Characterization of a Microbial Consortium That Converts Mariculture ﬁsh Waste to Biomethane

Aquaculture 453 (2016) 154–162

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Characterization of a microbial consortium that converts mariculture ﬁsh waste to biomethane

Brigit M. Quinn a, Ethel A. Apolinario a, Amit Gross b,KevinR.Sowersa,⁎ a Department of Marine Biotechnology, Institute of Marine and Environmental Technology, University of Maryland Baltimore County, Baltimore, MD 21202, United States b Department of Environmental Hydrology and Microbiology, Zuckerberg Institute for Water Research, Jacob Blaustein Institutes for Desert Research, Ben Gurion University of the Negev, Israel article info abstract

Article history: Environmentally responsible disposal of solid organic wastes from land-based brackish and marine recirculating Received 13 May 2015 aquaculture systems is critical for promoting widespread acceptance and implementation, but conversion Received in revised form 30 November 2015 efficiency of saline sludge to biomethane is generally low. We describe the development of a microbial consor- Accepted 1 December 2015 tium that can convert marine organic fish waste solids to biomethane at over 90% efficiency. The halotolerant mi- Available online 2 December 2015 crobial consortium, which was developed by sequential transfer in seawater with fish waste, is optimized for low fi Keywords: COD:N ratios typical of organic sh waste and does not require addition of amendments such as organic carbon or RAS nutrients. Temperatures for maximum rates of conversion range from 26 to 35 °C. Five predominant phylotypes Biomethane identified in the microbial consortium by denaturing HPLC were isolated. Two isolates included anaerobic fer- Saline waste mentative bacteria identified as a strain of Dethiosulfovibrio and a strain closely related to Fusobacterium spp., Waste reduction which both hydrolyze and ferment proteins, peptides and amino acids. The other three isolates included an Anaerobic acetate-utilizing methanogenic archaeon identified as a strain of Methanosarcina and two hydrogen-utilizing me- Microbial consortium thanogenic archaea identified as strains of Methanogenium and Methanoplanus. Bioconversion rates of sterile fish waste with the reconstituted microbial consortium containing all five isolates were equivalent to rates observed with the original enriched consortium after one sequential transfer. The results demonstrate unequivocally that halotolerant consortia of bacteria and archaea can be developed for bioconversion of saline organic solid waste with high efficiencies equivalent to those attained with non-saline waste systems. Understanding the microbial community composition is critical for management of solid organic waste from land-based marine aquaculture systems and to maintain or restore microbiota during start up and throughout the production process.

Statement of relevance

Appropriate disposal of solid organic wastes from land-based brackish and marine recirculating aquaculture systems is critical for promoting widespread acceptance and implementation. We demonstrate that halotolerant consortia of bacteria and archaea can be developed for bioconversion of saline ﬁsh waste with high efﬁciencies equivalent to those attained with non-saline waste systems. © 2015 Elsevier B.V. All rights reserved.

1. Introduction net-pen mariculture on the environment have been widely publicized and are currently being addressed by a number of approaches (Rust Marine fisheries have been in continuous decline globally and pro- et al., 2014). One such approach is recirculating aquaculture systems jections indicate that a collapse in the industry is imminent within a (RAS) that are under development as an eco-responsible alternative to few decades if current levels of trade continue (FAO, 2014). In order to traditional aquaculture technologies. However, there has been negligi- ease pressures on wild fisheries stocks, and to meet the growing global ble research on decreasing the environmental impact of saline organic consumption of seafood, there is a growing reliance on the aquaculture waste generated by RAS. of marine species (Campbell and Pauly, 2013). One of the major draw- A future shift from net-pen mariculture operations to more inland backs of marine aquaculture is the potential for localized eutrophication recirculating aquaculture systems will result in the generation of high due to the release of waste products. The potential adverse effects of volumes of saline sludge. The output from intensive RAS is primarily composed of suspended matter originating from uneaten feed and fish fecal material. An aquaculture facility growing 100 t of fish at a typical ⁎ Corresponding author at: Department of Marine Biotechnology, Institute of Marine – – and Environmental Technology, 701 E. Pratt St., Baltimore, MD 21202, United States. feed conversion ratio of 1.3 1.5, will use 130 150 t of feed and generate E-mail address: [email protected] (K.R. Sowers). 30–40 t of dry solid organic waste as total suspended solids (TSS). Hardy

(2001) calculated that a 100 t salmon farm releases an amount of ni- 2009). The system included two 12 m2 tanks each stocked with approx- trogen, phosphorus and fecal matter roughly equivalent to the nutri- imately 2100 fish that were grown from an average weight of 50 to ent waste in untreated sewage from 20,000, 25,000 and 65,000 450 g. Sludge samples consisting of approximately 2% solids were har- people, respectively. Most commonly used sludge treatments employ vested from a settling tank immediately upstream of a biogas reactor flocculation/coagulation processes to reduce sludge volume prior to and had an average chemical oxygen demand (COD) of 21 g l−1. Sam- composting it for land dispersal. However, unlike sludge from freshwa- ples were used immediately after harvesting or stored in sealed bottles ter RAS, the high salinity of brackish/marine sludge limits its use as at 4 °C prior to use. For culture medium sludge was concentrated by cen- fertilizer and creates a source of pollution in landfill sites and waste trifugation to 50% of its original volume to create a 2× stock and added outflows (Flaherty et al., 2000; Naylor et al., 1998). to an equal volume of medium to achieve a final COD of 21 g l−1. One solution for treating saline organic waste from an intensive marine RAS and achieve a near-zero discharge is by converting it to 2.2. Enrichment of sludge digesting consortium biomethane and carbon dioxide gases in an anaerobic digester. Bioreac- tors containing methanogenic consortia of bacteria and archaea can Growth medium consisting of buffered artificial seawater was digest high organic loads at low operating costs and with relatively prepared anaerobically under a N2–CO2 (4:1)atmosphereasde- low initial investment. Furthermore, the end product of anaerobic bio- scribed previously (Sowers and Noll, 1995). Artificial seawater mass conversion, biomethane, can offset operational costs as a combus- (Zohar et al., 2005)dilutedto15gl−1 with deionized water was −1 −1 tible energy source for heat or generation of electricity. Since the carbon amended with 1 g l Na2HCO3 as a buffer and 1 mg l resazurin dioxide generated from biomethane combustion is from an organic (7-Hydroxy-3H-phenoxazin-3-one 10-oxide) as a redox indicator. The non-petroleum source, there is no net release of greenhouse gas into pH was adjusted to 7.4. The medium was dispensed (250 ml) into a the atmosphere. Partial substitution of biomethane for petroleum- 700-ml safety coated reagent bottle and sealed under 101 kPa N2–CO2 based fuels to power or heat the RAS would effectively reduce the (4:1) with a screw cap containing a butyl rubber septum core. An carbon footprint of the system. However, several characteristics of equal volume (250 ml) of 2× concentrated fish waste sludge solids concentrated fish waste from recirculating mariculture systems can ad- was added to the medium as inoculum and substrate in the primary en- versely affect biomass conversion by methanogenic consortia, includ- richment culture; thereafter 250 ml of 2× concentrated fish waste ing: 1) high NaCl concentrations associated with seawater requiring solids was added as substrate to 250 ml buffered seawater immediately microorganisms adapted to growth in high extracellular solute concen- prior to inoculation with 50 ml of inoculum from the previous enrich- trations; 2) accumulation of toxic levels of sulfide from the reduction of ment culture. Bottles were incubated in a rotary shaking incubator at the high sulfate levels in seawater by sulfate reducing bacteria; and 3) 26 °C and 25 rpm. Enriched inoculum was maintained by sequential accumulation of ammonia from catabolism of highly proteinaceous transfers every 2–3months. fish feed by fish and fermentative bacteria (Mirzoyan et al., 2008; Zhang et al., 2013). Prior studies on anaerobic digestion of marine fish 2.3. Isolation and reconstitution of microorganisms from consortium waste using inoculum from non-marine sources such as municipal or industrial sludge digesters or pig manure were subject to long adapta- Microorganisms were isolated from enrichment cultures by plating on tion periods and results were mixed (Gebauer, 2004; Omil et al., 1995, agar-solidified anaerobic medium as described previously (Apolinario 1996). In contrast, Aspe et al. (1997) reported that inoculum from and Sowers, 1996) with modifications described below. Fermentative marine sediments developed more rapidly and was more effective at bacteria were isolated in medium that contained the following compo- treating marine fisheries wastewater than inoculum from pig manure. nents in g l−1 of artificial seawater: peptone, 5; cysteine, 0.25; and These results are consistent with a report by Sowers and Ferry (1984), resazurin, 0.0001. Oxygen was removed from the medium by sparging which described the development of a stable consortium of marine fer- with N2–CO2 (4:1) through a sintered glass gas distribution tube. The menters, acetogens and methanogens enriched from marine sediments degassed medium was then transferred to an anaerobic glove box with marine kelp (Macrocyctis pyrifera) that converted over 90% of the (COY Laboratory Products) containing an atmosphere of N2–CO2–H2 biomass to methane and carbon dioxide in seawater. Isolates in the mi- (16:4:1) and filter sterilized with a 0.45 μm disposable filter unit crobial consortium had equivalent roles to those in freshwater methan- (Nalgene). Aliquots of 150 ml deionized water degassed by sparging ogenic consortia, but were uniquely adapted to growth at marine saline with N2 were dispensed into 700 ml safety coated reagent bottles levels. Several reports describe the microbiota involved in aerobic and (Wheaton) each containing 3 g Bacto™ agar (Becton Dickinson). The anaerobic nitrogen processing (Rurangwa and Verdegem, 2014)butin- bottles were sealed under a N2 headspace as described above and ster- sights into the population dynamics of microbiota in the biogas reactors ilized by autoclaving at 121 °C for 20 min. After autoclaving the agar was of a marine RAS have not been reported. Understanding the microbial cooled to 55 °C in a water bath. Immediately prior to use the agar was community composition is critical for system management in order to transferred to an anaerobic glove box, combined with 150 ml of medi- maintain or restore crucial microbiota during start up and throughout um and poured into 15 × 100 mm petri plates. The final salinity of the the production process. In this report we describe the enrichment and solidified medium was 15 ppt. Plates were dried in the glove box for characterization of a stable marine consortium of fermentative and me- two days in a relative humidity of 30–35%. Ten-fold serial dilutions of thanogenic microorganisms developed from the solid waste digester of inoculum from an enrichment culture were created in 9 ml aliquots of a fully contained, land-based, marine RAS. This microbial consortium is liquid medium and 1 ml of each dilution was inoculated onto solidified capable of reducing over 90% of digestible proteinaceous marine fish medium by spreading. Plated cultures were incubated at 30 °C in stain- waste biomass to methane and carbon dioxide at saline concentrations less steel anaerobe jars (Torbal) under N2–CO2 (4:1) and 0.003% (v/v) −1 of 15 g l . H2S generated from Na2S as described previously (Apolinario and Sowers, 1996). Isolated colonies were further purified by streaking on 2. Materials and methods solidified medium. Methanogenic archaea were isolated on agar-solidified ECl medi-

2.1. Fish waste sludge um (Berkaw et al., 1996) with a reduced Na2S concentration. Oxygen was removed from the medium by sparging with N2–CO2 (4:1) Fish waste sludge solid samples used for developing and maintain- through a sintered glass gas distribution tube. Aliquots of 100 ml ing inoculum were obtained from the sludge collection tank of a were transferred to 125 ml serum vials each containing 1.25 g 3 24 m recirculating marine aquaculture system supporting growth of Bacto™ agar and 0.0025% (w/v)Na2S⋅9H2O. For isolation of aceticlastic gilthead seabream (Sparus aurata) as described previously (Tal et al., methanogens 0.1 M sodium acetate was added to the vial. The medium 156 B.M. Quinn et al. / Aquaculture 453 (2016) 154–162

was sealed under a N2–CO2 (4:1) headspace with butyl rubber septa se- plus COD (0–15,000 ppm range) test kit following manufacturer's cured with aluminum crimp seals and sterilized by autoclaving at directions. Samples were diluted with water to 25% of the original 121 °C for 20 min. After autoclaving the agar was cooled to 55 °C in a concentration prior to analysis. Total nitrogen (TN) was determined water bath. Immediately prior to use the agar medium was transferred using a High Range Total Nitrogen HACH test kit (Hach Method to an anaerobic glove box, and poured into 15 × 100 mm petri plates. 10072, 10–150 mg l−1 range). Samples were diluted with water to The plates were dried as described above. Ten-fold serial dilutions of 6.7% of the original concentration prior to analysis. Total organic carbon 1 ml inoculum from an enrichment culture were created in 9 ml (TOC) was measured using a Shimadzu TOC analyzer with a solids aliquots of liquid medium prepared anaerobically in Balch-style sample module (TOC-5000A and SSM-5000A) by combustion at 900 anaerobe tubes and 1 ml of each dilution was inoculated onto solidified °C after removal of inorganic carbon with hydrochloric acid. medium by spreading. Plated aceticlastic cultures were incubated in stainless steel anaerobe jars as described above. For hydrogen- 2.7. DNA extraction and amplification utilizing methanogens the N2–CO2 atmosphere was replaced with H2– CO2 (4:1) to 202 kPa. H2–CO2 was replenished when depleted to DNA was extracted by adding 0.25 g of culture containing suspended 101 kPa or less as indicated by a pressure gauge. Isolated colonies sludge particles to a PowerBead microfuge tube of a Power Soil® DNA were further purified by streaking on solidified medium containing Isolation Kit (MOBIO Laboratories, Inc.) as described previously (Payne 100 mg l−1 vancomycin. Isolates were maintained in liquid ECl medium et al., 2011). The total bacterial community was monitored by amplifica- under an atmosphere of 101 kPa N2–CO2 (4:1) or 202 kPa H2–CO2 (4:1). tion of the 16S rRNA genes with universal primers 341F/907R (Lane The microbial consortium was reconstituted by transferring the iso- et al., 1985; Muyzer et al., 1993). PCR amplification conditions were as lates to ECl medium containing 5 g l−1 Brain Heart Infusion broth follows: denaturation at 95 °C for 3 min; 35 cycles at 95 °C for 45 s; an- (Difco) for three sequential transfers. To compare the efficiency of the nealing at 45 °C for 45 s; extension at 72 °C for 1 min, followed by a final reconstituted consortium with the enriched consortium 5 ml of culture extension at 72 °C for 5 min. The 0.56 kb PCR product was purified on a was inoculated into 50 ml ECl medium containing fish waste sludge at a 1.2% agarose gel. Methanogenic archaea were monitored by amplifica- final COD concentration of 21 g l−1.Efficiency was determined by tion of methyl-coenzyme M reductase (MCR) with primers ME1/ME2 measuring the biochemical methane potential as described below. (Hales et al., 1996). PCR amplification conditions were as follows: denaturation at 95 °C for 5 min; 35 cycles at 95 °C for 45 s; annealing 2.4. Enrichment characterization at 50 °C for 30 s; extension at 72 °C for 1 min and a final extension at 72 °C for 3 min. The PCR product (0.75 kb) was purified on a 1.2% aga- The effect of selected treatments on biological methane potential was rose gel. DNA was eluted from the excised band with 50 μlofPCR tested by amending the buffered growth medium described above. To grade water and quantified with a NanoDrop 1000 Spectrophotometer determine the effect of trace nutrients the following components were (ThermoScientific). Extracted DNA samples had an A260/280 ratio of added to buffered artificial seawater: cysteine (0.025% w/v), trace metals ≥1.6 and an A260/230 ratio of ≥2.0. All DNA samples were diluted to stock (1% v/v) and B-vitamin stock solutions (1% v/v)(Wolin et al., 1963). 2ng/μlinTEbuffer. To determine the effect of trace metals specifically required by methanogenic archaea the following components were added to buffered arti- 2.8. Community identification using denaturing HPLC

ﬁcial seawater: FeSO4 (5 μm), CoCl2 (0.5 μm) and NiCl2 (0.5 μm). To reduce the sulfate concentration of the medium magnesium sulfate A WAVE 3500 HT denaturing gradient HPLC system (Transgenomic,

(MgSO4 ⋅7H2O) was substituted with the molar equivalent of magne- Inc.) equipped with a fluorescence detector was used to separate and sium chloride (MgCl2⋅6H2O). To increase the COD:N ratio of the sludge identify different 16S rRNA gene products as described previously carboxymethyl cellulose (CMC) was added (1% w/v;CMC=6560ppm). (Payne et al., 2011). PCR amplification product peak fractions from universal 16S primers 341F/907R and MCR primers ME1/ME2 were collect- 2.5. Biochemical methane potential (BMP) ed and sequenced as described previously (Kjellerup et al., 2008). Sequences were submitted to the National Center for Biotechnology The efficiency of biomass conversion by the sludge enrichment was Information's (NCBI) Basic Local Alignment Search Tool (BLAST) determined using a modification of the BMP assay (Moody et al., 2009). (Altschul et al., 1990) to determine similarity with other 16S rDNA Buffered artificial seawater (50 ml) was dispensed into 160-ml serum and MCR gene sequences. bottles and sealed under 101 kPa N2–CO2 with butyl rubber stoppers se- cured with aluminum crimp seals. Medium was inoculated with 50 ml 2.9. Nucleotide sequence accession numbers 2× fish waste sludge solids and 5 ml of enriched inoculum, then re- sealed under N2–CO2 (4:1). After equilibration for 30 min the headspace The 16S rRNA gene sequences from strains isolated in this study was sampled for methane using a 100 μl gas-tight syringe with valve as have been submitted to GenBank under the following accession num- described below. Bottles were incubated in a rotary shaking incubator at bers: Fusobacteriacea EA-F3, KT799837; Dethiosulfovibrio sp. EA-F2, 26 °C and 25 rpm. During the first three weeks after transfer the head- KT799836; Methanogenium sp. EA-M3, KT799838; Methanoplanus sp. space was sampled without subsequent purging. Thereafter, the bottles EA-M7, KT799839; and Methanosarcina sp. EA-M15, KT799840. were purged with N2–CO2 after each sampling to prevent changes in pH as a result of excess carbon dioxide. The methane reading was added to 3. Results the prior methane value to determine total methane production over the course of the experiment. 3.1. Characterization of solid waste from the RAS

2.6. Analytical methods The RAS used as a source of sludge for this study was described previously (Tal et al., 2009). Sludge collected with a microscreen drum filter Methane was assayed with a HP5890 gas chromatograph (Hewlett (60 μm screen mesh; Hydrotech, Model 801, Vellinge, Sweden) and Packard) equipped with a flame ionization detector and stainless steel backwash system that used tank water was collected from an 800 l con- column (0.32 × 182.88 cm) packed with silica gel (80/100 mesh; ical settling tank that had a hydraulic retention time of 4 h. Sludge for Supelco). The column oven was operated at 110 °C with He as the carri- the development and feeding of microcosm digesters was collected er gas. Purified methane (methane, ultra-high purity, Matheson Tri Gas) from the bottom of the settling tank and used within 1 day for all exper- was used as a standard. COD was determined using a HACH High Range iments. The sludge consisted of fish feces (69% average digestibility B.M. Quinn et al. / Aquaculture 453 (2016) 154–162 157

Table 1 Characteristics of sludge from recirculating aquaculture system.

Parameter Value

COD 20.8 ± 1.3 g l−1 TN 1.4 ± 0.3 g l−1 TOC 7.3 ± 0.0 g l−1 pH 7.4 C:N 5.2:1 COD:N 14.7:1 Salinity 15 g l−1

COD:SO4 20.5:1

coefﬁcient for seabream) (Lupatsch and Kissil, 1998), and uneaten Europa 15 and 18 pellets (3, 4, 6 mm; Skretting's) consisting of not less than 50% protein and 18% ﬁsh oils. The characteristics of the sludge are shown in Table 1.

3.2. Development of methanogenic microbial consortium for biomass Fig. 1. Effect of sequential transfers of fish sludge enrichment on rates of methane produc- conversion of waste material tion. Graph shows methane production for the initial (▲), third transfer (●) and seventh transfer (■) of the enrichment culture. Dashed line represents estimated maximum The rates and extent of biomass conversion to biogas in 500 ml methane yield from total COD of sludge. Values are means and standard deviations for three replicate culture samples. enrichment cultures are shown in Fig. 1. Prior to digestion the sludge was nearly black in color and had a thick consistency with particles remaining suspended in liquid. After digestion the sludge was light of 26 °C. However, methane production increased by 9.3% at 30 °C and brown in color with a sandy consistency that settled out of the liquid by 11.8% when at 35 °C, compared to 26 °C. phase. The rate of conversion in the initial inoculum was only −1 −1 41.2 mmol CH4 kg COD day , but increased 10-fold to a maximum 3.4. Characterization of the methanogenic microbial consortium −1 −1 rate of 428 mmol CH4 kg COD day after seven sequential transfers. The digestible fraction of fish waste averaged 90% of the total estimated DNA was isolated from the initial enrichment after incubation for yield based on COD. This high rate of activity and a reduced lag time of two months and from a culture enriched by six sequential transfers 5 days was consistent for subsequent 10% (vol/vol) transfers of enriched over a period of 12 months. Phylotypes were separated by denaturing inoculum. HPLC and DNA from individual peaks was collected and sequenced. Five predominant bacterial phylotypes with the highest identity to an- 3.3. Identification of factors affecting biological methane potential aerobic fermenters Odoribacter spp., Bacteroides spp. and Tindallia spp. were detected in the initial enrichment culture (Fig. 4). Since 16S The chemical composition of the solid waste was modified to identi- rRNA gene detection using universal primers is often less sensitive to ar- fy factors that might limit the rates and maximum extent of biomass chaea, a primer set was used that is specific for the gene encoding meth- conversion. Addition of a mixture of 12 trace metals required by bacteria yl coenzyme M reductase, which is shared universally by all described and ten-fold increase in Fe+2,Ni+2 and Co+2 required by methanogenic methanogens. Using the archaeal primers only two phylotypes with archaea for several cofactors and redox enzymes did not have an effect the highest sequence identities to acetate-utilizing Methanosarcina on the rate of methanogenesis or BMP. Addition of cysteine, added as an spp. were detected in the initial enrichment (Fig. 4). In the highly organic sulfur source, and eight B-vitamins did not increase methane enriched culture five predominant phylotypes were detected, but yield and actually slightly reduced the rate of methanogenesis from −1 −1 428 to 402 mmol CH4 kg COD day . Increasing COD:SO4 from 21:1 −1 −2 by substituting Cl for SO4 in the artificial seawater reduced the con- −1- version of biomass to methane from 428 to 316 mmol CH4 kg COD day−1. The effect of increasing the COD:N from 15:1 to 39:1 was also examined by addition of starch and methylcellulose. Starch had no observable effect. Increasing the COD with methylcellulose from 20,840 mg l−1 to 55,000 mg l−1 decreased the rate of methanogenesis −1 −1 from 428 to 185 mmol CH4 kg COD day and the final yields were 89 and 91%, respectively, of the estimated methane yield (Fig. 2). Temperature was shown to have an effect on the amount of methane produced (Fig. 3). Methane production decreased by 62.2% when bottles were incubated at 20 °C instead of the ambient system temperature

Table 2 Microorganisms isolated from ﬁsh waste enrichment culture.

Isolate Sequence similarity to reported strains Growth substrate

EA-F3 Fusobacteriacea strain SL-2013-9 (97%) Peptone EA-F2 Dethiosulfovibrio russensis sr-13 (99%) Brain heart infusion Fig. 2. Effect of temperature on rates of methane production by ﬁsh sludge enrichment. EA-M3 Methanogenium marinum AK-1 (99%) H –CO 2 2 Graph shows methane production during incubation at 20 (♦), 26 (■), 30 (▲) and 35 °C EA-M7 Methanoplanus sp. MobH (100%) H –CO 2 2 (●). Dashed line represents estimated maximum methane yield from total COD of sludge. EA-M15 Methanosarcina sp. WH-1 (100%) Acetate Values are means and standard deviations for three replicate culture samples. 158 B.M. Quinn et al. / Aquaculture 453 (2016) 154–162

Methanosarcina spp. and hydrogen-utilizing Methanogenium spp. and Methanoplanus spp. were detected.

3.5. Isolation and reconstitution of the methanogenic microbial consortium

Five microorganisms were isolated from the highly enriched culture sequentially transferred in medium with ﬁsh waste sludge (Table 2). A rod shaped bacterium (EA-F3) with high sequence similarity to species in the Fusobacteriacea was isolated on and subsequently maintained in E-Cl medium containing peptone. A vibrio-shaped bacterium (EA-F2) with high sequence identity to Dethiosulfovibrio spp. was isolated on solidiﬁed E-Cl medium containing peptone, but required brain–heart infusion medium for subsequent maintenance. Species in both genera are obligate fermentative bacteria capable of utilizing peptides and amino acids for growth (Staley and Whitman, 2011; Surkov et al., 2001). Two methanogenic archaea were isolated with hydrogen as the electron donor: a rod shaped archaeon with high sequence identity to Methanogenium spp. (EA-M3) and a disk-shaped archaeon with high se-

Fig. 3. Effect of carbon to nitrogen ratio on rates of methane production by ﬁsh sludge en- quence identity to Methanoplanus spp. (EA-M7). Species in both genera richment. Graph shows methane production during incubation with sludge with native utilize only hydrogen or formate for growth and methanogenesis COD:N ratio of 15:1 (■) and after increasing the COD:N ratio to 39:1 (●). Dashed line rep- (Sowers, 2009). An irregular coccus with sequence identity to resents estimated maximum methane yield from total COD of sludge and carbon amend- Methanosarcina spp. (EA-M15) was isolated with acetate as the growth ment. Values are means and standard deviations for three replicate culture samples. substrate. Species within all three genera have been isolated previously from saline environments (Sowers, 2009). there was a shift in the chromatograms (Fig. 4). Two bacterial The two fermentative bacteria and three methanogenic archaea phylotypes with the highest sequence identity to the anaerobic fermen- were reconstituted as a microbial consortium in E-Cl medium contain- ters Fusobacterium spp. and Dethiosulfobacter spp., and two archaeal ing brain–heart infusion broth. Successful reconstitution was conﬁrmed phylotypes with sequence similarity to Methanosarcina spp. were de- by production of methane as the methanogens cannot grow alone tected with 16S rRNA gene primers. Using MCR primers three archaeal in complex medium such as brain–heart infusion or peptone, but phylotypes with the highest sequence identities to acetate-utilizing require fermentative bacteria to generate hydrogen and acetate for

Fig. 4. Denaturing HPLC chromatogram of phylotypes detected after initial enrichment (red) and after seven transfers (black). Panel A shows phylotypes detected with primers specificfor the gene encoding methylcoenzyme M reductase in methanogenic archaea. Panel B shows phylotypes detected with primers specific for the gene encoding 16S rRNA in bacteria and archaea. Unlabeled peaks did not yield sequence. B.M. Quinn et al. / Aquaculture 453 (2016) 154–162 159 methanogenesis (data not shown). Conversion of sterile fish waste which are required for cofactors in methanogens, have been reported to sludge to methane was compared between enriched inoculum and the increase methane production in anaerobic digesters (Demirel and reconstituted microbial consortium to determine whether the principal Scherer, 2011). In addition B-vitamins are required by species of species required for optimal methanogenesis were identified (Fig. 5). Methanogenium and Methanosarcina (Jarrell and Kalmokoff, 1988). In The reconstituted microbial consortium transferred from artificial me- this system the addition of trace metals, B-vitamins and additional dium exhibited a greater lag and an overall reduction in total methane phosphate and organic sulfur, however, did not improve the rates or production by approximately 20% compared with enriched inoculum. total methane produced in batch experiments using fish waste. The However, one sequential transfer of the reconstituted microbial consor- results indicate that both fish waste and undigested fish meal likely tium in medium containing sterile fish waste completely restored the provide a rich medium with sufficient essential growth factors for mi- efficiency of bioconversion to that observed for the enriched inoculum. crobes. High sulfate concentration in marine sludge has the potential Profiles of the phylotypes in enriched inoculum and reconstituted to reduce methane production because sulfate reducing bacteria have microbial consortium with sterile fish waste were similar indicating a greater affinity for acetate and hydrogen as well as a small thermody- that the reconstituted microbial consortium included the critical species namic advantage, compared to the methanogens (Chen et al., 2008). required for efficient bioconversion of the marine fish waste (Fig. 6). In addition to substrate competition, the product of sulfate reduction,

H2S, can possibly reach the toxicity thresholds for methanogens 4. Discussion (Bhattacharya et al., 1996; Mirzoyan et al., 2008; Tal et al., 2009).

COD:SO4 ratios greater than 1.7–2.7 favor methanogens over sulfate re- The most critical barriers to widespread commercial development of ducing bacteria (Bitton, 2005). The COD:SO4 ratio of the seawater used marine RASs are the cost of saline solid waste removal and their low en- in this study was 21:1, which already favored methanogenesis and in- ergy efficiency (Martins et al., 2010; Van Rijn, 2013). Key advantages of creasing the ratio by eliminating the sulfate did not further stimulate treating saline fish waste by anaerobic methanogenic digestion are and even inhibited slightly bioconversion of fish waste to methane. A the requirement for minimal energy input and the production of COD:SO4 ratio that exceeds 10 is typical of marine/brackish RAS concen- biomethane as a product, which can be used as an energy source to fur- trated wastes (Bhattacharya et al., 1996; Mirzoyan et al., 2008). In this ther offset operating costs. A facility producing 130 t of seabream and study the results indicate that both fish waste and undigested fish 100 dry tons of sludge would generate up to 1200 Gj of energy annually meal likely provided a rich medium and essential growth factors were offsetting the initial capital costs of the bioreactor system within a few not limiting the rates and extent of biomethane production. years. Although anaerobic digesters are subject to the rate limiting Municipal systems typically operate between 25 and 40 °C with steps of hydrolysis and methanogenesis, they can handle relatively maximum activity at 35 °C (Bitton, 2005). As expected increasing the high organic loading rates with minimal energy input and space temperature of the fish sludge digesting microbial consortium did requirements. The process of anaerobic digestion typically requires an have a positive effect on the amount of methane produced by the interactive consortium of hydrolytic bacteria, fermentative acidogenic enriched consortium. However, an increase in temperature from 26 to bacteria, hydrogen-utilizing acetogenic bacteria and methanogenic 35 °C only increased the methane yield by 11.8% with no apparent in- Archaea (Bitton, 2005). In this study factors known to adversely affect crease in rate. The increase in methane production is not enough to off- the performance of methanogenic bioreactors were investigated to de- set the cost of having to increase the temperature of a commercial termine the maximum efficiency that can be achieved by methanogenic system by 10 °C. At 20 °C there was a significant reduction in the rate degradation of fish waste from a marine RAS. Fish sludge inoculum from of methane production. The results indicate that an RAS bioreactor can the marine RAS was sequentially transferred in seawater to develop a be operated optimally at temperatures from 26 to 35 °C with minimal biomethane generating microbial consortium that is indigenous to sa- changes in the hydraulic retention time. line fish waste. Trace elements such as iron, cobalt and especially nickel, A COD:N ratio of approximately 50–70:1 has been reported as optimal for stable performance of methanogenic digestion (Álvarez et al., 2010), but in the current study over 90% of theoretical total methane was produced at a COD:N ratio 15:1. Mshandete et al. (2004) reported an increase in methane yield by increasing the COD:N ratio of fish waste with sisal pulp. However, attempts to improve the efficiency further in the current study by increasing the COD over half to 39:1 with a soluble carbon source increased the amount of total gas produced but −1 decreased slightly the amount of methane kgCOD. The results indicate that the methanogenic microbial consortium was adapted to a low COD:N ratio characteristic of proteinaceous fish waste. There was a transition of the predominant species as inoculum originating from the RAS solid waste settling tank was sequentially transferred with marine fish waste under an anaerobic headspace. The predominant fermentative phylotypes in the initial methanogenic enrichment had the highest sequence identity to species of Bacteriodales, which commonly originate from the feces of humans and other species with high protein diets including fish (Cahill, 1990; Kabiri et al., 2010; Wu et al., 2011), and Clostridiales that include species of protein fermenting acetogens (Pikuta et al., 2003). Archaeal phylotypes closely related to hydrogen utilizing Methanogenium spp. and acetate utilizing Methanosarcina spp. were also detected. As the efficiency of Fig. 5. Conversion of fish waste to methane by enrichment culture and reconstituted mi- methanogenesis achieved a steady state rate beyond the seventh se- crobial consortium. Graph shows methane production from sterilized fish waste uninocu- quential transfer the composition of the microbial consortium changed, lated (▼) and inoculated with enriched culture (●), reconstituted consortium from BHI although several microorganisms were maintained throughout the medium (▲) and reconstituted consortium after one transfer in sterile fish waste (■). enrichment process. Among the fermentative strains EA-F2 has 99% Dashed line represents estimated maximum methane yield from total COD of sludge and carbon amendment. Values are means and standard deviations for three replicate sequence identity to Dethiosulfovibrio russensis, Dethiosulfovibrio culture samples. acidaminovorans and Dethiosulfovibrio marinus. These species were 160 B.M. Quinn et al. / Aquaculture 453 (2016) 154–162

Fig. 6. Denaturing HPLC chromatogram of phylotypes detected in medium with sterile fish waste inoculated with microbial enrichment consortium (red) and the reconstituted microbial consortium (black). Panel A shows phylotypes detected with primers specific for the gene encoding 16S rRNA in bacteria and archaea. Panel B shows phylotypes detected with primers specific for the gene encoding methyl coenzyme M reductase (mcrA) in methanogenic archaea. Strain designation are as follows: Fusobacteriacea EA-F3 ( ), Dethiosulfovibrio sp. EA-F2 ( ), Methanogenium sp. EA-M3 ( ), Methanoplanus sp. EA-M7 ( ), Methanosarcina sp. EA-M15 (M15). Unlabeled peaks did not yield sequence.

isolated from saline environments and all three species ferment pro- waste to acetate and hydrogen, which are substrates for acetotrophic teins, peptides and amino acids to acetate, hydrogen and carbon dioxide and hydrogenotrophic methanogenic archaea, respectively. In biocon- (Surkov et al., 2001). Strain EA-F3 has high sequence identity (up to version of waste in low saline waste treatment systems fatty acid- 97%) to species within the Fusobacteriaceae. Although the majority of utilizing acetogens oxidize butyrate and propionate to the methanogen- species within this phylum were isolated from humans or animals, ic substrates acetate and hydrogen. The absence of short chain fatty Ilyobacter spp. were isolated from marine sediments (Zhao et al., acid-utilizing acetotrophs in the microbial consortium suggests that 2009). Most species ferment proteins and amino acids to butyric, the hydrogen-utilizing methanogens create a thermodynamic shift to- propionic and acetic acids and other fermentation products. Strain EA- wards acetate production by the fermenters via inter-species hydrogen M15 has 100% sequence identity to Methanosarcina sp. WH1. This exchange (Sowers and Ferry, 2002). This thermodynamic shift would genus of methanogenic Archaea, which include halotolerant and obli- cause more complete conversion of the fish waste to the methanogenic gate marine species, utilize methylated amines, methylsulfides, acetate intermediates acetate and hydrogen. The efficiency of the process (up to and sometimes hydrogen for growth and methanogenesis (Sowers, 90%) combined with detection of these five predominant species 2009). Strain EA-M3 has 99% sequence identity to a Methanogenium supports the conclusion that this microbial consortium is highly sp., and strain EA-M7 has 100% sequence identity to a Methanoplanus adapted for direct conversion of fish sludge from an RAS to methane sp., which are hydrogen-utilizing methanogenic Archaea that include without additional pretreatment. It is interesting that acetate-utilizing marine species (Sowers, 2009). Microscopic examination revealed Methanosaeta spp., which are generally abundant in low-saline waste disk-shaped cells and irregular cocci characteristic of Methanoplanus treatment systems, were not detected in the fish waste enrichments and Methanogenium, respectively. Denaturing HPLC analysis of 16S (Sowers, 2009). One explanation could be the lack of visible granule for- rRNA genes for all of the pure cultures resulted in more than one mation resulting from high sodium concentration (Jeison et al., 2008), PCR fragment, an effect that was observed previously for analysis on which is critical for preventing washout of slow growing Methanosaeta. both DGGE gels and dHPLC (Wagner et al., 2009). Possible explana- However, this would not prevent growth of Methanosaeta spp. in batch tions include inaccuracy of polymerase with PCR amplification or se- culture. A more likely explanation is that acetate concentrations were quence variations within the template DNA (Coenye and Vandamme, high enough so that Methanosaeta, which have a lower KS for acetate 2003). Genome sequences of species for all five of the isolates have uptake, were overgrown by faster growing Methanosarcina spp. more than one 16S rRNA gene copy, which could also create multiple Although the reconstituted five member microbial consortium peaks. exhibited bioconversion rates that were equivalent to the enriched Overall, the fermentative species are well adapted for converting consortium, this does not rule out the possibility that other species highly proteinaceous fish feces and partially or uneaten food in fish have a role in the enriched consortium. Other proteinaceous fermenters B.M. Quinn et al. / Aquaculture 453 (2016) 154–162 161 might occur in low numbers that were not detected by denaturing HPLC Demirel, B., Scherer, P., 2011. Trace elements requirements of biogas digesters during biological conversion of renewable biomass to methane. Biomass Bioenergy 35, and were outgrown during enrichment and isolation. 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