Characterization of a Microbial Consortium That Converts Mariculture fish Waste to Biomethane
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Aquaculture 453 (2016) 154–162 Contents lists available at ScienceDirect Aquaculture journal homepage: www.elsevier.com/locate/aquaculture 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 sys- tems 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 http://dx.doi.org/10.1016/j.aquaculture.2015.12.002 0044-8486/© 2015 Elsevier B.V. All rights reserved. B.M. Quinn et al. / Aquaculture 453 (2016) 154–162 155 (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