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Article The Effect of Anaerobic and Aerobic Sludge Supernatant on Hydroponic

Simon Goddek 1,*, Zala Schmautz 2, Ben Scott 2, Boris Delaide 3, Karel J. Keesman 1, Sven Wuertz 4 and Ranka Junge 2,*

1 Biobased Chemistry & Technology, Wageningen University, P.O. Box 17, Wageningen 6700 AA, The Netherlands; [email protected] 2 Institute for Natural Resource Sciences Gruental, ZHAW Zurich University of Applied Sciences, Waedenswil CH-8820, ; [email protected] (Z.S.); [email protected] (B.S.) 3 Integrated and Urban Pathology Laboratory, Université de Liège, Avenue Maréchal Juin 13, Gembloux 5030, Belgium; [email protected] 4 IGB, Ecophysiology and , Müggelseedamm 310, Berlin 12587, Germany; [email protected] * Correspondence: [email protected] (S.G.); [email protected] (R.J.); Tel.: +31-61-122-8251 (S.G.); +41-58-934-5922 (R.J.)

Academic Editor: Mike McLaughlin Received: 12 May 2016; Accepted: 14 June 2016; Published: 21 June 2016

Abstract: The mobilization of from fish sludge (i.e., feces and uneaten feed) plays a key role in optimizing the resource utilization and thus in improving the sustainability of aquaponic systems. While several studies have documented the aerobic and performance of aquaculture sludge, the impact of the on plant growth has yet to be understood. The present study examines the impact of either an aerobic or an anaerobic digestion effluent on lettuce plant growth, by enriching a mixture of aquaculture and tap with supernatants from both aerobic and anaerobic batch reactors. The lettuce grown in the hydroponic system supplied with supernatant from an anaerobic reactor had significantly better performance with respect to weight gain than both, those in the system where supernatant from the aerobic reactor was added, as well as the control system. It can be hypothesized that this effect was caused by the presence of + NH4 as well as dissolved organic matter, plant growth promoting rhizobacteria and fungi, and humic acid, which are predominantly present in anaerobic effluents. This study should therefore be of value to researchers and practitioners wishing to further develop sludge remineralization in aquaponic systems.

Keywords: aquaponics; anaerobic digestion; aerobic digestion; ; sludge; integrated aquaculture; ; bio-

1. Introduction A primary concern of aquaponics is the efficient utilization of all nutrients that enter the system. Several studies have documented a high loss of nutrients from the recycling loop via the mechanical water treatment unit as well as unused Recirculating Aquaculture System (RAS)-derived sludge [1–6] that consists of feces and uneaten feed. Consequently, the reuse of sludge is gaining importance and plays a crucial role in the utilization of the supplied nutrients and thereby in reduction of emissions [2,7]. With respect to the usage of RAS water in hydroponic systems, Jijakli et al.[8] examined whether complementing the RAS water with mineral elements to levels usually targeted in hydroponics improves growth. They observed an increased plant growth of the complemented aquaponic solutions by up to 39% compared with the hydroponic control nutrient solution, which was composed of an equivalent nutrient composition. Their work shows that plants in a highly

Agronomy 2016, 6, 37; doi:10.3390/agronomy6020037 www.mdpi.com/journal/agronomy Agronomy 2016, 6, 37 2 of 12 concentrated RAS nutrient solution had better growth. However, their study has only a limited relevance with regard to the impact of aerobic and anaerobic supernatants from fish sludge digestion on plant growth performance. Although some research was carried out on the impact of domestic and industrial wastes on plant growth [9–11], to date, the impact of aerobically and anaerobically treated RAS-derived sludge on plant growth has not yet been experimentally investigated. Effluents from anaerobic digestion generally have high concentrations of nutrients, as sources of carbon are metabolized preferentially during treatment [9,11]. However, most anaerobic effluents are characterized by high chemical oxygen demands (CODs), hydrogen sulfides (H2S) content, and low dissolved oxygen (DO) concentrations, and thus can be considered phytotoxic. In this case, a post-treatment (e.g., aerobic detoxification) for the anaerobic effluent is required before being directed to the plants [10–12]. The primary aim of this study was to comparatively assess the effect of effluents originating from anaerobic and aerobic batch digesters on to plant growth performance. Even though simple digestion techniques were used that did not exploit the entire potential of both treatments, this study offers some important insights into the role that remineralization practices may play in aquaponic systems with respect to plant growth performance.

2. Materials and Methods

2.1. Experimental Setup The experiment was conducted in a in Wädenswil from 16 October to 20 November 2015. Three one-cycle aquaponics systems were used for this experiment, each built of a sump, a settling tank, and a biofilter, and three nutrient film technique (NFT) channels (Figure1) were run in parallel and were planted with 36 lettuce plants (Lactuca sativa yacht variety Salanova®). The total volume of one system was approximately 400 L. Aeration into the sump and the biofilter was provided via compressed air (AL-80, Alita Industries, Arcadia, CA, USA) and air stones to assure sufficient DO levels as well as proper mixing of biochips in the biofilter. A water pump (Aquarius Universal Eco 4000, Oase, Germany) with an approximate flow rate of 62 L/h directed water from the biofilter to the NFT channels. A heater (NEWA Therm pro 250 W, NEWA Tecno Industrial Srl, Loreggia, Italy) was installed in the biofilter maintaining constant temperatures in the system (22 ˘ 1.5 ˝C). No additional lighting was used. For two months (since July 2015), 4 L of RAS sludge of Nile culture fed with Hokovit Tilapia Vegi feed were added weekly to two reservoirs. One was constantly aerated and the other one was kept under anaerobic conditions. No additional sludge was added to the reservoirs during the experiment (i.e., after 16 October). To gather the supernatant, the sludge was stirred and centrifuged (Eppendorf Centrifuge 5430, Fisher Scientific, Waltham, MA, USA), 2.5 min at 7000 rcf. Supernatant nutrient composition was determined in the second week of the experiment (Table1). At the start of the experiment, all systems were filled with 85% tap water and 15% RAS water (Table1). After that, one liter of supernatant from each aerobic and anaerobic reservoir was added to the aerated system (AER) and unaerated system (ANA) three times a week, respectively. System RAS, acting as a control, was given one liter of RAS water instead.

Table 1. Nutrient composition (mg/L) of the Recirculating Aquaculture System (RAS) and tap water, as well as AER and ANA supernatants, at the beginning of the experiment.

Cl NO2 NO3 PO4 SO4 Na NH4 K Ca Mg AER 1 Supernatant 7.9 18.5 51.8 2.7 19.7 4.4 0.7 17.2 14.2 5.1 ANA 2 Supernatant 6.1 0.0 0.0 2.7 0.5 3.6 56.4 16.3 11.7 2.5 Tap 0.3 0.0 0.2 0.0 0.8 4.9 0.0 1.0 59.8 16.3 RAS 3 6.4 0.2 88.2 2.0 6.9 3.4 0.5 9.0 10.8 5.6 1 supernatant from the aerated reactor; 2 supernatant from the anaerobic reactor; 3 water from a recirculating aquaculture system. Agronomy 2016, 6, 37 3 of 12 Agronomy 2016, 6, 37 3 of 12

Figure 1. Schematic outline of the experimental systems comprising a sump (max. 280 L), a settling Figuretank (70 1. L), Schematic and a biofilter outline (110 of the L). Extensiveexperimental aeration systems with comprising an air pump a ensuredsump (max. reduction 280 L), of a chemical settling tankoxygen (70 demandsL), and a (CODs) as (110 well L). as optimalExtensive dissolved aeration oxygen with an (DO) air inpump the process ensured water reduction for plant of chemicalgrowth. Undilutedoxygen demands AER and (CODs) ANA as supernatants well as optima as welll dissolved as Recirculating oxygen (DO) Aquaculture in the process System water (RAS) for plantwater growth. entered Undiluted the system AER via theand sumps ANA supernatants of the respective as well systems as Recirculating three times Aquaculture a week. Samples System for (RAS)measurements water entered were takenthe system in the via sump the atsumps least of 24 th he after respective the supernatants systems three or RAS times water a week. were Samples added. for measurements were taken in the sump at least 24 h after the supernatants or RAS water were 2.2. Supernatantadded. and Water Analysis

2.2. SupernatantSamples of and both Water aerobic Analysis and anaerobic supernatants were taken at the end of the first week of the experiment. Macronutrients (NO2, NO3, PO4, SO4, NH4, K, Ca, and Mg) and micronutrients (Cl and Na)Samples were of analyzed both aerobic with anand IC anaerobic (930 Compact supernatants IC Flex, Metrohm, were taken Zofingen, at the end Switzerland) of the first and week other of themicronutrients experiment. (B,Macronutrients Cu, Fe, Mn, Mo, (NO and2, NO Zn)3, PO with4, SO an4, ICP-AES NH4, K, (VarianCa, andVista Mg) AXand CCDmicronutrients Simultaneous (Cl andICP-AES, Na) were Agilent analyzed Technologies, with an Santa IC (930 Clara, Compact CA, USA). IC PriorFlex, toMetrohm, these analyses, Zofingen, homogenous Switzerland) anaerobic and otherand aerobic micronutrients sludge was (B, taken Cu, from Fe, bothMn, reservoirsMo, and andZn) centrifugedwith an ICP-AES for 7.5 min (Varian at 7000 Vista rcf and AX filtrated CCD Simultaneouswith 0.22 µm syringeICP-AES, filters Agilent (Table 1Technologies,). Santa Clara, CA, USA). Prior to these analyses, homogenousThe initial anaerobic nutrient and concentration aerobic sludge within was the take threen from aquaponics both reservoirs systems and was centrifuged derived from for the7.5 mininitial at RAS7000 andrcf and tap waterfiltrated values with that 0.22 were μm syringe analyzed filters with (Table the IC 1). Flex 930 Metrohm (Table2). The same equipmentThe initial was nutrient used to concentration determine the within water the nutrient three concentrationaquaponics systems in the was sump derived of each from system the initialthree timesRAS (onand Days tap water 19, 27, values and 36) that throughout were analyz the 5-weeked with experiment. the IC Flex These 930 Metrohm samples were (Table taken 2). fromThe samethe sump equipment one day was after used tap to water determine was added the water to compensate nutrient concentration for water losses indue the tosump evapotranspiration of each system threeto make times sure (on the Days water 19, 27, was and well 36) mixed. throughout DO, pH,the 5-week EC, and experiment. temperature These were samples measured were twice taken a fromweek withthe sump a portable one multi-parameterday after tap water meter was (HQ40d added Portable to compensate Multi-Parameter for water Meter, losses Hach due Lange, to evapotranspirationDüsseldorf, Germany). to make sure the water was well mixed. DO, pH, EC, and temperature were measured twice a week with a portable multi-parameter meter (HQ40d Portable Multi-Parameter Meter,2.3. Lettuce Hach Lange, Düsseldorf, Germany). Six weeks before the beginning of the experiment, single pelleted lettuce was seeded into rock 2.3. Lettuce wool. The initial weight of the lettuce seedlings when transferred into the aquaponic system was 10.3 gSix˘ weeks0.2 g, whichbefore wasthe beginning determined of bythe pre-weighting experiment, single the rock pelleted wool. lettuce After 36was days seeded of cultivation, into rock wool.nine lettuce The initial plants weight (front, of middle, the lettuce back) seedlings laid down when in advance transferred were into sampled the aquaponic from each system system was for 10.3further g ± analysis.0.2 g, which The shootswas determined were separated by pre-weighting from the roots the by rock cutting wool. it just After above 36 days the rockwool of cultivation, block, ninewhereas lettuce the rootsplants were (front, cut middle, off at the back) bottom laid side down of the in rockwooladvance were block. sampled The shoots from were each dried system at 80 for˝C furtherfor 48 h analysis. for dry weight The shoots determination. were separated The content from ofthe P, roots S, Mg, by Ca, cutting K, Fe, it Cu, just Mn, above Zn, B,the Mo, rockwool and Na block,was determined whereas the with roots an were ICP-OES cut off (5100 at the VDV bottom ICP-OES, side of Agilent the rockwool Technologies, block. The Santa shoots Clara, were CA, dried USA) atafter 80 °C the for dried 48 h biomass for dry wasweight pulverized determination. and acid The mineralized. content of TheP, S, C,Mg, H, Ca, and K, N Fe, content Cu, Mn, was Zn, measured B, Mo, and Na was determined with an ICP-OES (5100 VDV ICP-OES, Agilent Technologies, Santa Clara, CA, USA) after the dried biomass was pulverized and acid mineralized. The C, H, and N content

Agronomy 2016, 6, 37 4 of 12

Agronomy 2016, 6, 37 4 of 12 on three samples of dry lettuce plants per system using an elemental analyzer (TruSpec CHN Macro Analyzer,was measured LECO, Sainton three Joseph, samples MI, USA). of dry lettuce plants per system using an elemental analyzer (TruSpec CHN Macro Analyzer, LECO, Saint Joseph, MI, USA). 2.4. Statistical Analysis 2.4. Statistical Analysis Data are presented as mean of n replicates. Analysis of statistical significance was conducted in R softwareData [13 ],are using presented analysis as mean of variance of n replicates. (ANOVA) Analysis test and of poststatistical hoc multiple significance comparison. was conducted Here, in the parametricR software Tukey’s [13], using HSD testanalysis (p < 0.05)of variance was performed. (ANOVA) test and post hoc multiple comparison. Here, the parametric Tukey’s HSD test (p < 0.05) was performed. 3. Results & Discussion 3. Results & Discussion During the experiment DO levels in the sump and the recorded water temperatures were stable (Table2).During Even thoughthe experiment all three DO systems levels werein the started sump an withd the the recorded same initial water mix temperatures of tap water were with stable RAS outflow,(Table the 2). ECEven in though the sump all ofthree System systems AER were was starte initiallyd with higher the same than ininitial the othermix of two tap sumpswater with (Figure RAS2a; Tableoutflow,2). This the difference EC in the is sump probably of System due to AER the was fact initially that the higher systems than were in the used other for two experiments sumps (Figure before. Ca2a; and Table K residues 2). This indifference both, the is pipes,probably and due the to biofiltration the fact that media the systems might were have used caused for experiments these slightly higherbefore. values. Ca and An K indication residues forin both, that isthe the pipes, higher and Ca the and biofiltration K content media in System might AER have (see caused Figure these3d,e), slightly higher values. An indication for that is the higher Ca and K content in System AER (see while the Ca and K concentrations of both supernatant solutions did not reveal similar differences Figures 3d,e), while the Ca and K concentrations of both supernatant solutions did not reveal similar (Figure4; Table1). The pH values during the trial did not show major symmetric differences in the differences (Figure 4; Table 1). The pH values during the trial did not show major symmetric three systems and fluctuated between 8.1 and 8.7 (Figure2b; Table2). differences in the three systems and fluctuated between 8.1 and 8.7 (Figure 2b; Table 2).

Table 2. Means and standard deviation of main measured water parameters during the 35 days of the Table 2. Means and standard deviation of main measured water parameters during the 35 days of experiment (n = 11). the experiment (n = 11).

SystemSystem AER AER1 1 SystemSystem ANA ANA2 2 System RASRAS3 3 (Control)(Control) 4 DODO4 (mg/L) (mg/L) 8.5 8.5˘ ±0.3 0.3 8.4 8.4˘ ± 0.30.3 8.6 8.6˘ ± 0.30.3 WaterWater Temperatures Temperatures (˝C) (°C) 22.9 22.9˘ ±1.3 1.3 22.6 22.6˘ ± 1.61.6 21.2 21.2˘ ± 1.61.6 ECEC5 (5µ (S/cm)μS/cm) 895.5 895.5˘ ±59.8 59.8 766.6 766.6˘ ± 41.241.2 725.1 725.1˘ ± 49.249.2 pHpH 8.4 8.4˘ ±0.2 0.2 8.5 8.5˘ ± 0.20.2 8.5 8.5˘ ± 0.20.2 1 supernatant1 supernatant from from the aeratedthe aerated reactor; reactor;2 supernatant 2 supernatant from the from anaerobic the anaerobic reactor; 3 waterreactor; from 3 water a recirculating from a 4 5 aquaculturerecirculating system; aquaculturedissolved system; oxygen; 4 dissolvedelectrical oxygen; conductivity. 5 electrical conductivity.

FigureFigure 2. Variation 2. Variation of of (a )(a electric) electric conductivity conductivity (EC) (EC) and and ((bb)) pHpH (on(on a scale from from 8.1 8.1 to to 8.7) 8.7) in in the the sump sump of theof the aerobic aerobic (AER), (AER), anaerobic anaerobi (ANA),c (ANA), and and RAS RAS systems systems overover thethe 35-day35-day experimental period. period.

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FigureFigure 3. 3.Concentrations Concentrations ofof ((aa)) ; ( (bb)) ; sodium; (c (c) )phosphate; phosphate; (d ()d potassium;) potassium; (e) ( ecalcium;) calcium; and and (f) (f)sulfate sulfate in in the the sump sump of of the the aerobic aerobic (AER), (AER), an anaerobicaerobic (ANA), (ANA), and and RAS RAS systems systems over over the 35-day experimentalexperimental period. period.

FiguresFigures4 and 4 and5 show 5 show the macro- the macro- and micronutrient and micronutrient concentrations concentrations in the aerobic in the and aerobic the anaerobic and the supernatantanaerobic supernatant at the beginning at the of beginning the experiment. of the It expe mustriment. be mentioned It must that,be mentioned due to ICP that, analysis, due to we ICP do analysis, we do not have any information about the form of the analyzed metals (i.e., to what degree not have any information about the form of the analyzed metals (i.e., to what degree they are chelated). they are chelated). The results in Figure 5 show an increased manganese (Mn) concentration in the The results in Figure5 show an increased manganese (Mn) concentration in the aerobic supernatant. aerobic supernatant. According to Resh [14], the optimum Mn in hydroponic solutions ranges According to Resh [14], the optimum Mn in hydroponic solutions ranges between 0.5 and 0.8 mg/L, between 0.5 and 0.8 mg/L, which is in compliance with the concentration in the aerobic supernatant which is in compliance with the concentration in the aerobic supernatant before being diluted in the before being diluted in the experimental system. Upon dilution, deficiencies of Mn would be experimental system. Upon dilution, deficiencies of Mn would be expected in both systems, but to expected in both systems, but to a higher degree in System ANA. Micronutrients in the RAS water a higher degree in System ANA. Micronutrients in the RAS water were not measured. were not measured.

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Figure 4. Nutrient composition of both undiluted AER and ANA supernatants as well as Recirculating Figure 4. Nutrient composition of both undiluted AER and ANA supernatants as well as AquacultureFigure 4. SystemNutrient (RAS) composition water at theof beginningboth undiluted of the experiment.AER and ANA supernatants as well as Recirculating Aquaculture System (RAS) water at the beginning of the experiment.

FigureFigure 5. Micronutrients5. Micronutrients of of the the undiluted undiluted AER AER and and ANA ANA supernatantsupernatant additives at the the beginning beginning of of thethe experiment. experiment.

The observed AER and ANA supernatant nutrient values (Figures 4 and 5) were relatively The observed AER and ANA supernatant nutrient values (Figures4 and5) were relatively stable stable throughout the experiment. However, four days before the end of the experiment, we throughout the experiment. However, four days before the end of the experiment, we observed observed an unexplainable drop in the redox potential in the aerobic reservoir—the sludge an unexplainable drop in the redox potential in the aerobic reservoir—the sludge blackened within. blackened within. Additional IC data showed that only NO3 and NO2 levels were affected. The Additional IC data showed that only NO3 and NO2 levels were affected. The corresponding drop corresponding drop in NO3 concentration can be seen in Figure 3a, where the graphs of System AER in NO concentration can be seen in Figure3a, where the graphs of System AER and ANA are not and3 ANA are not congruent anymore within the last couple of days. Apart from the different forms congruent anymore within the last couple of days. Apart from the different forms of nitrogen, the of nitrogen, the remineralization performance of both reservoirs was similar. However, we remineralizationhypothesize that performance a better remineralization of both reservoirs perfor wasmance similar. can be However, expected we when hypothesize using other that methods a better remineralization performance can be expected when using other methods of anaerobic digestion

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Agronomy 2016, 6, 37 7 of 12 such as upflow anaerobic sludge blanket (UASB) treatments with granular sludge [15–17], including a longerof anaerobicstart-up digestion time that such ensures as upflow the presence anaerobic of sludge sufficient blanket anaerobic (UASB) , treatments which with tend granular to grow slowersludge than [15–17], aerobic including bacteria. a longer start-up time that ensures the presence of sufficient anaerobic bacteria,Another which interesting tend to findinggrow slower is that than SO 4aerobicwas present bacteria. in the sump of System ANA (Figure3f) at the beginningAnother of the interesting experiment, finding but was is that not SO found4 was in present the anaerobic in the sump supernatant of System (Figure ANA4 ).(Figure These 3f) results at showthe beginning that the aerobic of the remineralizationexperiment, but was performance not found in was the slightly anaerobic more supernatant effective than(Figure the 4). anaerobic These results show that the aerobic remineralization performance was slightly more effective than the treatment. It can clearly be seen that the main nitrogen species in RAS water was NO3, compared anaerobic treatment. It can clearly be seen that the main nitrogen species in RAS water was NO3, with the aerobic and especially the anaerobic supernatant additives (Figure4). Here, in the anaerobic compared with the aerobic and especially the anaerobic supernatant additives (Figure 4). Here, in supernatant, NO3 was already reduced to NH4. It should also be mentioned that the aerobic effluent the anaerobic supernatant, NO3 was already reduced to NH4. It should also be mentioned that the contained almost three times as much SO4 than the RAS water, whereas the anaerobic effluent did aerobic effluent contained almost three times as much SO4 than the RAS water, whereas the not contain any SO4 at the time the sample was taken. This, however, can be explained by the fact anaerobic effluent did not contain any SO4 at the time the sample was taken. This, however, can be that sulfate gets converted to H2S—which has not been measured—under anaerobic conditions [18]. explained by the fact that sulfate gets converted to H2S—which has not been measured—under Thisanaerobic assumption conditionsis supported [18]. This by Krayzelovaassumption etis alsupported.[19], who by report Krayzelova oxidation et al of. [19], H2S who to SO report4 under aerobicoxidation conditions. of H2S to Thereby, SO4 under H2 S,aerobic which conditions. is hazardous Thereby, to plants, H2S, iswhich eliminated. is hazardous Congruently, to plants, in is our study,eliminated. a SO4 peak Congruently, was observed in our on study, Day a 18 SO (Figure4 peak3 wasf). The observed decrease on ofDay SO 184 (Figurein System 3f). ANAThe decrease cannot be directlyof SO4 explained, in System butANA we cannot hypothesize be directly that explained, bacteria and but fungi we hypothesize might have that stimulated bacteria itsand uptake fungi by themight plants. have Furthermore, stimulated its SO uptake4 accumulated by the plants. to a concentration Furthermore, SO of about4 accumulated 8 mg/L to in a System concentration AER due of to oxidationabout 8 mg/L of all sulfurin System components. AER due to oxidation of all sulfur components. ConcentrationsConcentrations of of six six nutrients—NO nutrients—NO33, Na, PO 44, ,K, K, Ca, Ca, and and SO SO4—within4—within the the Systems Systems AER AER and and ANAANA over over the the experimental experimental period period ofof 3535 daysdays are shown in in Figure Figure 4.4. In In contrast contrast to to the the anaerobic anaerobic supernatant,supernatant, NH NH4 4was wasnot not foundfound inin anyany of the systems, whic whichh is is plausible, plausible, as as aeration aeration assures assures the the oxidationoxidation of of NH NH4 to4 to NO NO3.3. Unexpectedly,Unexpectedly, lettuce lettuce had had betterbetter growthgrowth inin SystemSystem ANA (Figure (Figure 6).6). The The opposite opposite effect effect was was observedobserved with with regard regard to to the the root root growthgrowth ofof thethe plantsplants (Figure (Figure 7),7), leading to to a a higher higher shoot-to-root shoot-to-root ratioratio of of System System ANA ANA (2.14) (2.14) compared compared withwith SystemsSystems AER AER (1.39) (1.39) and and RAS RAS (1.30). (1.30). With With respect respect to tothe the generalgeneral nutrient nutrient uptake uptake of of plants, plants, LiebigLiebig1′ss lawlaw of the minimum minimum must must be be considered, considered, which which indicates indicates that the nutrient that is least present determines the maximum growth rate. Still, Figures 3–5 do not that the nutrient that is least present determines the maximum growth rate. Still, Figures3–5 do not indicate that there was any nutrient that was not available in System AER but present in System indicate that there was any nutrient that was not available in System AER but present in System ANA. ANA. However, Lynch et al. [20] report that an increased shoot-to-root ratio is particularly marked However, Lynch et al. [20] report that an increased shoot-to-root ratio is particularly marked with an with an increased N supply, which was not observed in our experiment. increased N supply, which was not observed in our experiment.

Figure 6. Lettuce shoot fresh weight in the aerobic (AER), anaerobic (ANA), and RAS systems after Figure 6. Lettuce shoot fresh weight in the aerobic (AER), anaerobic (ANA), and RAS systems after 35 days. Different letters indicate significant differences (Tukey’s HSD, n = 12, p < 05). Descriptive 35 days. Different letters indicate significant differences (Tukey’s HSD, n = 12, p < 05). Descriptive statistical data of this box plot can be found in Table3. statistical data of this box plot can be found in Table 3.

Agronomy 2016, 6, 37 8 of 12 Agronomy 2016, 6, 37 8 of 12

FigureFigure 7. 7. LettuceLettuce root root fresh fresh weight weight in in the the aerobic aerobic (A (AER),ER), anaerobic anaerobic (ANA), (ANA), and and RAS RAS systems systems after after 35 1 days.35 days. Different Different letters letters indicate indicate significant significant differences differences (Tukey (Tukey′s HSD,s HSD, n n== 12, 12, pp << 05). Descriptive statisticalstatistical data data of of this this box box plot plot can can be found in Table 3 3..

TableTable 3. 3. DescriptiveDescriptive statistical statistical data data for for shoot shoot and and root fresh weight in g.

Shoots Roots Shoots Roots SystemSystem AER AER System System ANA ANA System System RAS RAS System AER AER System System ANA ANA System System RAS RAS Min. 77.1 105.0 49.0 62.3 56.9 58.0 Min. 77.1 105.0 49.0 62.3 56.9 58.0 1st Quantile1st Quantile 81.8 81.8 120.9 120.9 78.2 78.2 71.171.1 61.961.9 68.368.3 MedianMedian 97.8 97.8 132.6 132.6 97.7 97.7 75.775.7 64.464.4 71.871.8 MeanMean 104.8 104.8 137.4 137.4 92.4 92.4 75.575.5 63.963.9 70.870.8 3rd 3rdQuantile Quantile 116.4 116.4 151.1 151.1 100.2 100.2 78.078.0 66.666.6 73.573.5 Max. 154.4 182.5 153.5 94.7 68.8 83.9 Max. 154.4 182.5 153.5 94.7 68.8 83.9

ThereThere are are several plausible explan explanationsations for for the the increased increased shoot-to-root shoot-to-root ratio ratio observed observed in in the the systemsystem ANA. ANA. Firstly, Firstly, the utilization of NH 4 providedprovided by by the the ANA ANA is is energetically energetically more more favorable favorable for for mostmost plants, sincesince in in most most cases cases NO NO3 has3 has to beto convertedbe converted to NH to4 NHby the4 by plant the [plant18]. Moreover, [18]. Moreover, the uptake the uptakeof one ofof these one of two these N-sources two N-sources also seems also to beseems pH-dependent. to be pH-dependent. Jones [21] reportsJones [2 that1] reports plants that prefer plants NO3 preferin acidic NO environments,3 in acidic whereasenvironments, NH4 is thewhereas favored NH nitrogen4 is the source favored in alkaline nitrogen environments. source in Sincealkaline the environments.pH values in the Since three the systems pH values were betweenin the three 8.1 and systems 8.7, NH were4 is the between preferred 8.1 nitrogen and 8.7, source NH4 foris the the preferredplants. In addition,nitrogen severalsource authorsfor the plants. [21–23] In have addi suggestedtion, several or reported authors that [21–23] NH4 boostshave suggested plant growth or reportedif it represents that NH a small4 boosts proportion plant growth of the if nitrogen it represents available. a small For proportion instance, it of has the been nitrogen observed available. that a Forproportion instance, of it 30% has ofbeen NH observed4 increased that the a lettuceproportion harvest of 30% in a drippingof NH4 increased the system lettuce [24 harvest]. This isin ina drippingaccordance irrigation with Sonneveld system [24]. [22] This and Jonesis in accordance [21], who state with that Sonneveld a supply [22] of 10%–15% and Jones and [21], 25% who of NHstate4 , thatrespectively, a supply is of optimal 10%–15% for mostand plants25% of grown NH4, onrespectively, substrates. is Contrary optimal tofor standing most plants aerated grown systems, on substrates.even 5% of Contrary NH4 could to stimulate standing NOaerated3 uptake systems, in constantly even 5% operated of NH4 systemscould stimulate like the ones NO3 used uptake in this in constantlyexperiment. operated This is attributedsystems like to the the ones fact thatused the in specificthis experiment. nitrogen uptakeThis is attributed is water-flow-dependent. to the fact that theThat specific is, the nitrogen lower the uptake NH4 concentration, is water-flow-dependent. the higher theThat flow is, the should lower be the [21 NH]. In4 concentration, our case, the NH the4 highercontent the of theflow anaerobic should be supernatant [21]. In our was case, high the (Figure NH4 content4) and couldof the haveanaerobic contributed supernatant to an increasedwas high (Figureuptake. 4) However, and could since have the contributed system was to an highly increased aerated, uptake. nitrification However, must since have the system occurred was very highly fast. aerated,Measurements of the must water have were occurred always taken very onefast. day Measurements after the supernatants of the water were were added. always This taken could onebe the day reason after the why supernatants we had not measuredwere added. NH This4 in could the system. be the Secondly,reason why the we sodium had not level measured in ANA NH was4 inslightly the system. lower Secondly, (Figure3b). the The sodium sodium level levels in ANA most likelywas slightly did not lower slow down(Figure the 3b). growth The sodium performance levels mostof the likely lettuce did plants, not slow as the down concentration the growth was performance far below the of the lettuce threshold plants, [25 as,26 the]. concentration was far below the salinity threshold [25,26]. Even though pH values between 8.2 and 8.7 were measured in the sump of System ANA, the possibility that the pH in the of System ANA was temporarily lower than in the other

Agronomy 2016, 6, 37 9 of 12

Even though pH values between 8.2 and 8.7 were measured in the sump of System ANA, the possibility that the pH in the rhizosphere of System ANA was temporarily lower than in the other two systems cannot be excluded. This could have been caused by the addition of the ANA supernatant. To restore the electrochemical balance in root cells and nutrient solution, the uptake of cations (i.e., + NH4) is balanced by a release of protons (H ) that decrease the rhizosphere pH [27–30]. A lower pH than the observed one in the sump, most likely would have caused an increase in nutrient uptake. The uptake of most macronutrients is optimal between pH values 6 and 8, whereas the uptake of micronutrients is better below pH 6 [31,32]. In commercial hydroponic systems, the pH is typically around 5.5 and 6.0 [33], which is much lower than the pH values reported here. With respect to the plant growth performance, there are several arguments that could explain an opposite effect. SO4 concentration could have restricted the growth of lettuce in System AER. However, just like the temporary H2S availability in system ANA, the observed SO4 concentration is much lower than recommended for hydroponic practice [33,34]. Another factor that decreases plant growth is a high COD level [9], which is much higher in anaerobic effluents than in aerobic effluents. Since our systems were highly aerated and showed a saturated DO level over the entire experimental period, we can reject this assumption. Besides COD, volatile fatty acids (VFAs) are present in high concentrations in anaerobic effluents, inhibiting shoot and root growth [35]. All these factors are enforced in low pH environments, which was not the case in our experiment with pH between 8.1 and 8.7 in the sump. Another factor that could explain the results is that the anaerobic effluent contained substances that promote the shoot growth and nutrient uptake (Table1). Literature indicates that these substances could be (1) dissolved organic matter (DOM) and (2) plant growth promoting rhizobacteria and/or fungi (PRPR and/or PGPF). Haghighi [36] showed that the addition of humic acid to a hydroponic solution improved N metabolism and photosynthesis activity of the lettuce plants, which led to a higher yield. These findings are supported by Ruzzi and Aroca [37], who state that PGPR can release phytohormones or induce hormonal changes within plants that stimulate plant cell elongation and division. Even though the results show better growth in the ANA treatment, this study did not look into human pathogens that might occur on the roots and leaves. This question could be addressed with metagenomic analyses of the present microbiota.

4. Conclusions This study examined the differences in lettuce plant growth performance comparing the addition of anaerobic and aerobic sludge effluents and RAS effluent to the hydroponic system. Our findings provide strong empirical confirmation that anaerobic effluents generally have a positive impact on plant growth. We hypothesize that the enhanced growth of lettuce in System ANA is a result of the added NH4 from the anaerobic fish sludge supernatant (Figure4), which improves the nutrient uptake and at least temporarily lowers the pH in the rhizosphere that contributes to the same effect. Moreover, DOM, PRPR/PGPF, and humic acid that occur in anaerobic effluents could play an important role in the lettuce’s nutrient uptake and utilization. Although the scope in this study was limited in terms of regular measurements of the supernatant composition that might have changed within this period, the study clearly indicates enhanced growth performance when exposing the plants to anaerobic supernatant enriched RAS water. Furthermore, anaerobic digestion generally needs a start-up period of several months to reach full efficiency, which was not fully realized. Further studies should focus on a better understand of the factors that led to these results, as well as the determination of the optimal dilution of anaerobic supernatant with RAS water. Within the framework of decoupled aquaponic systems, this information is encouraging with regard to the development and implementation of anaerobic nutrient recycling from sludge, improving resource utilization and reducing nutrient emissions in aquaponics. It would also be interesting to see whether a cumulative effect can be observed when exposing plants to a hydroponic solution that contains ANA supernatant as well as RAS water. Agronomy 2016, 6, 37 10 of 12

Acknowledgments:Agronomy 2016, 6, 37 The authors would like to acknowledge networking and publication support by COST10 Actionof 12 FA1305—The EU Aquaponics Hub—Realising Sustainable Integrated Fish and Vegetable Production for the EU as well as ZHAW, Institute of Natural Resource Sciences in Wädenswil for both funding this publication and publication and providing research facilities. Furthermore, a big “Dankeschön” to Aquaponik Manufaktur GmbH providing research facilities. Furthermore, a big “Dankeschön” to Aquaponik Manufaktur GmbH for co-funding thisfor publication. co-funding this publication. AuthorAuthor Contributions: Contributions:Simon Simon Goddek, Goddek, Ranka Ranka Junge, Junge, and Zala and Schmautz Zala Schmautz conceived conceived and designed and thedesigned experiments, the whichexperiments, were performed which were at the performed Zurich University at the Zurich of Applied University Sciences, of Applied Wädenswil, Sciences, Switzerland; Wädenswil,Simon Switzerland; Goddek, BenjaminSimon Goddek, Scott, and Benjamin Zala Schmautz Scott, and performed Zala Schmautz the experiments; performed Simon the experiments; Goddek and KarelSimon Keesman Goddek analyzedand Karel the data;Keesman Simon analyzed Goddek, the Sven data; Wuertz, Simon Boris Goddek, Delaide, Sven Zala Wuer Schmautz,tz, Boris andDelaide, Ranka Zala Junge Schmautz, wrote the and paper. Ranka Junge Conflictswrote the of paper. Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix Table A1. Mean values (˘ SD) of nutrient content (in mg/g) of the lettuce shoots of each system. Table A1. Mean values (± SD) of nutrient content (in mg/g) of the lettuce shoots of each system. Macronutrient System AER System ANA System RAS Macronutrient System AER System ANA System RAS ˘ a ˘ a b NN 53.7 53.7 ± 0.10.1 a 54.654.6 ± 0.0. 44 a 56.756.7 ± ˘0.50.5 b C 345.8 ˘ 6.9 a 338.8 ˘ 7.2 a 361.0 ˘ 8.2 b C 345.8 ± 6.9 a 338.8 ± 7.2 a 361.0 ± 8.2 b H 42.9 ˘ 3.1 a 42.9 ˘ 2.0 a 40.0 ˘ 3.2 a a a a PH 201.3 42.9 ˘± 3.10.7 a 42.9329.5 ±˘ 2.01.3 b 40.0191.7 ± ˘3.21.10 c a b c KP 1473.33 201.3 ±˘ 0.74.5 a 2134.67 329.5 ± ˘1.34.0 b 191.71437.7 ± 1.10˘ 4.16 c CaK 1757.001473.33˘ ± 1.74.5a a 2134.672504.33 ±˘ 4.02.3 bb 1437.71878.7 ± ˘4.161.46 c c MgCa 1757.00 324.20 ˘ ±0.8 1.7a a 2504.33502.20 ˘± 2.31.7 b 1878.7395.6 ±˘ 1.460.78 c c SMg 202.47324.20˘ ± 0.70.8a a 502.20215.73 ±˘ 1.70.8 bb 395.6199.5 ± ˘0.780.36 c c a b c NaS 135.33202.47˘ ± 0.30.7 a 215.73306.47 ±˘ 0.80.9 b 199.5231.9 ± ˘0.360.46 c Different letters indicateNa significant135.33 differences ± 0.3 (ANOVA a 306.47 with Tukey’s± 0.9 b HSD231.9post hoc± 0.46test, cp < 0.05, n = 3). Different letters indicate significant differences (ANOVA with Tukey’s HSD post hoc test, p < 0.05, n = 3).

(a) (b) (c)

FigureFigure A1. A1.Close-up Close-up images images of of lettuce lettuce grown grown in in (a )( Systema) System AER, AER, (b )( Systemb) System ANA, ANA, and and (c) System(c) System RAS. RAS.

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