site to site, depending primarily upon release origin
and TPH degradation. Furthermore, soil properties, such as
fraction of organic carbon used in calculating risk-based
standards, vary from site to site.
Regarding TPH in groundwater, there is no human healthbased justification for requiring non-detect as a cleanup
standard. Even if you treat the TPH mass as consisting
entirely of pyrene (a very conservative assumption), the
resulting risk-based standard for human ingestion in a
residential setting is approximately 1 mg/L. However,
ecological risk issues, such as endangerment of critical
habitats, or aesthetic concerns, such as taste or odor, may
result in more stringent cleanup standards.

From Focht, 1997. I note that scientists have found concentrations that are quite variable. We have found that diesel
fuel-contaminated soil gives an excellent fit to a fractile lognormal distribution plot (27 samples). My question therefore
has less to do with science, but rather with law.
In California the legal limit for TPH-contaminated soil is 100
ppm. But what does this mean? A log-normal mean, or all
samples being less than 100 ppm? I have found no one to
give an answer. The few attorneys who do understand
statistics have little appreciation for the meaning of a lognormal distribution. The general rule of thumb among soil
scientists is that contaminantsincluding natural ones such
as nitrateare log-normally distributed, while most other
intrinsic soil properties (organic matter, texture, mineral
constituents) are normally distributed. Even this is a
questionable generality if one samples soil located on
terminal moraines.
In lieu of recognizing spatial variability of contaminants, I
maintain that we are chasing a rainbow, with no pot of gold
nearbyexcept to snake oil peddlers and their remedies.

From Miller, 1997. Concentrations are quite variable.

But many books, articles, and guidance documents are
available on the subject of environmental statistics. Perhaps
most important are the guidance documents published by
the USEPA. In the absence of any specific state guidelines
on the subject, these documents will provide the recommended rules. The following are some
of the most applicable:
U.S. EPA, Office of Policy, Planning and Evaluation,
Methods for Evaluating the Attainment of Cleanup
Standards, Volume 1: Soils and Solid Media, Statistical
Policy Branch, Washington D.C., 1989

May/June 1998

U.S. EPA, Office of Solid Waste, Statistical Analysis

of Ground-Water Monitoring Data at RCRA Facilities,
Interim Final Guidance, Washington D.C., 1989
U.S. EPA, Office of Solid Waste, Statistical Analysis
of Ground-Water Monitoring Data at RCRA Facilities,
Addendum to Interim Final Guidance, Washington D.C.,
1992
U.S. EPA, Office of Emergency and Remedial
Response, Guidance for Data Usability in Risk
Assessment (Part A), Washington D.C., 1992
U.S. EPA, Office of Policy, Planning and Evaluation,
Methods for Evaluating the Attainment of Cleanup
Standards. Volume 3: Reference-Based Standards for
Soils and Solid Media, Environmental Statistics and
Information Division, Washington D.C., 1994
A very useful textbook on the subject is R.O. Gilberts
Statistical Methods for Environmental Pollution Monitoring,
Van Nostrand Reinhold, New York, 1987. The concentrations of foreign chemicals (xenobiotics) in the soil and
groundwater frequently do follow a log-normal distribution.
Many exceptions exist, however, so it is crucial to determine
the distribution of your data for each measured parameter.
Such tools as probability plotting, Lilliefors normality tests,
and Shapiro-Wilk normality tests can be used here. Once
the distribution has been determined for each chemical,
the appropriate measure of the datas central tendency
(e.g., mean, geometric mean, median) can be calculated.
You will find that the usual regulator-accepted value is an
upper confidence limit for that calculated central tendency.
Note that in your calculations you must use only data from
the contaminated area in your calculationsno averaging
in values from any clean background areas.
On the subject of environmental law: many state regulations
discuss very elementary statistical methods that are
acceptable for evaluating environmental data. (I am not
familiar with California laws.) In the absence of any such
guidance, it is usually acceptable to use the EPA procedures (see references above), but get approval from your
regulators. This may be heresy, but you can read and
interpret the state environmental laws yourselfyou dont
necessarily need a lawyer. I am an environmental chemist,
and I have been interpreting for years the relevant environmental laws of the federal government and of Massachusetts, New Jersey, New York, Pennsylvania, Ohio, and
Illinois. A little patience is required to plow through the
verbiage to find the applicable portions of the law.

How to classify pyrene and its relationship

to RBCA
From Willits, 1997. Maybe pyrene is lumped in with TPH in
Texas, but here in New Jersey, pyrene is grouped with
PAHs with an entirely different standard. In fact, I think that
one of the few things we use TPH analysis for is diesel
contamination. On another note, RBCA certainly seems to
have enabled a lot less actual action and cleanup.
From Cooley, 1997. Pyrene, when detected, is treated as a
separate PAH constituent in Texas and other states that I

May/June 1998

Underground Tank Technology Update

have worked in. From a risk standpoint, pyrene can be used

as a conservative surrogate for an entire TPH mass. In
other words, when calculating risk, treating 1,757 mg/kg
TPA as 1,757 mg/kg pyrene is conservative. Therefore, a
RBCA where TPH is treated as pyrene is protective of
human health and the environment.
I wish there were enough money in the world to remediate
every spill to background concentrations, but there isnt.
Therefore, we have to make intelligent choices on where
and how to spend remediation dollars. The best available
tool is RBCA, which is designed to achieve cleanups
protective of human health and the environment.

From Rothstein, 1997. Concerning what final hydrocarbon

concentration is considered as acceptable?: this is a
loaded question! It depends what compounds are in the
hydrocarbon. Benzo(a)pyrene is only allowed to be in
residential soil to 2.5 ppm while naphthalene is allowed to
3,000 ppm in Pennsylvania. If the oil is fairly innocuous,
such as mineral oil or food grade oil, then any concentration
that does not permit free product to cause a sheen when it
rains would be acceptable. This concentration (when no
sheening will occur during rain) would be the residual
concentration, which for mineral oil ranges from 12,400 ppm
in sand to approximately 50,000 ppm.
Pennsylvania has risk-based standards for a large number
of compounds. The list is contained in the Land Recycling
Act, also known as Act 2. This act has soil- and waterbased numbers. The soil numbers are different for residential and commerical sites; they also differ with proximity to
the aquifer. The big problem with Act 2 is that it doesnt
address oils, such as transformer mineral oil, that contain
some of the compounds listed but all of which are below
PQLs in the oil itself. In this case we had the state of
Pennsylvania agree that we would simply reduce the oil
concentration to below residual saturation so that there
would be no sheen. Another problem with Act 2 is that
virtually all of the methods require EPA GC/MS techniques
such as 8270B, which are very expensive.

From Willits, 1997. Mr. Cooley appears to believe that

certain cleanup criteria are unrealistic when balanced
against costs and risks. I believe that the main reason
we are at this juncture in accepting lowered standards
for environmental quality is that we have propounded a
massive failure in accomplishing cleanups over the past
20 years. It seems that both greed on the part of the
environmental industry as a whole and failure to exercise
proper caution in evaluating and applying technologies has
brought us to this sorry point. Our clients are exhausted
and fed up with spending millions of dollars for systems to
clean up soils and groundwater that are still not cleaned to
acceptable standards. No amount of statistical analysis is
adequate to prevent accidental contact or seepage to some
higher risk location by an unidentified preferential pathway.
Yet what we are doing has had such a high percentage of
failure: pump-and-treat systems, vapor extraction systems,
peroxide treatments, etc.
From Oppenheimer, 1997. We are a company who has
pushed inexpensive applied bioremediation with near 100%

success in cleanup. We are continuously in competition

with large funded companies that have the capability of
selling inefficient systems that can never reach criteria.
Now they are pushing for criteria reduction through risk
assessment or just plain politics.
Our governments are responsible for the health of the
population. It is inexcusable for any government unit to
reduce criteria when potential health hazards are present,
especially in groundwater. Bioremediation, involving proper
use of selected natural microbial populations, is a natural,
sensible way of removing pollutants from soil, air, and
water. We have proven it cost-effective for all natural
hydrocarbons and many chlorinated hydrocarbons in-situ.
It is currently estimated that in the U.S. we use approximately 35% of the GNP for pollution abatement.
A large company in Japan has spent $5-10 million on
research to produce microorganisms that degrade TCE,
and they will spend more on developing mass production.
I have been producing TCE organisms in mass amounts for
two years and have used them successfully in groundwater
cleanup. If the Japanese company (who had been contacted by our Japanese partner) had spent their millions on
the application of our proven microorganisms, they could
have cleaned up a great amount of TCE pollution generated by industry in Japan. Sometimes I think our scientists
are more interested in salaries than cleaning up our
polluted environment. This also applies to certain government agencies. Bioremediation using applied microorganisms and appropriate application technology can and will
turn a pollutant into a resource.

From Cooley, 1997. Certainly using a default-conservative

pyrene standard for evaluating risks due to TPH needs to
be done within the framework of a risk-based corrective
action (RBCA). For example, if you are following the ASTM
RBCA standard, generic non-site-specific tier 1 risk-based
screening levels (RBSLs) would be established for pyrene
for the various media and exposure pathways. If the TPH
values are less than the tier 1 RBSLs, no further action
would be required for TPH. If the TPH values exceeded the
tier 1 pyrene RBSLs, then you would have several options:
1. Speciate the TPH and develop tier 1 RBSLs for the
TPH, then compare the TPH concentrations to the
TPH RBSLs
2. Develop tier 2 site-specific target levels (SSTLs) for
pyrene; compare TPH concentrations to pyrene SSTLs
3. Speciate TPH and develop tier 2 SSTLs for TPH;
compare TPH concentrations to TPH SSTLs
4. Perform a tier 3 evaluation using numerical methods
(i.e. modeling); develop tier 3 SSTLs and compare
TPH concentrations to tier 3 SSTLs for TPH; tier 3
SSTLs could be based on pyrene or speciated TPH
The concept behind RBCA is to do sufficient analysis on
which to base decisions. For example, if you have a small
TPH release and you can live with cleaning up to tier 1
RBSLs for pyrene, then why spend the money performing a
detailed risk assessment? On the other hand, if you have a
very large release, it is worthwhile to develop tier 3 SSTLs
based on TPH speciation.

10

Underground Tank Technology Update

Please note that even a tier 3 based on speciated TPH is

protective of human health and the environment. All RBCA
programs that I am familiar with, including the ASTM
RBCA, are based on the EPA Risk Assessment Guidance
for Superfund (RAGS). Toxicity factors are typically taken
from the integrated risk information system. Yes, there is
conservatism built into the toxicological data, and yes,
there is some redundancy in conservatism with pathway
analysis. In the end, however, we have a system that is
protective of human health and the environment without
burdening the private and public sector with unreasonable
remediation costs.

From Ketcheson, 1997. Im from Canada, and I thought the

majority of cleanup guidelines were based on toxicological
evaluation using a generic, yet conservative set of exposure conditions. The established value actually represents
a no-effects limit for the most sensitive receptor, which has
then been reduced by some factor of safety to establish the
guideline value. I understand that the value represents an
upper concentration limit that will be tolerated in the
environment. Thus any decision above the limit represents
an unsatisfactory condition in the environment that conceptually needs to be mitigated.
I would be concerned if contaminant levels were close to
this value because the sampling is suggesting levels close
to an unsatisfactory limit. Given the heterogeneity at most
sites, I have no confidence that there would not be TPH
levels above the sampled value in close proximity to the
sampling location. This is where geostatistics may prove
useful.

Testing contaminated soil

From Smith, 1997. Regarding TPH cleanup standards, in
many places where large quantities of fuel have spilled into
the subsurface, there are large subsurface fuel pockets that
are difficult to detect with soil sampling and conventional
soil gas sampling. Ive been on several sites where soil and
soil gas have tested clean, but high methane levels and low
oxygen levels indicated TPH pockets somewhere. At one
Air Force base, these pockets supported a soil vapor
extraction system for several years without TPH ever
being detected in soil samples, and TPH being detected
in soil gas only after several hours of vapor extraction. Yet
methane concentrations were high, and low oxygen
concentrations were found in the original samples of
undisturbed soil gas.
Most investigations dont look for methane or measure
oxygen concentrations in the soil gas. In the San Francisco
Bay area some environmentalists and citizens are beginning to insist that these measurements be made before
they will agree to no action. The methane at Naval Air
Station in Alameda, California, constitutes 60 percent of the
soil gas under parking lots. This constitutes a very real
explosive hazard if it leaks into utility trenches or buildings.

May/June 1998

Yet methane is not regulated by federal or state toxics laws,

nor accounted for in quantitative risk assessments. Quantitative risk assessments have a very narrow scope, and
qualitative risks such as transformation into more toxic
products and explosive hazards should be taken into
account. In general, if oxygen is present in the subsurface
where hydrocarbons are found, some Bay Area citizens are
more comfortable with higher TPH cleanup levels.

Original inquiry?
From Morgan, 1997. Some time ago an individual asked a
question regarding permissible or legal limits of hydrocarbon contamination in soil. Our discussion group has
proceeded to give that individual all sorts of information
about what is wrong (or at least inconsistent) with permissible levels of contamination, but little to answer his or her
basic question. The original posting seemed to be asking a
general question and hoping, I think, to receive responses
from various geographic regions regarding cleanup criteria
used in their area. If my premise is correct, I suggest that
the people scan the ASTM document, DS64-Cleanup
Criteria for Contaminated Soil and Groundwater, edited by
A. Buonicore. The document is a summary of criteria used
by various states as well as other countries; it should not,
however, be viewed as the definitive or final document on
this subject. I offer it here only to assist the original poster in
getting an overview of the topic.

Acknowledgments
UTTU thanks the following for their comments:
Austin Cooley, Brown and Caldwell, Houston, Texas,
acooley@brwncald.com; Dr. Dennis Focht, 909-787-3446,
focht@citrus.ucr.edu; University of California, Riverside;
Jim Willits, BioActive Remediations Technologies, Inc., New
Jersey, willits@bioactive.com, http://www.bioactive.com;
Mike Miller, Camp Dresser & McKee Inc., Cambridge,
Massachusetts, millerme@cdm.com; Dr. Carl Oppenheimer,
carlo@mail.utexas.edu; Tony Morgan, hydrogeologist,
LGI, Inc., 909-390-2833, quatinvest@earthlink.net;
William Smith, chair of the Sierra Clubs East Bay Military
Conversion Task Force, Fremont, California, 510-490-3008,
WJASmith@AOL.com; David Ketcheson, dketches@
niagara.com; Mark Rothstein, Peco Energy Co., 215-8414868, mrothstein@legal.peco.com. Comments expressed
by these individuals are not necessarily those of their
affiliated organizations.
UTTU also graciously thanks Richard Schaffner, P.G.,
technical specialist, GZA GeoEnvironmental Inc., moderator
of the Bioremediation Discussion Group. For information on
the BioGroup, please visit the BioGroup home page, http://
biogroup.gzea.com) or send an electronic message to
rschaffner@gzea.com.

May/June 1998

Underground Tank Technology Update

occurring bugs work just fine (particularly indigenous

microbes)and theres no need to incur the expense of any
extensive research program to improve them. To the extent
that genetic engineering will be useful in bioremediation, it
will be for recalcitrant compounds, or for compounds where
natural degradation pathways dont exist.
To the best of my knowledge, transgenic bacteria have not
been used at all in commercial bioremediation, although
some academic groups, notably Gary Sayres at the
University of Tennessee, have obtained government
approval for outdoor testing of engineered microbes in
bioremediation experiments. Under current EPA regulations,
a company could use engineered microbes in a bioreactor
without needing a permit for field trials (experiments), but
EPA approval would be needed for commercial use. There
may have been some experimental, but unlikely commercial,
uses of engineered microbes in reactors.
You asked about the special environmental risks of using
transgenic microorganisms tailored for bioremediation: how
do strains derived from a contaminated site differ from those
derived from a lab culture (with adapted degradation
capability)? This question is probably too complicated to
answer in a brief e-mail. There are likely no unique risks
from transgenic microbes, but there are several risks one
should assess before the large-scale introduction of ANY
non-indigenous microbe, including the ability to survive and
disseminate beyond the plot, and any adverse effects on
nontarget organisms. With respect to the use of
bioaugmentation with natural bugs vs. engineered bugs:
lab cultures often cannot compete with indigenous populations, and most studies do not show any lasting effect of
introduced populations (e.g., in increasing rates of degradation). Thus, introduced populations dont persist long enough
to do any good, and they probably dont last long enough to
do any harm either.

From LaMountain, 1997. Another BioGroup member

expressed similar views in that shed heard that the new
organism could neither survive nor compete in the world
outside the lab. I have never heard that transgenic bacteria
have actually been used to successfully treat anything. In
most cases, if you talk to the authors who published the
wonderful paper about the creation of a strain that could
degrade new compounds, they will tell you that it did not
survive the competitive environment of a treatment process.
Although Gary Sayre has shown that some genetically
altered organisms can survive in the environment for at least
short periods of time, in most cases the organisms released
do not survive long. The major changes in their genome are
in degradative pathways, so it is unlikely that this would
affect their pathogenicity. The largest risk involves wasting a
lot of money.
Organisms that derive their degradative properties naturally through exposure to the compound or a similar
compound are much more stable and usually the best
competitors for that substrate. If they are soil organisms,
and not kept in the lab too long, they will thrive back in the

soil where they came from. If you keep the organisms

happy in the lab too long, they will not be as effective when
put back into the soil.

Acknowledgments
UTTU thanks the following for their comments: Dr. John
Rie, CBRS, Inc., Meriden, Connecticut, 203-237-1382,
cbrs@snet.net; Dr. Carl Oppenheimer, www.obio.com,
carlo@mail.utexas.edu; Dr. David J. Glass, D. Glass
Associates, Inc., Needham, Massachusetts, 617-726-5474,
DGlassAssc@aol.com; Dr. Debbie Roberts LaMountain,
Department of Civil and Environmental Engineering,
University of Houston, 713-743-4281, djroberts@uh.edu;
and Brian Wrenn, Rochester, New York, 715-787-0502,
bawrenn@rpa.net. The comments expressed by these
individuals are not necessarily those of their organizations.
UTTU graciously thanks Richard Schaffner, P.G., technical
specialist, GZA GeoEnvironmental Inc., moderator of the
Bioremediation Discussion Group. Please visit the
BioGroup home page (http://biogroup.gzea.com) for
information or send a message to rschaffner@gzea.com.

Acceptable hydrocarbon
concentrations in soils
A member of the BioGroup posed the following question:
For bioremediation of hydrocarbons in soils, what final
concentration is considered as acceptable? The comments
received from the BioGroup follow.

From Willits, 1997. To accurately respond to the question of

acceptable limits for soils, you need to know the potential
for impact to groundwater and subsequent food chain
consumption. In general, levels in the United States range
between 100 and 1,000 ppm for soils and virtually nondetect for groundwater. Different states have different levels
for standards.
From Cooley, 1997. I must take issue with this response, or
at least part of it. Soil standards for total petroleum hydrocarbons (TPH) have, in the past, been typically set to
arbitrary base-10 numbers (e.g. 100, 1,000 or 10,000 ppm).
Some states are moving toward using risk-based standards
for TPH. These standards usually are based on both
protection of groundwater from cross-media contamination
and consideration of dermal exposure, soil ingestion,
inhalation of volatiles or particulates, or a combination of
these exposure routes. For example, the Texas risk-based
standards take soil ingestion and inhalation of particulates
into account in establishing near-surface soil standards and
potential for leaching to groundwater for all impacted soil.

Underground Tank Technology Update

We screen all sites by a simple method. Homogenize

thoroughly a 500 g soil samplewater is easyand run
a baseline analysis on a composite of at least five grab
samples. Separate the soil into two 200 g samples. Place
the samples in shallow glass trays to a depth of one inch.
One is the control. To the second add your product. Moisten
the two samples to a mud consistency. Place on the bench
and allow to dry, stirring once a day. If working with high
volatiles, use a closed sealed glass pan. There will still be
sufficient oxygen because only two atoms of oxygen are
required to oxidize one hydrocarbon molecule. The open
trays will dry in about seven days. Residual water in the
covered trays will require adjustment.
Mix thoroughly all samples and take a composite of at least
five small samples from each tray. Mix the composites for
analysis. The results will give you information on potential
biodegradation rate, and you dont have to bill your customer
some outlandish fee. If the test is negative, go back to the
lab bench or change microorganisms. Old sites that have no
history of contamination are always suspect. The basic
problem is to select the right bacteria and understand the
environmental conditions to keep the living system viable.

From Wrenn, 1997. Although this method can probably tell

you if bioaugmentation will not work at a particular site
(assuming an adequate number of replicates are used), it
almost certainly will not give you enough information to
determine that it will. Two processes are important in
determining whether bioaugmentation will be effective:
bacterial transport and survival. The extensive and frequent
mixing that Dr. Oppenheimer uses simulates landfarming
reasonably well, but most other bioremediation processes
(especially in-situ processes) are much less well mixed.
Transport of bacteria from an injection well into a contaminated formation is difficult, because bacteria are sticky
particles that will be filtered out of suspension by the soil
particles. Also, if some type of inducer (for a desirable
enzyme), or solubility-enhancing compound (to improve the
bioavailability of the contaminants) is included in the product
being tested, these materials will remain in close proximity to
the added bacteria in the laboratory microcosms; however,
they probably will not move through the subsurface at the
same rate as the bacteria. This will obviously change the
way the microbial product performs in the field relative to
the laboratory. Furthermore, there is no reason to assume
that survival of an introduced microbial population in a
well-mixed and aerated laboratory microcosm is an indicator
of how well those organisms will survive in the subsurface,
where the concentration gradients of oxygen and hydrocarbons can be steep.
Bioaugmentation should always be evaluated in the field. It
should work in the laboratory. If it doesnt, the microorganisms in the product are poorly matched to the target contaminants or there is a problem with their viability. Success
in the lab, however, doesnt indicate that the product will
work in the field. The factors that determine success in the
field cannot be evaluated in simple laboratory microcosms.
Field studies must be carefully designed to distinguish
between the effects of bioaugmentation and those due to

May/June 1998

pumping water that contains oxygen, nutrients, and/or

surfactants.
One minor correction on the subject of laboratory treatability
studies: more than two atoms of oxygen are required to
oxidize most hydrocarbon molecules. Two moles of O2 (i.e.,
four oxygen atoms) are required to completely oxidize one
mole of methane, the simplest hydrocarbon. In general,
between 1.2 and 1.5 moles of O2 are required per carbon
atom to completely oxidize the hydrocarbons of interest in
bioremediation (e.g., BETX and above). Most target hydrocarbons have at least six carbon atoms and will require at
least 7.5 molecules of O2 for complete oxidation of each
molecule. The rule of thumb is 3 mg O2 per mg of hydrocarbon, which is equivalent to 11 ml of air per mg hydrocarbon.
Obviously, the volume of air required to completely mineralize the hydrocarbons in 200 g of soil depends on the degree
of contamination. If the soil has 5,000 ppm of degradable
hydrocarbons, 11 liters of air are required to provide sufficient oxygen for complete biodegradation. Most treatability
studies wont be conducted long enough to achieve that
degree of mineralization, but it would be foolish to draw
conclusions based on less than 10% mineralization, and
even that minimal level of treatment requires more than 1
liter of air. Also, the biodegradation rate will become oxygen
limited long before the O2 in the headspace is completely
exhausted. So, it isnt valid to assume that enough O2 will be
available, unless you do a few quick calculations on the size
of your system relative to the amount of hydrocarbon
degradation that you want to observe.

Questions raised concerning transgenic bugs

Another member wanted specific information on a genetically modified microorganism with hydrocarbon-degrading
capabilities that was patented in 1981. He also wanted to
know if transgenic bacteria were used for ex-situ bioremediation purposes. Finally, he wanted to know what risks
transgenic microorganisms presented to the environment.

From Glass, 1997. I have a great deal of experience with

U.S. regulation of genetically engineered microbes, including
nine years in the agricultural biotech industry when field tests
were first conducted in the mid to late 1980s. My company
conducted early field tests of genetically engineered
rhizobia for nitrogen fixation. I have been involved with the
bioremediation industry since 1990, and I have been a
proponent of using engineered microbes for bioremediation,
when their use made sense technically and economically.
Most individuals believe that transgenic microbes pose no
additional risk and that the regulatory process to gain
approval for their use is manageable and achievable. Many
bioremedial professionals, however, are apprehensive about
using engineered microbes because of possible adverse
public or governmental reaction; the simple fact is, for most
currently utilized applications of bioremediation, engineered
bugs are just not needed.
With respect to the microorganism that A.M. Chakrabarty
patented in 1981, I believe these microbes were never used.
There is generally no reason to improve hydrocarbondegrading bacteria through genetic engineeringnaturally

May/June 1998

Underground Tank Technology Update

History of bioremediation
references
The following references offer a glimpse of bioremediation
history.
Alexander, M., Introduction to Soil Microbiology, Wiley, N.Y.,
472 p., 1961.
American Petroleum Institute, Federal Water Pollution Control
Administration, Proceedings to the Joint Conference on
Prevention and Control of Oil Spills, 345 p., 1969.
American Petroleum Institute, Annual Oil Spill Conference,
Washington D.C., 1969 to present.
Atlas, R.M., Microbial Degradation of Petroleum Hydrocarbons: an Environmental Perspective, Microbiological
Reviews, No. 45, p. 180-209, 1981.
Azoulay, E. and J.C. Senez, Degradation bacterienne des
hydrocarbures parafinniques II. Determination des produits
intermediares par la methode des adaptations
simultanees, Ann. d. Inst. Pasteur, No. 8, p.868-879, 1960.
Baas Becking, L. G. M., Kaplan, I. R. and D. Moore, Limits of
the Natural Environment in Terms of pH and Oxidation
Reduction Potentials, Journal of Geology, No. 68, p. 243284, 1960.
Baas Becking, L. G. M., Geology and Microbiology, Contr.
Marine Microbiology, New Zealand, New Zealand Oceanographic Institute, Memoir 3, p. 48-64, 1959.
Beerstecher, E., Petroleum Microbiology, Elsevier Press,
375 p., 1954.
Blank, M., Ed., Chemistry of Biological Systems, Advanced
Experimental Medicine and Biology, Vol. 7, Plenum Press,
New York, 1970.

11

Clerk, R.B., Marine Pollution, Oxford, Clarendon Press,

168 p., 1992.
Colwell, R.R. and J.D. Walker, Ecological Aspects of Microbial
Degradation of Petroleum in the Marine Environment,
Critical Reviews in Microbiology, No. 5, p. 423-445, 1977.
Conservation Foundation, State of the Environment, An
Assessment Mid-Decade, Conservation Foundation,
Washington D.C., 586 p., 1984.
Corredor, J.E., Morell, J.M. and C.D. Castillo, Persistence of
Spilled Crude Oil in a Tropical Intertidal Environment,
Marine Pollution Bulletin, No. 21, p. 358-388, 1990.
Council on Environmental Quality, Environmental Quality 9th
Annual Report, Washington D.C., 599 p., 1978.
Davis, J.B., Petroleum Microbiology, Elsevier Publications,
Amsterdam, 604 p., 1967.
DeRosa, M., Gambacorta A. and A. Gliozzi, Structure,
Biosynthesis and Physicochemical Properties of
Archaebacterial Lipids, Microbiology Review, No. 50,
p. 70-80, 1986.
Dobell, C., Antonie van Leeuwenhoek and his Little Animals,
London Staples Press, 1932.
Driver, J.I., The Geochemistry of Natural Waters, Prentice Hall,
Englewood Cliffs, New Jersey, 437 p., 1988.
Drucker, H. and R.E. Wildung, Biological Implications of
Metals in the Environment, Proceedings Symposium,
Technical Information Center, Energy Research and
Development Administration, 682 p., 1975.
Ehrlich, H.L., Geomicrobiology, Marcell Dekker, New York,
393 p., 1981.
Englemann, T.W., Bacterium photometricum, Ein Beitrag
zurverglichenden Physiologie des Licht und Farbensinnes,
Pflugers Arch., No. 30, p. 95, 1883.
Fenchel, T. and T.H. Blackburn, Bacteria and Mineral Cycling,
Academic Press, London, 225 p., 1979.

Blumer, M. and J. Sass, Oil Pollution Persistence and

Degradation of Spilled Fuel Oil, Science, No. 176,
p. 1120-1122, 1972.

Ford, B.J., Microbe Power, Stein and Day, New York, 181 p.,
1976.

Bowan, H.J.M., Trace Elements in Biochemistry, Academic

Press, New York, 241 p., 1966.

Garrels, R.M., Mineral Equilibria, New York, Harper Brothers,

254 p., 1960.

Boylan, D.B. and B.W. Tripp, 1971, Determination of Hydrocarbons in Sea Water: Extracts of Crude Oil and Crude Oil
Fractions, Nature, No. 230, p. 44-47, 1971.

Gabler, R.E., Sager, R.E., Brazier, S.M. and D.L. Wise,

Essentials of Physical Geography, Saunders College
Publication, Philadelphia, 550 p., 1987.

Brock, T.D., Smith, D.W. and M.T. Madigan, Biology of

Microorganisms, Prentice Hall, Englewood Cliffs, New
Jersey, 847 p., 1984.

Gibson, D.T., Cardini, G.E., Maseles, F.C. and R.E. Kallio,

Incorporation of Oxygen-18 into Benzene by Pseudomonas Putida, Biochemistry, Vol. 9, p. 1631, 1970.

Brown, S.O., Microbial Oxidation of Crude Oils and Water

Soluble Fraction of Crude Oils as Shown by the B.O.D.
Method, Texas A&M Research Foundation, Project 9, p. 2,
1950.

Gliozzi, A., Rolandi, R., DeRosa, M., Gambacorta, A. and B.

Nicolaus, Membrane Models of Archaebacteria, in
Transport in Biomembranes, Edited by R. Anolini, Raven
Press, New York, 1982.

Bushnell, L.D. and H. F. Haas, The Utilization of Certain

Hydrocarbons by Microorganisms, Journal of Bacteriology,
No. 41, p. 653-673, 1941.

Gortner, R.A. and W. Gortner, Outlines of Biochemistry, John

Wiley, New York, 1078 p., 1949.

Calvin, M., Chemical Evolution, Oxford University Press,

278 p., 1969.
Campbell, R., Microbial Ecology, Blackwell Science Publication, Oxford, 191 p., 1983.
Carey, F.A., Organic Chemistry, McGraw-Hill, New York,
1219 p., 1989.

Gundlach, E.R., Boehm, P.D., Atlas, R.M., Ward, D.M. and

D.A. Wolfe, The fate of AMOCO CADIZ Oil, Science,
No. 221, p. 122-129, 1980.
Gunkel, W., Gassmann, G., Oppenheimer, C.H. and I. Dundas,
Preliminary Results of Baseline Studies of Hydrocarbons
and Bacteria in the North Sea, 1975, 1976 and 1977,
Spanish Conference on Hydrocarbon Pollution, Santiago de
Compostela, 38 p., April 1977.

12

Underground Tank Technology Update

Haas, H.F., Yantzi, M.F. and L.D. Bushnell, Microbial Utilization of Hydrocarbons, Trans. Kansas, Academic Science,
No. 44, p. 39-45, 1941.
Hildebrand, H.H. and G. Gunter, Deposition of Petroleum Tars
and Asphalts, Beaches of the Northern Gulf of Mexico,
UTMSI Report, University of Texas, Port Aransas, Texas,
88 p., 1955.
Hitzman, D.O., Petroleum Microbiology and the History of its
Role in Enhanced Oil Recovery, in E.C. Donaldson and
J.B. Clark (Eds.), Proceedings, 1st International Conference
on Microbial Enhanced Oil Recovery, May 16-21, 1982,
Afton, Oklahoma, p.162-218, 1983.
Horvath, R.S., Microbial co-Metabolism and the Degradation of
Organic Compounds in Nature, Bacteriological Reviews,
No. 36, p. 146-155, 1972.
Hunt, John M., Petroleum Geochemistry and Geology, W.H.
Freeman and Company, San Francisco, 617 p., 1979.
Hutner, S.H., Nutrition of Protists, in This is Life, Johnson and
Steere, Editors, Holt Reinhart and Winston, New York,
1962.
James, A.M., The Electrochemistry of the Bacterial Surface,
Proceedings of Biophysics and Biophysical Chemicals,
No. 8, p. 98-144, 1957.
Johnson, F.H., Goodale, W.T. and J. Turkevich, The Bacterial
Oxidation of Hydrocarbons, Journal Cell. Comp. Physiol.,
Vol. 19, No. 163-172, 1942.
Kator, H.I., Utilization of Crude Oil Hydrocarbons by Mixed
Cultures of Marine Bacteria, Unpublished PhD Thesis, the
Florida State University, Department of Oceanography,
1972.
Kator, H.I., Oppenheimer, C.H. and R.J. Miget, 1971, Microbial
Degradation of a Louisiana Crude Oil in Closed Flasks and
Under Simulated Field Conditions, Proceedings of the Joint
Conference on Prevention and Control of Oil Spills,
American Petroleum Institute, EPA, and U.S.C.G.,
p. 287-296, 1971.
King, J.W. and D.A. Stevens, Proceedings of the First International MEOR Workshop, Department of Energy, April 1-3,
1986, Bartlesville, Oklahoma, NTIS DOE/BC/10852-1,
(DE87001216), 373 p., 1987.
Kinghorn, R.R.F., An Introduction to the Physics and Chemistry
of Petroleum, Wiley and Sons, New York, 420 p., 1983.
Krumbein, W.E., Editor, Microbial Geochemistry, Blackwell
Science Publications, 330 p., 1983.
Kuznetsov, S.I., Ivanov, M.V. and N.N. Lyalikova, Introduction
to Geological Microbiology, Trans. 1963, McGraw-Hill,
New York, 1962.
La Riviere, J.W.M., The Production of Surface Active Compounds by Microorganisms and its Possible Significance
in Oil Recovery II, Antonie v. Leeuwenh, Journal of
Microbiology Serol., No. 21, p. 9-27, 1955.
Lederberg, J. and E.M. Lederberg, Journal Bacteriology,
63:399, 1952.
Lee, C.C. and W.K. Craig, Water Soluble Hydrocarbons
from Crude Oil, Bulletin Environmental Contaminated
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Leeuwenhoek, A. van, 1694, Ondervindingen en
Beschouwingen der Onsigtbar Geschapene Waarheden,
2nd Ed., p 45., Delft, in Dobell, 1932.

May/June 1998

McKenna, E.J. and R.E. Kallio, The Biology of Hydrocarbons,

Annual Reviews of Microbiology, Vol. 19, p.183, 1965.
Marr, E.K., The Bacterial Oxidation of Benzene, Dissertation,
Pennsylvania State College, 1959.
Margulis, L., Early Life, Jones and Bartlett, Boston. 260 p.,
1984.
Mason, S.F., Chemical Evolution, Origin of the Elements,
Molecules and Living Systems, Clarendon Press, Oxford,
317 p., 1992.
Master, M. and C.H. Oppenheimer, On the Solution of Quartz
and Precipitation of Dolomite in Sea Water During
Photosynthesis and Respiration, Zeit. F. Allgemeine
Mikrob, Vol. 5, p.48-51, 1965.
Meinschein, W.G., Origin of Petroleum, Bulletin of the
American Association of Petroleum Geologists, Vol. 43,
p. 925, 1959.
Meinschein, W.G., Biological Markers and n-Alkanes as
Geological Agents, in Organic Geochemistry of
Contemporaneous and Ancient Sediments, Ed. Meinschein,
Great Lakes Section Society of Economic Paleontologists
and Mineralogists, Bloomington, Indiana, Chapter 2,
p. 29, 1983.
Miget, R.J., Oppenheimer, C.H., Kator, H.I. and P.A. LaRock,
Microbial Degradation of Normal Paraffin Hydrocarbons
in Crude Oil, Proceedings for the Joint Conference on
Prevention and Control of Oil Spills API-FWPCA,
December, p. 327-331, 1969.
Montgomery, C.W., Fundamentals of Geology, W.C. Brown,
357 p., 1989.
National Academy of Sciences, Productivity of World
Ecosystems, NAS, Washington D.C., 166 p., 1975.
Oppenheimer, C.H., Thesis, University of California at
Los Angeles, Effect of High Pressure on Marine
Microorganisms, 1951.
Oppenheimer, C.H. and L. Kornicker, Effect of Microbial
Production of Hydrogen Sulfide and Carbon Dioxide on the
pH of Recent Sediments, Publication of the Institute of
Marine Science, The University of Texas, Vol. 5, p. 5-15,
1958.
Oppenheimer, C.H., Bacterial Production of Hydrocarbon-like
Materials, Zeitschrift fur Allgemeine Mikrobiologie, Vol. 5,
No. 4, p. 284-307, 1965.
Oppenheimer C.H., Eh and pH of Marine Sediments, in
Encyclopedia of Earth Sciences, Reinhold Publications
Company, New York, 1966.
Oppenheimer, C.H., Testimony Presented to the Oversight
Hearings on the Effect to the United States on the Blowout
of the Pemex IXTOC Oil Well, September 11, 1979.
Oppenheimer, C.H., Personal Paper, Collection of Papers
Prepared for the Bureau of Land Management, Offshore
Oil Lease Hearings, Louisiana, Mississippi, Florida, New
York, 1980.
Oppenheimer, C.H., Oil Ecology, Chapter 1, Marine Environmental Pollution,1, Hydrocarbons, R. Geyer, Editor, p. 2135, Elsevier Oceanography Series, Amsterdam, 1980.
Oppenheimer, C.H. and W. Drost-Hansen, A Relationship
Between Multiple Temperature Optima for Biological
Systems and the Properties of Water, Journal of
Bacteriology, Vol. 80, p.21-24, 1960.

May/June 1998

Underground Tank Technology Update

hydrogen peroxide. We and all other aerobes also have

catalase, which converts H2O2 to O2 and H2O.
Microaerophiles have SUD, but not catalase, while obligate
anaerobes have neither.
Clinical bacteriologists readily observe that it is quite easy to
grow hemolytic streptococci on blood agar plates, but not on
other agar plates. So guess where microaerophiles are
most commonly found, and what is so unique about blood:
the mammalian mouth and the lining of other body cavities,
which contain lots of catalase. Not surprisingly, most
microaerophiles that reside at low oxygen concentrations
are pathogens (hemolytic streptococci, diplococci) while
others that reside in anaerobic environments (lactic acid
bacteria) are not pathogenic. Based on what we know
about the ecology of soil bacteria, the significance of
microaerophiles is obscure and not likely to be important vis
a vis biodegradation because other bacteria would be better
competitors under microaerophillic or anaerobic conditions.

Additional information
For additional information on definitions of anaerobes or
associated metabolic processes, consult the issues of The
American Society of Microbiology News published in the
early 1980s. A number of definitions were proposed and
debated in the Letters to the Editor (Liss, 1997).

Acknowledgments
UTTU thanks the following for their contributions to this
article: Dr. Martin Whittaker, Ph.D., Golder Associates Ltd.,
905-567-4444, mwhittaker@golder.com; Dr. Debbie Roberts
LaMountain, Department of Civil and Environmental
Engineering, University of Houston, 713-743-4281,
djroberts@uh.edu; Mike Barden, Geoscience Resources
LTD, Albuquerque, New Mexico, 505-821-5508,
mike-barden@ibm.net; Robin Gerlach, Center for Biofilm
Engineering, Montana State University, Bozeman, Montana,
406-994-4770, robing@erc.montana.edu; Dr. Steven Liss,
Ryerson Polytechnic University, Toronto, Ontario, 416-9795000, sliss@acs.ryerson.ca; Dr. Allan Zhang, OConnor
Associates Environmental, Inc., Langely, British Columbia,
604-513-1005, allan-zhang@oconnor-associates.com;
Dr. Jean Pelmont, Universite Joseph Fourier, Grenoble,
France, 33-0-476-51-48-05, jean.pelmont@ujf-grenoble.fr;
Dr. Dennis Focht, University of California, 909-787-3446,
focht@citrus.ucr.edu. The views of these individuals are not
necessarily those of their organizations.
UTTU also thanks Richard Schaffner, P.G., technical
specialist, GZA GeoEnvironmental Inc., moderator of the
Bioremediation Discussion Group. For administrative
information on the BioGroup, please visit the BioGroup
home page (http://biogroup.gzea.com); for additional
information, send a message to rschaffner@gzea.com.

Engineered microbe effectiveness,

bioaugmentation in lab vs. field
and transgenic microorganisms
This article is based on a November 1997 discussion within
the BioGroup. The exchange began when a member
wanted to verify a claim that a catalyst that contained
facultative anaerobes and microaerophiles would draw
oxygen down into the substrate and would complete
petroleum hydrocarbon degradation in 60 days. The
member wanted to know if there were any methods to
demonstrate the superbugs effectiveness.

Assessing microbe effectiveness

From Rie, 1997. Several methods are available to check
the effectiveness of the additions. First, a word of caution:
I have yet to find a refereed, peer-reviewed article in any
publication that shows any more than a brief (30-day or
less) acceleration of natural biodegradation when microbes
are added. This is in comparison to equal treatment of a
control plot without microbe addition. Normally, some
combination of moisture, nutrient, and/or air addition is
enough to stimulate existing microbes. See, for instance,
the numerous Air Force studies.
As for assuring the effectiveness of the native or foreign
(indigenous or added) microbes, several methods exist.
Probably the best cheap method is a simple respiration test,
measuring oxygen and carbon dioxide in test wells that are
already in place. Water samples could be checked for
dissolved oxygen and pH, which will decrease with increasing carbon dioxide levels.
A second method would be a relatively inexpensive lab
count of hydrocarbon-degrading microbes, using core
samples, carefully taken during soil sampling for hydrocarbons. Such testing would have to be carefully planned,
but it could yield total heterotrophic plate counts as well
as hydrocarbon degraders for a cost of less than $2,000
$3,000. We recently developed a method for specifically
measuring hydrocarbon degraders, which, when combined
with TPH data, can give an excellent picture of what is
going on. To complete the picture, TPH measurements
can be taken for the same series of samples. Several
laboratories, including ours, provide the biological testing
and planning of sample taking at nominal fees.

Potential biodegradation: the lab vs. the field

From Oppenheimer, 1997. A quantitative jump exists
between the ideal lab and the field. Believe me, Ive taught
geo and marine microbiology for 40 years and for the past
7 years Ive been attempting to be a businessman. The
bottom line for the multitude of small projects is to get
results without an expensive procedure.

Underground Tank Technology Update

Anoxic-anaerobic, from Pelmont, 1997. Nitrate is just one

electron acceptor. Here we call denitrification anaerobic
respiration, using nitrate as an acceptor. This is dissimilatory reduction of nitrate, producing typically N2O and N2.
Assimilation of nitrate is reduction to ammonia. The
distinction between these two modes, however, is not
always clear. Nitrate-using respiration shifts easily to other
acceptors when nitrate is depleted: dimethylsulfoxide,
trimethylamine oxide, fumarate, and others. E. coli does so,
with a hierarchy of acceptors under control at the transcription level. I do not see the usefulness of this distinction
between anoxic and anaerobic. Other respirations involve
the reduction of Fe(III), Mn(II). Methanogenesis has been
shown to be a respiratory process producing energy with
CO2 or acetate as an acceptor.

May/June 1998

than in the gas phase, an oxic system can become anoxic

quite quickly, where there is lots of water. The next most
efficient electron acceptor commonly found in the
environment is nitrate, which has a redox equilibrium (Eh)
of 421 mV. Nitrate, nitrite and nitrous oxide are utilized, in
lieu of dioxygen, as oxygen acceptors by many common
aerobic soil bacteria, e.g. Pseudomonas, Bacillus,
Alcaligenes. Denitrifying bacteria may thus be considered
as facultative aerobes. They simply switch over to using
the next best electron acceptor when O2 is not available.
Ferric iron (Eh= 357, pH 7.0) is the next best electron
acceptor. Several books give an erroneous value of 770 mV,
which is the value at pH 0. Many, but not all, denitrifying
bacteria can use Fe+3 as an electron acceptor.

May/June 1998

Underground Tank Technology Update

Oppenheimer, C.H., Miget, R.J. and H.I. Kator, Ecological

Relationships Between Marine Microorganisms and
Hydrocarbons in the O.E.I. Study Area, Louisiana, Rice
University Studies, Vol. 65, p. 287-325, 1979.

Stotzky, G., Influence of Clay Minerals on Microorganisms:

III Effect of Particle Size, Cation Exchange Capacity and
Surface Area on Bacteria, Canadian Journal of Microvvbiology, Vol. 12, p. 1235-1246, 1966.

Oppenheimer, C.H. and F.K Hiebert, Microbially Enhanced

Oil Production Field Tests in Texas, Proceedings of the
Symposium on Applications of Microorganisms to
Petroleum Technology, USDOE/NIPPER, Bartlesville,
Oklahoma, 1988.

Tausson, W.O., Bacterial Oxidation of Crude Oils,

Neftyanoe Khoz, Vol. 14, p. 220-230, 1928.

Oppenheimer, C.H. and F.K. Hiebert, Microbiological

Techniques for Paraffin Reduction in Producing Oil Wells,
Department of Energy Final Report, DOE/BC/14014-9
(DE89000741), 67 p., 1989.
Pasteur, L., Ann.Chim.Physics., Vol. 58, 323 p., 1860.

Fermentative. We here use a biochemical definition of

fermentation. Fermentative processes produce ATP at the
substrate level. Respiration is an energetic process building
directly a membrane potential, ATP being made with H+ or
Na+ translocating ATP syntheses. This is usually a clear-cut
definition, as proposed in the past by Slater . . . According
to this definition, acetic acid production from ethanol is not a
fermentation, just a respiration going to CO2 and H2O if not
stopped in time. Fermentation is a word commonly used in
industry, whatever the biochemical mechanisms are.

Obligate anaerobes (respiratory), from Focht, 1997.

These are the domain of the sulfidogens (sulfate reducers,
Eh =-190mV), which may be thought of as having a very
truncated respiratory system. This is a tough way to make a
living: note the small energy difference, about 200 mV, in
oxidizing H2 this way as opposed to coupling H2 oxidation
with O2, 1230mV. But if table scraps are all that remain, then
these organisms prevail under these meager conditions. The
sulfide producers and the methanogens use molecular H2 as
their energy source. H2 is generated by the other group of
anaerobes immediately below.

Pashley, R.M., McGuiggan, P.M., Ninham, B.W. and D.F.

Evans, Attractive Forces Between Uncharged Hydrophobic
Surfaces: Direct Measurements in Aqueous Solution,
Science, Vol. 229, p.1088-1089, 1985.

Since fermentation is often a lower energy-yielding process,

it is usually characterized by the making of large amounts
of organic by-products. Fermentation cannot be carried in
oligotrophic conditions. But organic compounds are not the
sole by-products: CO2 and H2 are also abundant sometimes, as in the case of some obligate anaerobes as
Clostridia doing efficient fermentation with the production
of a lot of gas. A bug like Clostridium thermoaceticum,
however, is a true acetogenic species, producing acetic
acid according to a mechanism that is interpreted as a
CO2-using respiration, according to the above definition.

Obligate anaerobes (fermentative), from Focht, 1997. So far,

we have been talking about inorganic electron acceptors
(respiration). Fermentation, by Pasteurs definition, is the use
of organic electron acceptors. The production of lactic acid
(e.g. yogurt production) from glucose by the lactic acid
bacteria (which are microaerophiles) results actually from the
reduction of pyruvate to give a stoichiometric balance of
protons and electrons in lieu of an inorganic electron
acceptor. The bacteria of the genus Clostridium actually
produce H2 gas during the production of protons and
electrons.

Science News Letter, Nov. p. 297, 1942.

Shabtai, Y. and D.L. Gutnik, Exocellular Esterase and
Emulsan Release from the Cell Surface of Acinetobacter
Calcoaceticus, Journal of Bacteriology, Vol. 161, p.11761181, 1985.
Sharpley, J.M., Elementary Petroleum Microbiology, Gulf
Publications, 256 p., 1966.
Shennan, J.L. and I. Vance, Microbial Enhanced Oil Recovery
Techniques and Offshore Oil Production, in Microbial
Problems in the Offshore Oil Industry, p. 73, Ed. E.C. Hill,
et.al., Wiley and Sons, Chinchester, 1987.
Sieburth. J.Mc.N., Microbial Seascapes, University Park Press,
Baltimore, 248 p., 1975.
Sieburth, J.Mc.N., Sea Microbes, Oxford University Press,
491 p., 1979.
Simanov, A.I., Nazarov, M.I., Gruzinov, N.V., Afanas-Eva,
M.A. and V.P. Andryukov, Meteorologiya i Gidrologiya,
Vol. 3, p. 64-72, 1984.
Smith, P.V., Preliminary Note of Origin of Petroleum, Bulletin
of American Association of Petroleum Geologists, Vol. 36,
p. 411, 1952.
Sondheimer, E. and J.B. Simeone, Chemical Ecology,
Academic Press, N.Y., 336 p., 1970.
Stephenson, M., Bacterial Metabolism, Longmans, Green
and Co., London, 398 p., 1949.
Stone, R.W., White, A.G.C. and M.R. Frenske, Journal of
Bacteriology, Vol. 39, p. 91, 1940.

Terms applied to bacterial physiology

and thermodynamics
The words describing aeration have unfortunately become
confusing due to incorrect usage, even among microbiologists. Lets consider these terms as they pertain to bacterial
physiology and thermodynamics.

Aerobic, from Focht, 1997. Aerobic refers to life with air,

ergo, molecular oxygen. We fall into this category. O2 is the
most efficient electron acceptor known because of the high
redox potential (816 mV, pH 7.0) for the O2/H2O couple at a
1:1 molar ratio, referred to as the Eh. The Eh for the H2/2H+
is 414 mV, pH 7.0). Rather than argue about how little
oxygen it takes to be anaerobic or anoxic (without molecular
oxygen), we should view oxidation/reduction reactions from
the perspective of the Nernst equation; that is the basis of
electro-chemistry. Energy can be evaluated and compared
by use of electro-chemical potentials.
Anoxic, from Focht, 1997. Consider what happens in the
environment as a result of respiration. Because diffusion of
molecular oxygen in water is about 100,000 times slower

Facultative anaerobes, from Focht, 1997. Microorganisms,

including yeasts and other eucaryotes as well as bacteria,
that can grow aerobically by respiration or anaerobically by
fermentation, fit this term as defined by Pasteur. Unfortunately, too many textbooks have corrupted this term by
incorrectly calling denitrifying bacteria facultative anaerobes.
Denitrifying bacteria are not fermentative: they are strictly
respiratory and are unable to use organic substrates as
electron acceptors. Hence, they (and iron and manganesereducing bacteria) should correctly be referred to as facultative anaerobes, i.e. they are obligate respiratory bacteria.
Microaerophiles, from Focht, 1997. These bacteria are
basically fermentative anaerobes. They are different,
however, from obligate anaerobes because they are able to
tolerate small quantities of oxygen during growth. How
small? That depends on the environment. Microaerophiles
have an incomplete cytochrome system, such that when
protons react with O2, hydrogen peroxide is produced. Even
more toxic are super oxide radicals (O2-) that are generated
during respiration. All aerobic organisms have superoxide
dismutase (SUD), which converts super oxide radicals to

13

Perfiliev, B.V. and D.R. Gabe, Capillary Methods of Investigating Microorganisms, Translated from Russian, University
of Toronto Press, J.M. Shewan, Editor, 1969.
Perry, J.J., Microbial Cooxidations Involving Hydrocarbons,
Microbiological Reviews, Vol. 43, p. 59-72, 1979.
Pinta, M., Detection and Determination of Trace Elements,
Translated from French, Ann Arbor-Humphrey Science
Publisher, Ann Arbor MI, 588 p., 1970.

Teal, J.M. and R.W. Howarth, Oil Spill Studies: A Review of

Ecological Effects, Environmental Management, Vol. 8,
p. 27-44, 1984.
Thimann, K.V., The Life of Bacteria, Macmillan, N.Y., 909 p.,
1964.
Twenhofel, W.H., Treatise on Sedimentation, Dover Publications, New York, Two Volumes, 926 p., 1961.
Vandermeulen, J.H., Some Conclusions Regarding Long-Term
Biological Effects of Some Major Oil Spills, Phil. Trans. R.
Society of London, Vol. 297, p. 335-351, 1982.
Van der Linden, A.C. and G.J.E. Thijsse, The Mechanisms of
Microbial Oxidations of Petroleum Hydrocarbons, Adv.
Enz., Vol. 27, 1965.
Ward, C.H., Bender M.E. and D.J. Reish, The Offshore
Ecology Investigation, Rice University Studies, Vol. 65,
Nos. 3&4, 589 p., 1979.
Warner, J.S., Determination of Aliphatic and Aromatic
Hydrocarbons in Marine Organisms, Analytical Chemistry,
Vol. 48, p. 576-583, 1976.
Wentworth, C.K., A Scale of Grade and Class Terms for
Classifying Sediments, Journal of Geology, Vol. 30,
p. 377-391, 1922.
Yen, T.F., University of Southern California, A State of the
Art Review on MEOR, NSF Grant OIR-8405134, 1988.
Young, L.Y. and C.E. Cerniglia, Eds., Microbial Transformation
and Degradation of Toxic Organic
Chemicals, Wiley-Liss Inc., New York, 1995.
ZoBell, C.E., The Effect of Solid Surfaces Upon Bacterial
Activity, Journal of Bacteriology, Vol. 46, p.39-56, 1943.
ZoBell, C.E., Marine Microbiology, Chronica Botanica,
Waltham, MA., 240 p., 1946.
ZoBell, C.E., U.S. Patent #2,413,278, Described a process by
which bacteria release oil from geological formations, 1946.
ZoBell, C.E., Studies on Redox Potential of Marine Sediments, Bulletin of American Association of Petroleum
Geologists, Vol. 30, p. 477-513, 1946.
ZoBell, C.E., Assimilation of Hydrocarbons by Microorganisms, Advance Enzym., Vol. 10, p. 443-486, 1950.
ZoBell, C.E., Bacterial Activities and the Origin of Oil, World
Oil, Vol. 130, p. 128, 1950.
ZoBell, C.E., U.S. Patent # 2,742,398, described a process to
reduce paraffin in oil wells, 1956.
UTTU graciously thanks Dr. Carl Oppenheimer,
carlo@mail.utexas.edu., for sending us his reference list.

14

Underground Tank Technology Update

1998 national RNA survey

by Mike Martinson
The May/June 1997 UTTU gave results from a national
survey of states on the use of remediation by natural
attenuation (RNA). The survey, using written questionnaires
or phone calls, gathered information from UST programs.
By late January 1997, all 50 states, the District of Columbia
and Guam had responded to the survey.
In January 1998, in cooperation with UTTU, Mike Martinson
of Delta Environmental Consultants, Inc. completed a similar
telephone survey. The original respondents from the UTTU
survey (if available) were asked basically the same questions to facilitate comparison with the UTTU 1997 survey. In
addition, questions pertaining to upcoming RNA and methyl
tert-butyl ether (MTBE) issues were posed.
Both surveys contained questions that could be construed
as somewhat ambiguous. The 1998 Delta survey, however,
was undertaken completely over the phone so as to assist in
clarifying questions. Authors of each survey acknowledge
that responses were subject to human bias: that is, even
regulators from the same state might not give the same
answer to the same question. In addition, the 1998 survey
queried other staff experts as suggested by the initial
regulatory contacts.
The telephone poller began the survey by specifying that the
topic was RNAs use in state UST programs, specifically for
groundwater petroleum hydrocarbon cleanup; thus,
responses were focused on the major chemical constituents
(i.e., BTEX, PAHs, and MTBE) of gasoline and diesel, rather
than on the rarer UST chlorinated compounds. In addition,
the focus of the 1998 survey was on groundwater contamination; very little information was obtained concerning soil
contamination.
The respondents were familiar with the definition of natural
attenuation: biological, physical and chemical transformations that can make contaminants less mobile and toxic, as
well as reduce their mass, volume and concentration. RNA
is regarded as a passive remediation technique whereby
natural processes are used to clean up a site; in contrast,
active remediation involves an engineered intervention that
expedites or enhances natural processes.
Comparison of results from both surveys indicate the
following:
all states continued in their previous assessment that
RNA was acceptable in combination with other remedies
to clean up petroleum-contaminated sites
the use of RNA as a sole groundwater cleanup remedy
increased from 1997 results (to about 75%), with only 7
states (<14%) in 1998 specifying that RNA was not
allowed as a sole remedy

May/June 1998

the 1997 survey indicated that most states had or

planned to establish statutes, regulations or guidance on
RNA; the 1998 survey further clarified this data:
4 states had statutes specific for RNA
1 state would add statutes by 1999
11 states had RNA-specific regulations
1 state planned to have new regulations for RNA
17 states currently had RNA-specific guidance
14 states planned to develop or consider guidance
the 1998 requirements for implementing RNA on a
groundwater remediation project were even more
consistent than those reported in 1997:
site characterization was mandatory for all states
all states favored free-phase removal to either
minimal measurement levels or until technical
infeasibility was demonstrated
the majority of the states required or preferred
groundwater monitoring (49 states) and source control
(45 states)
as stated in the 1997 survey, a stable or shrinking
groundwater plume extent (47 states) was considered
the most important (or primary) line of evidence to
demonstrate RNA effectiveness; decreasing concentration trends, according to 44 states, were also a primary
line of evidence
Additional RNA trends emerged from the 1998 survey:
groundwater RNA (including monitoring-only policies)
was considered an acceptable or site-specific remedy
alternative for most states gasoline/BTEX-impacted
sites (50 states) and diesel fuel/PAH-impacted sites
(49 states)
only 13 states required a contingency plan prior to RNA
implementation; 34 states did not require such a plan
risk assessment requirements to implement RNA were
approximately the same in 1997 (24 states) compared to
1998 (25 states); in addition to the states requiring risk
assessment, 9 states required site-specific
consideration for risk assessment in 1998, compared
to 4 states in 1997
groundwater cleanup goals for petroleum hydrocarbon
contamination were set at generic levels for 44 states
and as site-specific levels for 46 states
states increased their use of required and site-specific
use of geochemical indicators (i.e., secondary lines of
evidence) to support plume extent and concentration
trend data; geochemical indicators were required by
19 states in 1997; 20 states in 1998 had requirements
while 11 additional states were evaluating site-specific
cleanup requirements for secondary lines of evidence

May/June 1998

Underground Tank Technology Update

Microaerophillic bacteria, from Zang, 1997. I have seen some

of these products under different names. They may contain
nutrients like nitrate, which can be used as the electron
acceptor, for instance.
Degradation/transformation pathways. I really cannot see
how a lesser degradable PAH (higher molecular weight) can
result in an increase in BTEX. For one thing, when the
aromatic rings are broken, the molecule would be destabilized. The thing would essentially fall apart. More importantly, the resulting BTEX would be more biodegradable than
the parents.
Having said all that, I have even seen BTEX generating out
of thermal desorption processes. I have also seen a graduate
of UBC tell me that he saw increased BTEX through slurry
phase biotreatment. I think we should go back to the laboratory. If a sample has high hydrocarbons other than BTEX,
they will mask the BTEX because in order to perform GC
analyses, the laboratory will have to dilute the sample to a
certain degree. Thus, the BTEX may become non-detectable
in the diluted extract. In the treated sample, when background hydrocarbons are reduced to a lower level, BTEX
stands out.

Microaerophillic bacteria, from Gerlach, 1997. By the way,

cant we try to define the terms aerobic, anaerobic, anoxic,
microaerophillic . . . so that we can use them consistently
throughout our discussions? From a microbiological standpoint, you can always group bacteria into either aerobic or
anaerobic species; however, many bacteria have the
metabolic capability to grow aerobically or anaerobically,
depending on the conditions they discover; they are called
facultative aerobes, or facultative anaerobes.
In my opinion (I am an engineer) and the opinion of many
microbiologists:
aerobic means that oxygen (dissolved oxygen)
is available
anaerobic means that no (dissolved) oxygen is available.
For metabolism (biodegradation) to occur, other electron
acceptors must be available. Electron acceptors in
anaerobic environments can be nitrate, sulfate, iron,
manganese, carbon dioxide, or organics (fermentation);
and there may be a few more that are of little relevance
with respect to bioremediation
Saying no oxygen, however, has an inherent problem: Is
no oxygen absolutely no oxygen, or is oxygen just below
the detection limit of whatever method is being used? People
started using the term microaerophillic, or anoxic, for
environments where only little oxygen is available, usually
less than 0.2 mg/L, which is coincidentally the detection limit
of many oxygen tests! In my opinion anoxic has mostly
been used for environments where no, or little, oxygen is
available and nitrate is the dominant electron acceptor.
Anoxic is most widely being used in wastewater treatment.
For biodegradation to occur, electron acceptors must be
available. Thus, bacteria in microaerophillic environments
have to use other electron acceptors to biodegrade contaminants efficiently. Id be really careful if somebody tells me that

his bugs do not need any nutrient-addition while others do,

something the vendor might not explicitly state, but suggest.
Nutrients are required for bacterial growth and for efficient
bioremediation. If the required nutrients are not available in
the system (bioreactor, aquifer), they must be supplied.
There is no direct relationship between nutrient supply
(usually N, P) and electron acceptor; however, there might
be electron acceptors that can also serve as a nutrient, e.g.
nitrate. What your sources might be meaning to say is that
the relative abundance of BTEX and other aromatics is
increasing. That indicates that easily degradable compounds such as straight-chain aliphatic hydrocarbons are
being degraded more readily than aromatics or branched
aliphatics. Thus, the overall TPH concentration can decrease, but the relative abundance of persistent compounds
can increase . . . unless there is a mobilization mechanism,
such as biosurfactant production, that increases the
concentration of dissolved contaminants. I do not have an
idea why low-molecular-weight alkanes should increase.
Alkanes are usually degraded via carboxylic acids; these
concentrations can increase but are usually of little
regulatory concern. However, as mentioned above, the
relative abundance of branched alkanes can increase,
e.g., pristane and tristane in a diesel contamination.

Terminology consensus
From LaMountain, 1997. I agree that a consensus on
terminology would be great. I have found the following to be
a general consensus among anaerobic microbiologists, and
consequently this is what I teach to my students:
aerobicwhen O2 is present
microaerophilliclimiting O2 (less than 1 mg/L);
this describes organisms, rather than environmental
conditions; the organisms have specific requirements
of low O2
anoxicnitrate is the main electron acceptor
anaerobicno O2, no nitrate, usually sulfate or
CO2 as electron acceptor
fermentativeno electron acceptor available, characterized by production of low-molecular-weight acids and
solvents; this is unusual in engineering reactors or soils
but common in industry
I havent heard of any opinions or new names for ironreducing conditions; I suppose they would have to go in
with anoxic.

Descriptions of aerobic, anoxic-anaerobic and

fermentative organisms
Aerobic, from Pelmont, 1997. Aerotolerant organisms
should be mentioned, for instance, Lactobacilli. They are
anaerobes with no respiratory chain and no use of O2.
However, they still do fermentation in the presence of
some O2, i.e. in aerobic conditions. Other anaerobic bugs
such as some sulfate-reducing species have been shown
to stand low levels of O2. This is very important in the
environment since they can thrive at the limit between
aerobic conditions and oxygen-depleted media in water,
mats and even biofilms.

Underground Tank Technology Update

Discussion of
bioremediation terms
A member of the GZA GeoEnvironmental Inc., BioGroup
was approached by a vendor selling treated microbes. The
member wanted information on microaerophillic bacteria
and degradation/transformation pathways. Here are the
responses the member received.

Microaerophillic bacteria, from Whittaker, 1997. Used in

bioaugmentation, these guys purportedly need just the
minutest amounts of oxygen (no specifications provided)
and can biodegrade just about anything. One vendor claims
that they are neither aerobic nor anaerobic (rather,
microaerophillic) and do not require any nutrient addition. I
am not familiar with the term microaerophillic (though I can
make the obvious assumptions based on the name itself); in
the absence of any technical information provided by the
vendor, I wondered if anyone out there could enlighten me.
Degradation/transformation pathways. I have been told from
unreliable sources that during bioremediation, one should
expect an increase in the abundance of low molecular
weight alkanes/aromatic compounds (including BTEX)
because they are produced from the transformation of
larger molecules, although this will pass as these compounds are themselves degraded. I expressed deep
scepticism about this, since from my understanding, aerobic
biotransformation proceeds via carboxylic acid/catechol
formation, not the discrete alkane/aromatic compounds.
Questions: Can biotransformation really result in an
increase in lower-molecular-weight compounds (in whatever
form) to the extent that BTEX (for example) concentrations
would go up? Or more likely, can the actual lowermolecular-weight intermediates be produced in such
abundance that they could be mistaken for alkane/aromatic
compounds during analysis?
Microaerophillic bacteria, from Schaffner, 1997. It doesnt
matter whether they are microaerophillic, or winged pixies,
for that matter. The primary rate-limiting factor for biodegradation of contaminants such as petroleum hydrocarbons
(PHC) is the electron acceptor. If the superbugs are viable
using a lower electron acceptor concentration, I believe that
means theyll require a longer time to degrade targeted
compounds. The bottom line, as always, remains reaction
stoichiometry, i.e., you must meet the electron acceptor
demand exerted by the PHCs.
Degradation/transformation pathways. I believe you are
correct regarding PHC metabolites, i.e., functional groups
are not merely stripped from compounds. Regarding
increased BTEX concentrations, however, it is possible
that stimulation may induce biosurfactant production which
may temporarily mobilize sorbed constituents. I have not
observed this myself, but I recall that this mechanism has
been evoked in some of the early literature to explain
increases in contaminant concentrations following
biostimulation.

May/June 1998

Microaerophillic bacteria, from Barden, 1997. It sounds like

another case of overselling things. Microaerophillic refers
to conditions of very low dissolved oxygen (generally less
than 1 mg/L) where dissolved oxygen is not completely
absent, hence the description of the condition as not
anaerobic, but not fully aerobic. These microbes are capable
of various degradation pathways but primarily can utilize
organic substrates such as BTEX. They are not superbugs
and have essentially the same nutrient requirements as
other microbes. Very likely they are using nitrate as a
terminal electron acceptor (nitrate reduction). If your vendor
cant provide you with specific factual technical information
supporting their product, I would tell them to take a hike.
Degradation/transformation pathways. The biodegradation/
biotransformation does not produce BTEX compounds
(complete aromatic rings). In some cases, degradation of
straight- or branched-chain alkanes could potentially
produce a slight increase in low-molecular-weight alkanes,
but youd be unlikely ever to see it. It is possible that some
degradation intermediates (organic acids) from anaerobic
degradation could show up in the same range as BTEX in
a GC scan. The lower-molecular-weight hydrocarbons are
generally more biodegradable and dont tend to accumulate
in the system.

May/June 1998

states did not widely accept using RNA for MTBE

contamination:
only 5 states permit an RNA remedy for MTBE
25 states consider RNA of MTBE to be a
site-specific decision
24 states have MTBE regulations
3 states require that MTBE cleanup requirements
be site-specific
24 states currently have no MTBE regulations
9 states expect to adopt MTBE regulations in
1998-99
6 states anticipate adopting regulations soon after
the U.S. EPA issues its final health risk advisory
and/or establishes an MTBE maximum contaminant
level (MCL)
The following section lists the questions posed to each of
the 51 state contacts and their responses.
1. Does your state allow the use of natural attenuation (often
termed remediation by natural attenuation, or RNA) as a
groundwater cleanup remedy for UST sites with petroleum
hydrocarbon contamination:
(a) in combination with other treatment technologies or
methods?
49 Yes
1 Yes site-specific
1 Yes post-remediation

Microaerophillic bacteria, from LaMountain, 1997. By

definition, these bacteria are oxygen-requiring organisms
that grow only at reduced oxygen concentrations, less than
one atmosphere. These organisms are supposedly aerobes
that do not have all of the protective enzymes to ward off
oxygen toxicity, so they can only grow at low levels of O2.
Degradation/transformation pathways. I think the vendors
are pushing organisms that will use nitrate as an electron
acceptor and use O2 only for the oxygenase enzymes. Ron
Olsen has performed some research on these. These
organisms would still require nutrient addition, depending on
the groundwater characteristics, e.g. nitrate or phosphates.
Olsen has said that if you add O2, then you will select for
other organisms that like O2 better, and these organisms will
never get a chance to operate. You are correct to assume
that the intermediates would be more polar than the original
hydrocarbons. You should see fatty acids and catechols or
carboxylic acids as degradation products. I think your source
of information was thinking lower molecular weight in
general, e.g. big multiple rings chopped into smaller ring
units, and not thinking about their substitution, e.g. having
hydroxyls or carboxylic acid groups on them.
I have dealt with many vendors of bioremediation techniques
or organisms, and many do not understand their products.
This is because they are salespeople. I usually ask them to
get their companys microbiologist to call me, so I can get
the straight story about the product.
If this is a major remediation project or a major expense for
your company, I would certainly ask for much more background than they have apparently given to you. Do not buyin to anything without a good deal of technical information
that a trusted microbiologist says is reliable; there are too
many people trying to make a quick buck in this business.

Underground Tank Technology Update

(c) Is a contingency plan required prior to RNA

implementation?
34 No
13 Yes
2 Yes afterwards
2 Site-specific
2. Pertaining to the specific use of natural attenuation or RNA,
has your state established:
(a) statutes
46 No
1 Planning to

4 Yes

(b) regulations
39 No
1 Planning to

11 Yes

(c) guidance
17 Yes
20 No
14 Considering/Developing
3. What cleanup goals are available for closure of groundwater
contamination in your state?
(a) Generic levels
44 Yes
1 Planning to
(b) Site-specific levels
46 Yes

6 No

5 No

4. To implement natural attenuation (or RNA, or monitoring

only), does your state require:
(a) site characterization
51 Yes
(b) source/hot spot control
43 Yes
2 Preferred
1 No

(b) as a sole remedy?

38 Yes
3 Yes site-specific
1 Yes not always
1 Yes as monitoring only
1 No site-specific possible
7 No

15

3 Site-specific
2 Yes not always

(c) free-phase/free-product removal

51 Yes to either minimal measurable levels or
to extent of technical feasibility

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Underground Tank Technology Update

16

(d) risk assessment

25 Yes
9 Site-specific
1 Optional
1 No response

10 No
4 Within RBCA
1 Planning to

(e) monitoring
48 Yes
1 No response

1 Site-specific

5. Is RNA an acceptable cleanup remedy for the following

contaminants?
(a) gasoline/BTEX compounds
46 Yes
4 Yes site-specific
1 No
(b) diesel fuels/PAH compounds
40 Yes
9 Yes site-specific
2 No
(c) MTBE
22 Site-specific
13 Not regulated
5 Yes
3 Yes possible w/acceptable risk
3 Probably no
3 No NA policy
2 No acceptable risk data available

Vol. 12, No. 3

May/June 1998

7. What evidence does your state require to demonstrate

RNAs effectiveness?
(a) shrinking or stable plume
47 Yes
1 Yes not always

Department of Engineering Professional Development The College of Engineering University of WisconsinMadison

2 No formal method/policy
1 Preferred models OK
(b) decreasing contaminant concentrations
44 Yes
3 No
2 No formal method/policy
1 Yes not always
1 Preferred models OK
(c) geochemical indicators
20 Yes
20 No
11 Site-specific
8. Does your state have groundwater regulations for MTBE
as of January 1998?

24 Yes
24 No

3 Site-specific

9. Of the 24 states without current MTBE regulations,

does your state anticipate adopting MTBE groundwater
regulations in the future?

9
8
1
6

(a) specify a minimum number of monitoring points?

28 No
15 Yes
8 Site-specific

(c) require a minimum duration of monitoring?

17 1-2 yrs
17 Site-specific
15 to <GW standards/goals
2 >2 yrs

Underground Tank
Technology Update

(d) specify a monitoring frequency (typical)?

38 1 to 4 events per year
13 Site-specific

6. For RNA, does your state:

(b) specify the number of points?

32 Site-specific
19 Other

May/June 1998

No change
Adopt regulations in 1998
Adopt regulations in 1999
Adopt regulation pending EPA issuing final
health advisory/MCL

UTTU thanks Mike Martinson, Delta Environmental Consultants,

Inc., for contributing this article. Mike can be reached at
612-697-5165 or mikema@deltaenv.com.
The next issue of UTTU will contain the specific data that Mike
Martinson obtained during the course of this survey.

Underground Tank Technology Update is

published bimonthly by the University of
WisconsinMadison, Department of
Engineering Professional Development.
UTTU supplies useful information to
federal, state, and local officials working
with groundwater technology and to other
interested technical specialists. For new
subscriptions or address corrections,
use the form on inside back page.
UTTU is funded by the U.S. EPA under
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Mention of trade names or commercial
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Comments and suggestions are welcome
and may be directed to John T. Quigley,
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If you have a problem locating a reference
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Advisory Board
Gilberto Alvarez, Environmental Engineer
OUST, U.S. EPA, Region 5, Chicago, Illinois
Bruce Bauman, Research Program
CoordinatorSoil and Groundwater, API
Washington D.C.

Article summaries
Discussion of bioremediation terms ............................... 2
This is the first of three articles summarizing bioremediation
issues originally debated in November 1997 by members of the
BioGroup on the Internet.

Engineered microbe effectiveness, bioaugmentation

in lab vs. field, and transgenic microorganisms ............ 5
In this article researchers give practical information on
bioaugmentation.

Acceptable hydrocarbon concentrations in soil ............ 7

This article gives technical and philosophical perspectives on what
is the acceptable concentration of hydrocarbons left in soil?

History of bioremediation references ............................ 11

For those interested in the history of bioremediation, a list is
provided.

1998 national RNA survey .............................................. 14

This article summarizes the states RNA practices.

Robert Hitzig, Geologist

OUST, U.S. EPA, Washington D.C.

Underground Tank Technology Update

Engineering Professional Development

432 North Lake Street
Madison, Wisconsin 53706

George Mickelson, Environmental Engineer

Wisconsin Department of Natural Resources
Madison, Wisconsin
Nonprofit
Organization
U.S. Postage
PAID
Madison, WI
Permit No. 658

Mark D. Millsop, Hydrogeologist

GME Consultants, Crosby, Minnesota
Phil OLeary, Professor
Department of Engineering Professional
Development, UWMadison
Gerald W. Phillips
U.S. EPA, Region 5, Chicago, Illinois
Matt Small, Hydrogeologist
U.S. EPA, Region 9
San Francisco, California
Staff
John T. Quigley
Pat Dutt Komor
Darrell Petska
Debbie Benell
Susan Kummer/Artifax

Project Director
Geologist/Writer
Copy Editor
Program Assistant
Graphics

EPAs internet chat room at www.epa.gov/swerust1/flags.htm

consists of EPA, New Mexico, Alaska, Arizona, Montana,
Minnesota and California sites. The Minnesota site, for instance,
lists documents relating to land treatment, composting and release
investigations in karst areas. For a list of state contacts, see the
site http://www.epa.gov/swerust1/states/statcon1.htm.
UTTUs home page, http://epdwww.engr.wisc.edu/uttu/, contains
an alphabetical and topical list of every article that has appeared
in UTTU since 1987.

For information on The Environment: The Cleanup and

Re-use of Brownfields, a workshop presented in Seattle,
Washington, May 11-12, send an e-mail to dwert@aipt.org
or see the web site http://aipt.org/environment.html.