sustainability

Article
Hydroponic Lettuce Production Using Treated
Post-Hydrothermal Liquefaction Wastewater (PHW)
Samuel D. Jesse 1 , Yuanhui Zhang 1 , Andrew J. Margenot 2 and Paul C. Davidson 1, *
1 Department of Agricultural and Biological Engineering, University of Illinois Urbana-Champaign,
1304 W. Pennsylvania Ave., Urbana, IL 61801, USA
2 Department of Crop Sciences, University of Illinois Urbana-Champaign, 1102 S. Goodwin Ave., Urbana,
IL 61801, USA
* Correspondence: pdavidso@illinois.edu; 217-300-3755




Received: 23 May 2019; Accepted: 28 June 2019; Published: 30 June 2019


Abstract: Post-hydrothermal liquefaction wastewater (PHW) is a byproduct of the hydrothermal

liquefaction (HTL) process. Previous research indicates that PHW is free of pathogens and contains
nutrients needed for crop growth, but may contain metal(loid)s. This study evaluated the ability of
differentially treated PHW for effective and safe hydroponic lettuce production. Water containing
only hydroponic fertilizer (Source Water 1) had the highest total dry yield of all five treatments;
3.1 times higher than Source Water 2 (diluted PHW with sand filtration), 3.5 times higher than Source
Water 3 (diluted PHW with sand + carbon filtration), 2.6 times higher than Source Water 4 (diluted
and nitrified PHW with sand filtration), and 1.3 times higher than Source Water 5 (diluted PHW
supplemented with hydroponic fertilizer). Findings also indicated that while PHW was below the
US Department of Agriculture Foreign Agriculture Service maximum levels for cadmium, lead,
and mercury in food, the concentration of arsenic was 1.6, 2.4, and 2.0 times higher than the maximum
level for Source Waters 2, 3, and 4, respectively. There was no detectable E. coli or fecal coliforms
in any of the treated PHW. While nitrogen was present in the raw PHW, only 0.03% was NO3 -N
and NO2 -N. Diluted PHW supplemented with hydroponic fertilizer had lower lettuce yield than
hydroponic fertilizer alone, indicating a potential non-nutrient inhibition of plant growth by PHW.
Therefore, this research demonstrates that treated PHW does not pose a biological contamination
risk for lettuce, but may entail levels of arsenic in edible leaf tissues that are in excess of safe levels.
Additional treatment of PHW can benefit crop production by allowing crop utilization of a greater
fraction of total nitrogen in the raw PHW.

Keywords: PHW; food safety; wastewater; pathogens; hydroponic

1. Introduction
The U.S. produces an estimated 79 million dry tons of sustainably collectable livestock manure
and food processing waste annually [1]. This has encouraged a growing interest in hydrothermal
liquefaction (HTL), a process by which organic matter, such as sewage or manure, is converted to
bio-crude oil under high temperatures and pressures [2]. Furthermore, this process produces an
aqueous byproduct that contains crop nutrients including nitrogen, phosphorus, and potassium [3].
The high temperatures and pressures needed for the HTL process destroy biological and organic
contaminants, such as pharmaceuticals, pathogens, and genetic material, which are present in the
HTL feedstock [4,5]. Therefore, there is high potential for post-hydrothermal liquefaction wastewater
(PHW) for food crop production.
Wastewater reuse for irrigation is a current topic of study, in particular on the effect of treated
wastewater on crops and soil [6,7]. Some concerns with the use of wastewater as irrigation are

Sustainability 2019, 11, 3605; doi:10.3390/su11133605 www.mdpi.com/journal/sustainability

Sustainability 2019, 11, 3605 2 of 16

metal(loid) contamination, drugs, and pathogens [8,9]. However, the high temperatures and pressures
in HTL are hypothesized to eliminate the drugs and pathogens from the PHW, which leaves a potential
risk for metal(loid)s in PHW. Studies have shown the benefits and limitations of non-PHW wastewater
sources in the hydroponic production. Hydroponic crop production entails plant production in the
absence of soil in which roots are directly exposed to nutrient solution. Compared to non-hydroponic
agriculture, hydroponic production requires 82 times more energy but 92% less water and produces
11 times more yield per area [10].
Treatment of wastewater has been shown to be possible over 7 days with a Pistia stratiotes
phytofiltration lagoon, with an average biomass production of 5.8 g of dry weight per square meter per
day [11]. Lettuce (Lactuca sativa) can successfully be produced with treated greywater without posing
a substantial health risk for pathogens or metal(loid)s, while also providing a 5.1 log10 reduction of
E. coli in the final effluent [12]. Hydroponic barley (Hordeum vulgare) production using wastewater
has been investigated [13] and found a decrease in yields of 47% and 17% for conventional activated
sludge and lagoon system water compared to half-strength Hoagland Solution, concluding that further
fertilization with wastewater would be necessary and that metal(loid) concentrations in plant tissue
increased, but did not exceed, toxic levels. Al-Karaki [14] explored using tertiary-treated sewage
wastewater to grow barley in controlled environment vertical hydroponic growing towers. Compared
to treatments of tap water alone, which was used without fertilizer for hydroponic green fodder due to
short growth period, wastewater, and wastewater mixed with municipal tap water improved both
yields and water efficiency. Furthermore, no pathogens were found in any hydroponic treatment,
compared to previous research in conventional agriculture.
Primary and secondary treated municipal wastewater was investigated for hydroponic cucumber
(Cucumis sativus) irrigation with a nutrient film technique [15]. It was found that primary treated
wastewater, with or without nutrient supplementation, decreased plant height and reduced leaf and
flower number, but this was not seen with secondary treated wastewater. However, the increased
fruit number and weight resulted in increased yields, but it was also found to spread plant disease
in plant roots and fruit. Chow et al. [16] cultivated lettuce and Chinese cabbage (Brassica rapa ssp
pekinensis) with primary and secondary municipal wastewater effluents and half-strength Cooper
nutrient solution. Chinese cabbage showed signs of phytotoxicity with both effluents, but lettuce did
not; both crops had lower yields than the control. However, da Silva Cuba Carvalho et al. [17] sought
to evaluate nutrient absorption and yields of lettuce cultivated hydroponically with wastewater from a
domestic sewage treatment station. Using nutrient film technique with treatments of tap water and
fertilizer, wastewater supplemented with fertilizers, and wastewater alone, they found that wastewater
supplemented with fertilizers had lettuce yields and nutrient uptake comparable to tap water with
fertilizers. However, treated sewage effluent alone did not have the nutrients necessary for lettuce
production and had a quarter of the mass compared to supplemented wastewater. Cui et al. [18]
treated septic tank effluent using vertical-flow wetlands followed by hydroponically growing spinach
(Spinacia oleracea) and lettuce, which resulted in removal efficiencies sufficient for a secondary biological
treatment plant discharge, with yields less than nutrient solution hydroponic control, but higher than
in field production. Norström et al. [19] investigated using hydroponics and microalgae production in
conjunction with conventional biological treatment to treat domestic wastewater. The system was able
to achieve a 90% chemical oxygen demand (COD) removal, 72% total nitrogen reduction, and 47%
phosphorus reduction, but was not sufficient for Swedish effluent standards.
It has been demonstrated that wastewater spiked with PHW can augment the growth of mixed
culture algae and rice. Phototrophic algae growth was quantified as Chlorophyll a concentration,
which was increased by over 30% when using a small percentage of PHW (0.5%). However, larger
concentrations of PHW had an inhibitory effect, perhaps due to high ammonia concentrations or organic
inhibitors [20–23]. In past work, phenol, toluene, benzene and a variety of nitrogenous heteroatomic
compounds have been identified in PHW [24] and are known to be toxic to some algae [25,26]. It is
also known that acclimated algae can tolerate up to 15% HTL aqueous product. In comparison to
Sustainability 2019, 11, 3605 3 of 16

conventional wastewater treatment processes like activated sludge, it has been shown that phototrophic
algae can enhance removal of wastewater nutrients, N and P, by 86% and 95%, respectively [27].
Average rice yields from plants grown using a blend of nutrients from swine manure and PHW
was approximately 18.5% higher than control plants grown in municipal water containing synthetic
nutrients [28]. However, one problem noticed in this study was that the rice irrigated with PHW had
higher levels of certain metals (i.e., Hg, Cd, Pb, with only Hg exceeding the current FDA standards
for rice), which is a topic of this proposed study.
The overall goal of this study was to assess the ability of treated PHW to effectively and safely
grow lettuce, hydroponically. Specifically, this study measured yield and shoot content of metal(loid)s,
nitrogen, and phosphorus of lettuce grown using five different source waters.

2. Materials and Methods

In this study, five different source waters were used to grow lettuce, hydroponically (Table 1);
one water source consisting of municipal tap water and hydroponic fertilizer and four water sources
consisting of treated PHW. The treated PHW are based on previous work [29]. The water sources
were used as an irrigation/fertigation source for the production of lettuce. A series of five identically
constructed hydroponic racks (Figure 1) were used for the hydroponic production of lettuce. Each rack
contained three growing bins and received water from the treatment group’s source water. Each bin
consisted of a batch reactor, in essence a static flood tray. Each bin contained a foam board layer to
keep the plant potted in organic media above the water, but allowed the root system to be submerged
in the nutrient-rich feed (e.g., diluted but otherwise untreated PHW, nitrified PHW, filtered PHW, etc.).
Each bin accommodated 18 lettuce plants.

Table 1. Descriptions of the five source waters used in this study.

Source Water Number Source Water Description

1 Hydroponic fertilizer
2 2.5% PHW with sand filtration
3 2.5% PHW with sand+carbon filtration
4 2.5% PHW with nitrification and sand filtration
5 2.5% PHW with sand filtration and supplemented with hydroponic fertilizer
Sustainability 2019, 11, x FOR PEER REVIEW 5 of 17

Figure 1. Photograph
Figure ofoffull
1. Photograph fullhydroponic systemduring
hydroponic system during lettuce
lettuce growing
growing cycle.
cycle.

Each grow bin consisted of a Sterilite 86 cm L × 47.6 cm W × 17.8 cm H quart plastic tote container
(Townsend, MA, USA), with a GreenGuard (Marietta, GA, USA) R3 Unfaced Polystyrene insulation
foam board cut with 18 circular holes for PonicsFarm NP3AB 3-inch Black Slotted Mesh Net Pots,
and filled with the respective treatment nutrient solution. The holes were spaced so that pots were
Sustainability 2019, 11, 3605 4 of 16

2.1. Experimental Design

Lettuce plants, in bins of 18 plants randomly selected from plug trays, received the five source
water treatments, as shown in Table 1.
Three replications of each of the five source water treatments were completed in a controlled
environment, for a total of 15 experimental units. The hydroponic fertilizer treatment served as
a control group for normal hydroponic lettuce production. For the individual experimental units,
the following parameters were measured:
1. Net change in fresh mass from planting to harvest (net fresh yield)
2. Total dry yield at harvest
3. Metal(loid) concentrations (Hg, As, Cd, and Pb) in harvested dry mass
4. Nitrogen and phosphorus contents of harvested dry mass

2.2. Materials

2.2.1. PHW Source

The PHW was produced in a hydrothermal liquefaction laboratory pilot reactor with a feedstock
of algae grown on swine manure. The PHW was undiluted in barrels at a storage facility prior to being
collected. The PHW was well mixed and then placed in five 5-gallon sealed plastic buckets.

2.2.2. Water Source and Dilutions

Due to the large volume of water needed, municipal tap water was used to prepare all dilutions in
55-gallon plastic barrels. Tap water was added to raw PHW to generate the 2.5% mixtures. This dilution
factor was chosen as an estimated dilution factor based on nutrients required for lettuce growth and
previous PHW analysis from this lab.

2.2.3. Filtration Equipment

Sand and sand+carbon filtration treatments were performed with a Sand Master Soft Sided
Above Ground Pool Sand Filter System (Swimline, Edgewood, NY, USA), filled with sand and a
sand-carbon-sand layering, respectively. The sand was Fairmount Minerals Pool Filter #20 Grade
Silica Sand (Fairmount Santrol, Benton Harbor, MI, USA), and the activated carbon source was Custom
Aquatic Premium Bulk Granular Activated Carbon (Custom Aquatic, Vista, CA, USA) [29].

2.2.4. Nitrification Equipment

Nitrification was performed in 55-gallon plastic barrels and was aided by ATM Aquarium Products
Colony Nitrifying Bacteria (ATM Aquarium Products, Las Vegas, NV, USA) true nitrifying bacteria
starter, a 45 L/min Hydrofarm AAPA45L Active Aqua Commercial Air Pump (Hydrofarm, Petaluma,
CA, USA), 6 VIVOSUN 4 × 2 Inch Large Air Stone Cylinders (Vivosun, Los Angeles, CA, USA),
and Aquatic Experts Rigid Latex-Coated Coarse Bulk Roll Pond Filter Media, 18x72x1 Inches (Aquatic
Experts, Greensboro, NC, USA) as biofilter material for the bacteria [29].

2.2.5. Hydroponic Fertilizer Formula

The hydroponic fertilizer was formulated from the General Hydroponics (Santa Rosa, CA, USA)
brand Flora Series hydroponic fertilizer for “aggressive vegetative growth” solution, using the three
component fertilizers Flora Gro, Flora Micro, and Flora Bloom. This fertilizer was chosen as it is an
easily obtained and consistent commercial hydroponic fertilizer. Flora Gro, Flora Micro, and Flora
Bloom are 2-1-6, 5-0-1, and 0-5-4 NPK fertilizers, respectively. The “aggressive vegetative growth”
formulation utilizes 396, 264, and 132 mL/100 liters of Flora Gro, Flora Micro, and Flora Bloom,
respectively. To prepare Source Water 1, the components were measured with graduated cylinders and
thoroughly mixed in a 55-gallon plastic barrel.
Sustainability 2019, 11, 3605 5 of 16

2.2.6. Growing Racks and Bins

Each of the five source water treatments were assigned a growing rack (Figure 1) with 3 grow bins
per rack. The racks were Husky (Denver, NC, USA) 78 in. H × 48 in. W × 24 in. D adjustable steel
wire shelf units that were modified to have four shelves. The top three shelves were used for holding
grow bins and lighting fixtures and the bottom was used for holding the air pump and electrical
equipment. Each lettuce growing shelf had a Lithonia (Atlanta, GA, USA) Lighting High Bay Industrial
6-light hanging fluorescent fixture, a grow bin, and two VIVOSUN (Los Angeles, CA, USA) 4x2 inch
cylinder air stones with connected tubing. The lighting fixtures of each rack were fitted with six 4 ft T8
32-Watt Daylight 6500K ALTO Linear Fluorescent Light Bulbs and connected to a GE (Boston, MA,
USA) Indoor 24 h Mechanical Timer to control photoperiod to 12 h per day. The air stones of each
rack were connected to a 20-Watt 45 L/min Hydrofarm (Petaluma, CA, USA) AAPA45L Active Aqua
air pump split with a 6-outlet manifold to provide diffused oxygen to the root zone of the grow bins.
The distance from the bottom of the lighting fixture to the top of the grow bin was 30.5 cm.
Each grow bin consisted of a Sterilite 86 cm L × 47.6 cm W × 17.8 cm H quart plastic tote container
(Townsend, MA, USA), with a GreenGuard (Marietta, GA, USA) R3 Unfaced Polystyrene insulation
foam board cut with 18 circular holes for PonicsFarm NP3AB 3-inch Black Slotted Mesh Net Pots,
and filled with the respective treatment nutrient solution. The holes were spaced so that pots were
spaced in five rows, with three rows of 4 pots and two rows of 3 pots staggered between them. The net
pots were held in the holes by the buoyancy of the foam board on the nutrient solution. Each net pot
contained the lettuce plug, surrounded by Hydroton lightweight expanded clay aggregate (LECA) to
hold the plug and allow room for root growth. The air stones were placed at either end of the length of
the grow bin, submerged under the nutrient solution.

2.2.7. Lettuce Plug Source

The lettuce plants used in this experiment were VF Lettuce Buttercrunch Plug 288 transplant plugs
sourced from Wenke Greenhouse through Ball Horticultural Company (West Chicago, IL, USA). Prior
to shipment at 4 weeks old, the seeds were germinated at 20 ◦ C and grown after germination at 15.6 ◦ C
with low humidity. The media used was a peat perlite mixture with no starter charge of fertilizer
but were fertilized with 15-3-16 NPK 2× micros at 100 mg kg−1 . Upon arrival, the lettuce plugs were
put under the laboratory conditions, allowed to soak in the hydroponic fertilizer solution for 1 day,
and then healthy plugs were randomly selected, weighed, and transplanted into the grow bins.

2.3. Procedure

2.3.1. Initial PHW & Dilutions

The lab ambient temperature was kept at 18.3 ◦ C. When not in use, PHW was sealed in the 5-gallon
plastic barrels to prevent contamination from the room environment. For each of the source water
treatments, the water was well-mixed with a powered stirring rod for 3 min before being added to the
grow bins [29].

2.3.2. Nitrification Process

Nitrification was stimulated with the aid of ATM Aquarium Products Colony Nitrifying Bacteria
(ATM Aquarium Products, Las Vegas, NV, USA) true nitrifying bacteria starter, a 45 L/min Hydrofarm
AAPA45L Active Aqua Commercial Air Pump (Hydrofarm, Petaluma, CA, USA), 6 VIVOSUN
4 × 2 Inch Large Air Stone Cylinders (Vivosun, Los Angeles, CA, USA), and Aquatic Experts Rigid
Latex-Coated Coarse Bulk Roll Pond Filter Media, 18 × 72 × 1 inches (Aquatic Experts, Greensboro,
NC, USA) as biofilter material for the bacteria. The nitrification was carried out in 55-gallon plastic
barrels [29].
For Source Water 4, prior to filtration, nitrification was performed over an 8 day period in order
to convert some of the ammonium and organic forms of nitrogen in the raw PHW into NO3 +NO2 .
Sustainability 2019, 11, 3605 6 of 16

A 100 mL volume of true nitrifying bacteria solution per 200 L of diluted PHW to introduce the bacteria
to the wastewater. The temperature was held at 26.7 ◦ C in closed-lid 55-gallon bins and supplied with
oxygen from 45 L/min air pumps using 6 air diffusing stones for 8 days. Additionally, 48 × 72 × 1
inches of filter pad media was submerged in the nitrifying solution to increase the surface area for
nitrifying bacteria to multiply on [29].

2.3.3. Filtration Preparation and Process, Sand and Sand + Carbon

Two pool filters were used, one for sand filtration and the other for sand+carbon filtration.
For sand filtration (Source Waters 2 and 4), filter-quality sand was poured into the filtration bed of
the pool filter to three-quarters of the height of the bed, according to the manufacturers instructions.
For sand+carbon filtration (Source Water 3), the middle third of the filter sand was replaced with
activated carbon. The filter bed was primed with tap water before use. The sand was Fairmount
Minerals Pool Filter #20 Grade Silica Sand (Fairmount Santrol, Benton Harbor, MI, USA), and the
activated carbon source was 4 × 8 US mesh size (particles were in the range of 0.25” to 0.125”) Custom
Aquatic Premium Bulk Granular Activated Carbon (Custom Aquatic, Vista, CA, USA). Between
nitrified and non-nitrified solutions, the filter media was removed, the filter bed was cleaned with
bleach and thoroughly rinsed, and fresh filter media was added to the filter bed. The 200 L of diluted
(both non-nitrified and nitrified) PHW were then passed through the pool filter at 5 psi at a flow rate of
75.7 L per minute (20 gallons per minute) for a single-pass filtration [29].

2.3.4. Fertilizer Supplementation

For Source Water 5, the procedure for preparing Source Water 1 was repeated, but using the sand
filtered 2.5% PHW mixture (Source Water 2) to prepare the formulation of hydroponic fertilizer instead
of tap water. Thus, this source water has both the hydroponic fertilizer and PHW.

2.3.5. Growth Period and Harvesting

After the source water mixtures and grow bins were prepared, the lettuce was grown for 28 days
before harvesting. Environmental conditions in the room were controlled and spatially consistent for
the experiment duration. During this time, grow bins were inspected daily to observe growth and
replace any water lost to evapotranspiration with the prepared source water stock solution. At harvest,
the leafy mass of the lettuce was weighed individually and then collected by the source water group
and shelf number to be dried for quantifying leaf tissue elemental concentration.

2.4. Analyses

2.4.1. Lettuce Yields

Lettuce yields were determined by measuring the mass of both initial lettuce plugs and final
aboveground lettuce tissue for each individual plant. Mass was measured with a Mettler Toledo
(Columbus, OH, USA) PL601-S Electronic Scale Balance. The difference in plant mass only accounts for
the “above ground” portion of the plant, though the plug measurements also contained the roots and
plug media for the mass calculations, as removing the roots and rooting media would damage the
plant. For a healthy and fully-grown lettuce plant, the initial mass added from the inclusion of the
roots and plug media was assumed negligible compared to the grown mass of the plant.
The net fresh yield is presented as the difference between the fresh mass at harvest minus the
fresh mass of the plug at the start of the study. The net fresh yield is important for highlighting how
much the lettuce plants grew after exposure to the water treatments, as well as illustrating treatments
where the lettuce plants decreased in mass or died. The total dry yield, however, is the average mass
across the three bins for each treatment of the dry lettuce at harvest. The plant tissue was dried in a
drying oven at 70 ◦ C until a constant mass was achieved. From this, the dry mass of the aboveground
plant tissue was recorded.
Sustainability 2019, 11, 3605 7 of 16

2.4.2. Metal(loid) Uptake by Lettuce

Dried plant leaf tissue from the harvested lettuce was gathered by grow bin unit and ground
for one minute using a blade grinder (80335R Hamilton Beach, Glen Allen, VA, USA) to homogenize
and achieve a consistent particle size of dry plant tissue. Ground tissues samples were digested and
analyzed for mercury (Hg), lead (Pb), cadmium (Cd), and arsenic (As) by inductively coupled plasma
mass spectrometry (ICP-MS) using a Perkin Elmer Nexion 350D at the Microanalysis Laboratory at the
School of Life Sciences and the University of Illinois.

2.4.3. Nutrient Element Assay

Nutrients in all aqueous samples were tested at the Water Quality Lab at the Agricultural
Engineering Sciences Building. Samples were analyzed for nitrogen (total N and NO3 + NO2 )
according to automated hydrazine reduction method (Standards Methods 4500-NO3-H, NEMI) and
for orthophosphate and total P using the ascorbic acid reduction method (Standard Methods 4500-P-F,
NEMI). A portion of the dried plant material was sent to Midwest Laboratories for determination of
total N and P.

2.4.4. E. coli/coliforms Testing

The coliform and E.coli indicator organisms were detected and enumerated using 3M PetrifilmTM
E.Coli/Coliform Count plates (Product Code 6414). These E.coli/Coliform plates contain Violet Red Bile
nutrients, a cold-water-soluble gelling agent, an indicator of glucuronidase activity, and a tetrazolium
indicator that facilitate colony detection and enumeration in food and aqueous samples. One milliliter
of water was taken from each sample, plated, and incubated at 37 ◦ C for 24 h.

2.5. Source Waters

Based on methods described above, the nutrient and metal(loid) concentrations of the source
waters were determined and are presented in Table 2.

Table 2. Average concentrations of nutrients and metal(loid)s in the water prior to lettuce being grown.

Source NO3 -N + Total N PO4 -P Total P

Cd µg/L Hg µg/L Pb µg/L As µg/L
Water NO2 -N mg/L mg/L mg/L mg/L
1 193.38 211.20 29.30 29.30 0.26 0.00 2.45 1.86
2 0.00 8.55 0.09 0.10 0.08 0.00 3.06 8.18
3 0.00 1.36 0.42 0.43 0.13 0.00 2.75 6.75
4 0.00 20.58 0.05 0.18 0.54 0.00 0.50 2.21
5 193.38 219.74 29.39 29.40 0.26 0.30 2.19 2.13

Across all source water treatments, no E. coli/coliforms were detected in the water, initially.
Therefore, E. coli/coliform testing was not conducted for the lettuce in this experiment, as any
contamination would not be due to the water.

2.6. Statistical Analysis

Differences between the treatments were analyzed for significance using a one-way analysis of
variance (ANOVA) with post-hoc Tukey Honestly Significant Difference (HSD) for comparing multiple
source water treatments. When sample sizes were unequal, the Tukey-Kramer method was used.

3. Results and Discussion

At the end of the 4-week growing cycle, all lettuce tissue was harvested and each individual plant
weighed. The plants were then combined according to bin, dried, and weighed again. The dried
Sustainability 2019, 11, 3605 8 of 16

plants were analyzed for nutrient content and concentrations of metal(loid)s. Table 3 summarizes the
concentrations of metal(loid)s, nutrient content, and yields of lettuce for all five source water treatments.

Table 3. Summary table of concentrations of metal(loid)s, nutrient content, and yield of lettuce for each
of the five source water treatments.

Source Concentrations of Metal(loid)s in Dry Leaf Nitrogen and

Water Mass (µg/kg) Phosphorus (%) Net Fresh Total Dry
Yield (g) Yield (g)
Cd Hg Pb As N P
1 4.20 2.74 57.86 30.56 5.45% 1.04% 127.34 93.67
2 15.60 3.19 136.50 81.81 1.93% 0.16% 3.49 30.60
3 16.45 2.63 77.01 118.32 1.84% 0.14% 0.78 27.00
4 7.76 1.21 32.86 99.85 1.53% 0.23% 7.19 35.57
5 0.81 0.93 3.64 42.69 5.44% 0.99% 78.25 70.20

Table 3 summarizes the average concentrations across the three bins for each source water treatment
of cadmium, mercury, lead, and arsenic, as well as the percentage of nitrogen and phosphorus in the
dry lettuce tissue at harvest. In addition, the net fresh and total dry yields are provided. The net fresh
yield is presented as the difference between the fresh yields at harvest minus the fresh mass of the
plug at the start of the study. Source Water 1 (control) had the highest net fresh yield of all five source
water treatments; 36.5 times higher than Source Water 2, 163.3 times higher than Source Water 3, 17.7
times higher than Source Water 4, and 1.6 times higher than Source Water 5. Source Water 1 also had
the highest total dry yield of all five source water treatments; 3.06 times higher than Source Water 2,
3.5 times higher than Source Water 3, 2.6 times higher than Source Water 4, and 1.3 times higher than
Source Water 5. It should be noted that the total dry yield is higher than the net fresh yield for Source
Waters 2, 3, and 4, due to desiccation of above-ground biomass of many of the individual lettuce plants.
Plants that had lower mass or died allowed for a decrease in net fresh yield, but still contributed to a
total dry yield since some plant matter remained. Differences in metalloid concentrations thus appear
to be driven by N and P contents of source water via biomass dilution.
The concentration of cadmium was the highest in the lettuce from Source Water 3, at nearly
four times higher than Source Water 1 (control). Lettuce from Source Water 3 also had the highest
arsenic concentration, which was nearly four times higher than Source Water 1. Lettuce from Source
Water 2 had the highest mercury concentration, but it was only slightly higher than Source Water 1.
The concentrations of cadmium, mercury, and lead were significantly lower for the lettuce from Source
Water 5, even though it was the diluted PHW supplemented with hydroponics fertilizer, which Source
Water 1 also received. Therefore, it is a possibility that a compound in the PHW prevented the lettuce
from taking up cadmium, mercury, and lead, or perhaps a mechanism of the plant that prevented
uptake. The concentrations of mercury and lead were lower for the lettuce from Source Water 4 than
Source Water 1.
Lettuce shoot nitrogen and phosphorus contents were nearly identical for Source Water 1 and 5
even though the total dry yield was 1.3 times higher for Source Water 1. The nitrogen content for Source
Water 1 was 2.8, 3.0, and 3.6 times higher than Source Waters 2, 3, and 4, respectively. The phosphorus
content for Source Water 1 was 6.5, 7.4, and 4.5 times higher than Source Waters 2, 3, and 4, respectively.
Figure 2 shows the net fresh yields of each bin and for each source water. Therefore, it seems that
Source Waters 2, 3, and 4 could have used more nitrogen and phosphorus.
The overall trend is that Source Waters 1 and 5 had relatively lower metal(loid) concentrations,
but higher nitrogen and phosphorus concentrations as well as higher fresh and dry weight. This can
likely be explained by Source Waters 1 and 5 having sufficient nutrients for normal lettuce growth
and thus the lettuce plants in those treatments were able to uptake more nitrogen and phosphorous
and produce biomass. Meanwhile, the deficiency in nutrients for Source Waters 2, 3, and 4 likely
limited plant growth and could have promoted uptake of metal(loid)s as a byproduct of plant stress.
 Sustainability 2019, 11, x FOR PEER REVIEW 9 of 17

The overall trend is that Source Waters 1 and 5 had relatively lower metal(loid) concentrations,
Sustainabilitybut
2019, 11, 3605
higher nitrogen and phosphorus concentrations as well as higher fresh and dry weight. This can 9 of 16
likely be explained by Source Waters 1 and 5 having sufficient nutrients for normal lettuce growth
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not uniformly
higher concentrations of contaminants and nutrients in the source water did not uniformly lead to a
higher uptake of those compounds in the plant tissue. However, it is difficult to draw full conclusions
higher uptake of those compounds in the plant tissue. However, it is difficult to draw full conclusions
from this fromphenomenon, as theasplants
this phenomenon, were
the plants wereseverely deficient
severely deficient in nutrients
in nutrients and already
and already had partially
had partially
desiccated, which could
desiccated, confound
which could thethe
confound findings
findingsfor thesesource
for these source water
water treatments.
treatments.

Figure
Figure 2. Net 2. Net
fresh fresh
yield yield ofduring
of lettuce lettuce experiment
during experiment
plottedplotted according
according to thetofertilizer
the fertilizer
used over the
4 weeks ofused over the
growth for4each
weeks of growth for
replication each replication
performed. performed.
The cross The cross
represents represents
mean, centermean,
bar represents
center bar represents median, upper and lower limits of the box represent the upper and
median, upper and lower limits of the box represent the upper and lower quartiles, and whiskers
lower quartiles, and whiskers represent the range of the data, with outliers outside of the
represent whiskers.
the range of the data, with outliers outside of the whiskers.

Within each source

Within eachwater
sourcetreatment, replications
water treatment, had similar
replications distributions
had similar forfor
distributions yields.
yields.Of
Ofthe
thetreatments,
treatments,
Source Water 1 hadSource Water 1 net
the greatest had fresh
the greatest
yields,netfollowed
fresh yields, followed
closely byclosely
Source by Water
Source Water 5.
5. Source Waters
2, 3, and 4Source Waters 2, 3, and 4 all had similar, but extremely poor yields. Table 4 shows the p-values for
all had similar, but extremely poor yields. Table 4 shows the p-values for all source water
all source water comparisons of net fresh yield. The significant differences (p < 0.05) are highlighted
comparisons of for
in red fresh yield. The significant differences (p < 0.05) are highlighted in red for convenience.
netconvenience.

Table 4. Significant differences in fresh mass (yield) between source waters. Significant differences (p <
0.05) are highlighted in red.

Source Water 1 Source Water 2 p-Value

1 2 0.001
1 3 0.001
1 4 0.001
1 5 0.001
2 3 0.900
2 4 0.900
2 5 0.001
3 4 0.900
3 5 0.001
4 5 0.001
 2 4 0.900
2 5 0.001
3 4 0.900
Sustainability 2019, 11, 3605 3 5 0.001 10 of 16
4 5 0.001

The statistical analysis confirmed that the Source Water 1 net fresh yield was significantly higher
The statistical analysis confirmed that the Source Water 1 net fresh yield was significantly higher
than all other treatments and the Source Water 5 net fresh yield was higher than Source Waters 2, 3,
than all other treatments and the Source Water 5 net fresh yield was higher than Source Waters 2, 3,
and 4. Figure 3 shows the total dry lettuce yield from each bin and for each source water. The average
and 4. Figure 3 shows the total dry lettuce yield from each bin and for each source water. The average
total dry yield is also displayed for all three bins for a given source water with the solid blue bar.
total dry yield is also displayed for all three bins for a given source water with the solid blue bar.

Total Dry Lettuce Yield at Harvest

120
Bin 1 Bin 2 Bin 3 Average
100

80
Yield (g)

60

40

20

0
1 2 3 4 5
Source Water

Figure3. 3.
Figure Total
Total dry yield
dry yield of atlettuce
of lettuce harvestatforharvest for the
the 5 different 5 different
source source
waters. The waters.
individual yieldThe
for
individual yield for each bin is provided, in addition
each bin is provided, in addition to the average of the three bins.to the average of the three bins.

The
The total
total dry
dry yield
yield of
of each
each individual
individual bin bin varied
varied slightly.
slightly. Source
Source Waters
Waters 1,1, 2,
2, and
and 33 had
had higher
higher
total
total dry
dryyields
yieldsfrom
frombinbin1,1,which
whichwaswasthethebin
binononthe
thetop
topshelf
shelfof ofthe
therack.
rack. In
In contrast,
contrast, Source
Source Waters
Waters
44 and
and 55 had
had higher
higher total
total dry
dry yields
yields from
frombin bin3,3,which
whichwaswasthe theshelf
shelfatatthe
thebottom
bottomof ofthe
therack.
rack. ItIt isis
possible
possiblethat
thatlocation
locationofofeach binbin
each within
within thethe
rackrack
could havehave
could impacted the yields.
impacted However,
the yields. since since
However, each
treatment had one
each treatment hadbin at bin
one eachatlevel
eachoflevel
the rack,
of thethis potential
rack, variation
this potential is accounted
variation for when
is accounted fortaking
when
the average
taking the of the three
average of bins. Tablebins.
the three 5 shows the5p-values
Table shows thefor all source water
p-values for alltreatment comparisons
source water treatment of
total dry yieldofwith
comparisons totalsignificant
dry yield differences (p < 0.05)
with significant highlighted
differences in red.
(p < 0.05) highlighted in red.

Table 5. Significant differences in total dry mass (yield) between source waters. Significant differences
(p < 0.05) are highlighted in red.

Source Water 1 Source Water 2 p-Value

1 2 0.001
1 3 0.001
1 4 0.001
1 5 0.060
2 3 0.900
2 4 0.900
2 5 0.002
3 4 0.755
3 5 0.001
4 5 0.006
 2 5 0.002
3 4 0.755
3 5 0.001
Sustainability 2019, 11, 3605 11 of 16
4 5 0.006

Total
Total drydry yield
yield from
from SourceWater
Source Water1 1and
and5 5was
wassignificantly
significantlyhigher
higherthan
thanSource
SourceWaters
Waters 2,2, 3,
3, and
and 4.
4. There was not a significant difference in the total dry yield between Source Waters 1 and 5. This is is
There was not a significant difference in the total dry yield between Source Waters 1 and 5. This
in in contrast
contrast to to Mazur
Mazur [28][28]
whowho did
did seesee a significant
a significant increase
increase in in yields
yields andand inferred
inferred that
that PHWPHW cancan
increase grain yields, whereas our lettuce yields with supplemented PHW decreased
increase grain yields, whereas our lettuce yields with supplemented PHW decreased overall yields. overall yields.
Figure
Figure 4 shows
4 shows thethe nitrogen
nitrogen andand phosphorus
phosphorus contents
contents in in
thethe total
total dry
dry lettuce
lettuce from
from each
each binbin
and and forfor
eachtreatment.
each treatment. Lettuce
Lettuce leaf
leafnutrient concentration
nutrient guidelines
concentration were provided
guidelines by Midwest
were provided by Laboratories
Midwest
as benchmarks of sufficiency for nitrogen and phosphorus.
Laboratories as benchmarks of sufficiency for nitrogen and phosphorus.

N and P in Dry Lettuce at Harvest

6%
1
5%
2

4% 3

4
3%
5
2%
Norm

1%

0%
Nitrogen Phosphorus

Figure
Figure4.4. Percent nitrogen
Percent nitrogen andand phosphorus
phosphorus in the
in the dry dry
lettuce forlettuce for water
each source each treatment.
source water
Norms
treatment.
are from Midwest Laboratories for sufficient element composition for adult lettuce plant tissue. for
Norms are from Midwest Laboratories for sufficient element composition
adult lettuce plant tissue.
The nitrogen and phosphorus contents for lettuce harvested from Source Waters 1 and 5 were
above
The the norms,and
nitrogen meaning there was
phosphorus sufficient
contents nitrogen
for lettuce and phosphorus,
harvested from Sourcewhile the lettuce
Waters 1 andharvested
5 were
fromthe
above Source
norms,Waters 2, 3,there
meaning and 4was
were below the
sufficient normsand
nitrogen for phosphorus,
nitrogen andwhilephosphorus, meaning
the lettuce there
harvested
wasSource
from a nitrogen
Watersand2,phosphorus
3, and 4 werelimitation.
below theTable 6 shows
norms the p-values
for nitrogen for all sourcemeaning
and phosphorus, water treatment
there
comparisons of nitrogen and phosphorus content. The significant differences (p < 0.05) are highlighted
in red for convenience.

Table 6. Significant differences in N and P content in the dry leaf mass between source waters.
Significant differences (p < 0.05) are highlighted in red.

Source Water 1 Source Water 2 N P

1 2 0.001 0.001
1 3 0.001 0.001
1 4 0.001 0.001
1 5 0.900 0.900
2 3 0.900 0.900
2 4 0.060 0.672
2 5 0.001 0.001
3 4 0.185 0.457
3 5 0.001 0.001
4 5 0.001 0.001

The statistical analysis confirmed that the nitrogen and phosphorus contents in the dry lettuce
was significantly greater for Source Waters 1 and 5 than Source Waters 2, 3, and 4. There was no
 3 4 0.185 0.457

3 5 0.001 0.001
4 5 0.001 0.001
Sustainability 2019, 11, 3605 12 of 16
The statistical analysis confirmed that the nitrogen and phosphorus contents in the dry lettuce
was significantly greater for Source Waters 1 and 5 than Source Waters 2, 3, and 4. There was no
significant different
significant differentininnitrogen
nitrogenor
orphosphorus contentbetween
phosphorus content betweenSource
Source Water
Water 1 and
1 and Source
Source Water
Water 5. 5.
Figure 5 shows the concentrations of metal(loid)s in the dry lettuce from each bin and for each source
Figure 5 shows the concentrations of metal(loid)s in the dry lettuce from each bin and for each source
water treatment.
water treatment.

Metal(loid)s in Dry Lettuce at Harvest

300
1 2 3 4 5 Max

250

200
Concentration (µg/kg)

150

100

50

0
Cd Hg Pb As
Metal

Figure
Figure 5. Comparison
5. Comparison of the
of the concentration
concentration of metal(loid)s
of metal(loid)s in theinlettuce
the lettuce at harvest
at harvest for thefor the 5
5 different
different source water treatments. The maximum allowable levels, as set by the
source water treatments. The maximum allowable levels, as set by the US Department of Agriculture US
Department
Foreign of Agriculture
Agriculture Foreign
Service, is also Agriculture
included Service,
for comparison is also included for comparison
[30].
[30].
The concentrations of cadmium, mercury, and lead were all below the maximum allowable levels
for food as determined by the US Department of Agriculture Foreign Agriculture Service [30] for all
five source water treatments. The concentration of arsenic in lettuce from Source Waters 2, 3 and 4,
however, were above the maximum allowable level. A study by Eregno et al. [12] involved growing
lettuce hydroponically with treated greywater with added human urine and found that lettuce leaves
across all treatments were below estimated Health Risk Index and Target Hazard Quotient values and
therefore implied to have low health risk for consumption. However, Eregno et al. [12] did report
that the HRI for As and THQ for As and Cr were relatively higher than the other metal(loid)s tested,
which is similar to our findings for arsenic. Table 7 shows the p-values for all source water treatment
comparisons of metal(loid)s content. The significant differences (p < 0.05) are highlighted in red
for convenience.

Table 7. Significant differences in metals concentrations in the dry leaf mass between source waters.
Significant differences (p < 0.05) are highlighted in red.

Source Water A Source Water B Cd Hg Pb As

1 2 0.042 0.900 0.669 0.073
1 3 0.029 0.900 0.900 0.003
1 4 0.804 0.498 0.900 0.014
1 5 0.830 0.350 0.880 0.900
2 3 0.900 0.900 0.834 0.264
2 4 0.210 0.277 0.453 0.797
2 5 0.009 0.181 0.242 0.213
3 4 0.146 0.561 0.900 0.784
3 5 0.006 0.408 0.714 0.008
4 5 0.303 0.900 0.900 0.042
Sustainability 2019, 11, 3605 13 of 16

The average concentration of cadmium across the three bins was significantly lower in Source
Water 1 than Source Waters 2 or 3 and significantly higher in Source Waters 2 and 3 than Source Water 5.
In addition, the average concentration of arsenic across the three bins was significantly higher in Source
Waters 3 and 4 than Source Waters 5. Figure 6 shows a regression analysis between the total dry lettuce
yield and metal(loid) concentrations.
Sustainability 2019, 11, x FOR PEER REVIEW 14 of 17

Sustainability 2019, 11, x FOR PEER REVIEW 14 of 17

Total Dry Lettuce Yield versus Metal(loid) Concentrations
100
Total Dry Lettuce Yield versus Metal(loid) Concentrations
90
100
80
90
70
80
60
Yield (g)

70
50 R² = 0.0197
60
Yield (g)

R² = 0.2165
40 R² = 0.6607
R² = 0.0197
50
30 R² = 0.2165
40 R² = 0.6607
20 R² = 0.8749
30
10
20 As Pb R² = 0.8749
Hg Cd
0
10
0 20 40 As 60 Pb 80 100
Hg 120 Cd 140
0 Concentration (µg/kg)
0 20 40 60 80 100 120 140
Figure 6. Correlation between dry lettuce yield and metal(loid)
Concentration concentrations. The R22
(µg/kg) concentrations.
Figure 6. Correlation between dry lettuce yield and metal(loid) The R values are
values are provided for each best fit line.
provided for each best fit line.
Figure 6. Correlation between dry lettuce yield and metal(loid) concentrations. The R2
There
values is are
a negative
providedcorrelation between
for each best fit line.lettuce leaf yield and leaf concentrations of all four
There is a negative
metal(loid)s, meaning correlation
a higher yield led between
to biomass lettuce leaf
dilution, yieldthe
in which and loadleaf concentrations
of metal(loid) elements of all four
metal(loid)s, meaning
is diluted
Thereinto a higher
is more
a negative yield led
tissue biomass.
correlation to biomass
A strong
between dilution,
correlation
lettuce existed
leaf inbetween
yield which
and leaf the
total loadlettuce
dry of metal(loid)
concentrations yield fourelements
of alland
is diluted intoconcentration
arsenic moremeaning
metal(loid)s, tissue(Rbiomass.
a2 =higher
0.8749). AAmoderate
yield strong
led correlation
correlation
to biomass existed
existed
dilution, between
between
in which total of
the load total
dry dryyield
lettuce
metal(loid) lettuce
and yield and
elements
cadmium
arsenic concentration (R2 =tissue
is dilutedconcentration
into more (R2 biomass.
= 0.6607),
0.8749). Aand relatively
strong
A moderate weak existed
correlation
correlation correlations
between
existed withtotal
between leaddry
(R2lettuce
total = dry
0.2165) and
yield
lettuce andyield and
mercury (R = 0.0197). Figure
2
arsenic concentration (R 2 7 shows
= 0.8749). a regression
A moderate analysis existed
correlation between the total
between drydry
total lettuce yield
lettuce and
yield and
cadmium concentration (R = 0.6607), 2 and relatively weak correlations with lead (R = 0.2165) and 2
nutrient
cadmium content.
concentration (R2 = 0.6607), and relatively weak correlations with lead (R2 = 0.2165) and
(R2 = 0.0197).
mercurymercury (R2 = 0.0197). Figure 7 showsa aregression
Figure 7 shows analysis
regression analysis between
between the total
the total dry lettuce
dry lettuce yield and yield and
nutrient nutrient
content.content.
Dry Lettuce Yield versus Nutrient Content
100
90
Dry Lettuce Yield
N versus Nutrient
P Content
100
80
90 R² = 0.9373 N P R² = 0.8928
70
Yield (g)

80
60 R² = 0.8928
R² = 0.9373
50
70
Yield (g)

4060
3050
2040
1030
020
10 0 1 2 3 4 5 6

0 Nutrient Content (%)

0 1 2 3 4 5 6
Figure 7. Correlation between dry lettuce yield and
Nutrient nutrient
Content (%) content. The R values are 2

provided for each best fit line.

Figure 7.Figure 7. Correlation

betweenbetween dry lettuce yield and nutrient content.
The RThe R values are 2
Correlation dry lettuce yield and nutrient content. 2 values are provided for
provided for each best fit line.
each best fit line.
Sustainability 2019, 11, 3605 14 of 16

There is a positive correlation between yields and both nitrogen and phosphorus, meaning the
total dry yield decreased as the concentrations of each nutrient increased. A strong correlation existed
between total dry lettuce yield and both nitrogen (R2 = 0.8928) and phosphorus (R2 = 0.9373).

4. Broader Impacts
Lettuce yields and nutrient concentrations indicate that PHW source waters without supplemented
hydroponic fertilizer (Source Waters 2, 3, 4) had significantly lower lettuce yields and nutrient content
due to N and P limitation. Therefore, diluted PHW alone is not a sufficient sole nutrient source
for meeting N and P needs of hydroponically grown lettuce. However, supplemented PHW and
conventional hydroponic fertilizer were not significantly different. Thus, PHW can be used in
hydroponic fertilizer formulation without a significant negative impact on yields and nutrition as a
source of nitrogen and irrigation water. Still, it was seen that supplemented PHW had overall lower
yields and nutrients than hydroponic fertilizer alone, indicating some inhibitory effect in PHW that
warrants further investigation. Moreover, N and P supplementation promotes significant increases in
the biomass of edible tissue, diluting concentrations of metal(loid)s and thus increasing the utility of
PHW for hydroponic crop production from a food safety standpoint.
While the plant tissue analysis shows that arsenic exceeded the maximum allowable concentration
for lettuce in Source Waters 2, 3, and 4, these treatments had vastly lower yields and showed
extreme symptoms of nutrient deficiency. In other words, one potential implication of this study is
that sufficient nutrient supplementation can be used to manage potential metal(loid) accumulation
in edible tissues. This means that PHW needs to be supplemented with other nutrients for lettuce
cultivation, but when PHW is supplemented, it can produce similar yields and nutrition to conventional
hydroponic production.
As HTL is already an attractive technology of biofuel production derived from wastewater,
producing hydroponic crops with the PHW byproduct only further increases the incentive to include
HTL in the Food-Water-Energy Nexus approach. HTL production facilities could be coupled with
hydroponic crop production facilities to address all three pillars of the Food-Water-Energy Nexus and
increase the sustainability of the HTL technology.
Further research on PHW as a crop fertilizer should focus on finding methods to convert the
nitrogen in PHW into plant-available inorganic forms (e.g., NO3 and NO2 ), identifying inhibitory
compounds in PHW, finding sources of wastewater rich in phosphorous that could complement PHW’s
nitrogen concentration, expanding this research to other cultivars of lettuce or other hydroponic crops,
and examining the overall nutrient, water, and energy efficiency of the hydrothermal liquefaction
process in conjunction with cultivation using PHW.

5. Conclusions
This study evaluated treated PHW source waters for the production of safe hydroponic lettuce.
Four PHW source water treatments were compared to synthetic fertilizer in cultivating lettuce. Water
containing only hydroponic fertilizer (Source Water 1) had the highest total dry yield of all five
treatments. Findings also indicated that while PHW was below the US Department of Agriculture
Foreign Agriculture Service maximum levels for cadmium, lead, and mercury in food, the concentration
of arsenic was higher than the maximum level for Source Waters 2, 3, and 4, respectively. There was
no detectable E. coli or fecal coliforms in any of the treated PHW. While nitrogen was present in the
raw PHW, only 0.03% was NO3 -N and NO2 -N. Diluted PHW supplemented with hydroponic fertilizer
had lower lettuce yield than hydroponic fertilizer alone, indicating a potential non-nutrient inhibition
of plant growth by PHW.
This study demonstrates the agronomic and food safety potential of PHW for hydroponic
production of nutritionally important crops such as leafy greens. Additionally, this study identifies
the importance of supplementing source waters with nitrogen and phosphorus for economic crop
production as well as to mitigate the risk of metalloids to consumers via biomass dilution.
Sustainability 2019, 11, 3605 15 of 16

Author Contributions: Conceptualization, S.J. and P.C.D.; Data curation, S.J. and P.C.D.; Investigation, S.J. and
P.C.D.; Methodology, S.J. and P.C.D.; Project administration, P.C.D.; Resources, Y.Z., A.J.M. and P.C.D.; Supervision,
P.C.D.; Writing—original draft, S.J.; Writing—review & editing, Y.Z., A.J.M. and P.C.D.
Funding: This research received no external funding.
Acknowledgments: The authors would like to thank Quintin Potthast and John (Jack) Bryant for their assistance
with laboratory data collection.
Conflicts of Interest: The authors declare no conflict of interest.

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