International Biodeterioration & Biodegradation 86 (2014) 248e251
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Short communication
Sulfide removal from livestock biogas by Azospirillum-like anaerobic phototrophic bacteria consortium M.L.B. da Silva a, *, M.P. Mezzari b, A.M.G. Ibelli a, K.B. Gregory c a
Embrapa Swine and Poultry, P.O. Box 21, 89700-000 Concórdia, SC, Brazil Federal University of Santa Catarina, Department of Chemical Engineering, P.O. Box 476, 88040-900 Florianópolis, SC, Brazil c Carnegie Mellon University, Department of Civil & Environmental Engineering, 5000 Forbes Avenue, 15213 Pittsburgh, PA, USA b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 August 2013 Received in revised form 16 September 2013 Accepted 18 September 2013 Available online 13 November 2013
Biogas production from anaerobic biodegradation of livestock waste is a potential source of renewable energy. In addition to methane, biodegradation of this high-strength waste also produces sulfide that must be removed in order to prevent costly corrosive impacts on infrastructure. In this work, an anaerobic, phototrophic microbial community enriched from the native population in a swine waste lagoon was evaluated for its potential to remove sulfide from swine waste biogas. Batch experiments with the consortium attained removal efficiencies greater than 97% for sulfide concentrations above 1200 ppm. 16S rRNA gene sequencing revealed that the dominant population was most closely related to the isolate Azospirillum strain C5 (similarity index of 99%). Photomicrograph of the enriched consortium revealed the presence of cells with intracellular globules resembling sulfur storage. The enrichment of Azospirillum-like and the concomitant sulfide consumption suggest that this microorganism played an important role in sulfide removal in the bioreactor. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Azospirillum sp. Biofilter Biogas Photobioreactor Sulfide Swine manure
1. Introduction The anaerobic biodegradation of livestock waste produces valuable methane as a source of renewable energy. However, hydrogen sulfide, a highly corrosive, toxic, and malodorous compound is coproduced with methane at concentrations as high as 1000 to 20,000 ppm of the biogas (Lastella et al., 2002; Tchobanoglous et al., 2003). Sulfide is produced from the biodegradation of proteins and other sulfur containing compounds present in the original waste. The most common and commercially available technologies to remove H2S from biogas streams are chemically based and expensive to operate (Gabriel and Deshusses, 2003). For example, farms in Brazil most commonly use iron oxide particles to remove sulfide from biogas streams. The recurring costs of the system undermine the economic sustainability of the renewable energy produced.
* Corresponding author. Tel.: þ55 49 34410456; fax: þ55 49 34410497. E-mail addresses:
[email protected] (M.L.B. da Silva), mmezzari@ gmail.com (M.P. Mezzari),
[email protected] (A.M.G. Ibelli), kelvin@ cmu.edu (K.B. Gregory). 0964-8305/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2013.09.013
Additionally, there are concerns about the pyrophoric properties of the iron sulfide that is produced during iron-based treatment that may cause unnecessary hazard and malfunctioning of the filter. Previous work has examined the potential for biofiltering with aerobic, chemotrophic (Su et al., 2013) and/or phototrophic microbial consortia for removal of sulfide from biogas (Syed et al., 2006). Research with anoxygenic phototrophs suggested that indigenous (to the bioreactor) populations may be advantageous for the removal of sulfide from biogas streams due to their ability to adapt to reducing conditions in the bioreactor. Moreover, the use of indigenous, well-adapted bacteria creates a community that is less susceptible to local variation of environmental conditions such as temperature that may cause ecological changes in the microbial community and give rise to detrimental changes in reactor efficiency. The objective of this work was to examine the potential for a phototrophic, sulfide-oxidizing bacterial consortium, enriched from a local swine waste lagoon, to remove sulfide from biogas. The work describes the enrichment of the consortium, characterization of the microbial community using 16S rRNA gene clone library, and an assessment of the community’s ability to remove sulfide from a biogas reactor.
M.L.B. da Silva et al. / International Biodeterioration & Biodegradation 86 (2014) 248e251
2.1. Anaerobic microbial enrichment The anaerobic sulfur-oxidizing bacteria consortium was isolated from a local swine waste pit located in southern of Brazil. The swine waste impoundment measured 10 m length 3 m wide 2.5 m deep. The pit has been used for over 30 years to store waste from an average of 30 finishing hogs, producing approximately 8 L waste animal1 d1. Sampling occurred during the winter. At the time of sampling, the impoundment water had a reddish hue, likely due to the proliferation of red, phototrophic sulfur and/or non-sulfur bacteria. The laboratory enrichment took place in a 2-L airtight bottle closed by a stopper. Reactor headspace was 100 mL. The reactor contained two sampling ports that enabled sterile, anaerobic collection of headspace and liquid samples. The sample was enriched on Pfennig’s modified media (Pfennig, 1974) with 0.0015% yeast extract and kept at room temperature and under autotrophic conditions. The photobioreactor was exposed to 40 W fluorescent lamp with a light intensity of 45.2 mmol m2 s1. The consortium was enriched for over a year prior to the experimental assays by replacing 80% v/v of the culture medium every 15 days. The transferred culture medium was previously deoxygenated by purging with nitrogen gas for 15 min. Sulfide removal experiments were performed using identical reactor configuration and conditions. 2.2. Sulfide removal Biogas was obtained directly from an anaerobic digester treating swine waste. Biogas were collected in 1-L aluminum plastic sampling bags (ALU-Verbundfollie A 30, Herman Nawrot AG, Germany) and utilized shortly after collection. Biogas was then purged for 5 min within the reactor to ensure contact time and transfer into water phase. The experiments were initiated when the consortium in the photobioreactor had reached exponential growth phase, within 24 h following inoculation (10% v/v inoculum). 5-mL headspace samples were collected over time using gas tight syringe. Sulfide concentration was determined by methylene blue method assay (4500-S2-D, APHA et al., 1995). An identical, biological negative control reactor was prepared to run in parallel to discern biotic sulfide losses. The negative control reactor was prepared by inoculation of a similar biomass of phototrophic, purple non-sulfur Rhodopseudomonas sp. that is incapable of sulfide oxidation. This bacterial strain was isolated from swine effluent wastewater through serial dilutions of the initial enrichment utilizing Pfennig’s culturing medium specific for non-sulfur bacteria (Pfennig, 1974). Identification consisted of cell morphological analysis under 1000 magnification using an optical light microscope (Zeiss A10 e Scope A1). 16S rRNA gene sequencing analyses were further conducted to aid bacteria identification (as described below) revealing 99% similarity with Rhodopseudomonas sp. 2.3. 16S rDNA gene sequencing The microbial community in the experimental reactor was examined using 16S rRNA gene sequencing. Whole community DNA was extracted using the MoBioÒ UltraClean Microbial DNA isolation kit according to manufacturer’s instructions (MoBio Laboratories, Solana Beach, CA). PCR amplification of the 16S rRNA gene fragments was performed in reactions containing 500 nmol of each universal bacteria primer (1055F 50 -ATGGCTGTCGTCAGCT-30 and 1392R 50 -ACGGGCGGTGTGTAC-30 primers (Ferris et al., 1996), 2 PCR Master mix (Ludwig Biotec, Brazil) and DNA template obtained from the consortium. Thermocycler conditions were
denaturation at 95 C for 5 min, followed by 40 cycles of 95 C for 15 s, annealing at 53 C for 15 s and a final extension step at 72 C for 40 s. PCR products were purified using PureLinkÒ PCR Purification Kit (InvitrogenÒ) and cloned into pGEMT Easy Vector Systems (PromegaÒ) according to manufacturer’s protocols. Cloned samples were inserted into E. coli DH5a (InvitrogenÒ) using heat shock and plated on selective LB medium. Colonies containing plasmids with insert were selected for sequencing and grown overnight in LB medium. Plasmids were isolated from the cultures using PureLinkÒ Quick Plasmid Miniprep Kit and DNA concentration and purity quantified by spectrophotometric analysis (NanodropÒ). A total of 20 colonies were randomly selected for sequencing. DNA sequencing was performed using 300e500 ng of DNA containing BigDyeÒ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and 2 mM M13 primers. Sequencing products were purified using isopropanol/ethanol precipitation method prior to analysis (ABI Prism 3130 Avant sequencer, Applied Biosystems). Trimmed sequences were aligned using Ribosomal Database Project (RDP) Infernal Aligner tool. Sequences were compared to each other using BLASTnÒ; www.blast.ncbi.nlm.nih. gov and a phylogenetic tree constructed using RDP treebuilder (Cole et al., 2009). Similar sequences within the clone library were assigned when they shared >98% sequence similarity. 2.4. Microscopy 0.5-mL liquid samples from the experimental reactor were transferred to 1-mL Eppendorf vials and centrifuged (3000 g, 1 min; Scilogex D3024R, Berlin CT, USA). The collected cell pellet was smeared onto glass microscope slides and observed under 1000 magnification using an optical light microscope (Zeiss A10 e Scope A1). Micrographs were taken using a Carl Zeiss Axiocam 1Cc3 camera equipped with the software Axiovision rel 4.8.1. 3. Results and discussion Sulfide concentration in the reactor enriched with phototrophic sulfide-oxidizing bacteria consortium decreased from 1200 to 30 ppm within 180 min (Fig. 1). The measured removal efficiency >97.5% was within typical values observed for suspended growth biofilters inoculated with photoautotrophs (i.e., 90e100%) (Syed
Sulfide-oxidizing consortium
Negative control
Re-spiked biogas
1200 1000 H2S (ppm)
2. Material and methods
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y = 8850.8e-0,012x r² = 0.99
800 600 y = 1262.4e-0,02x r² = 0.99
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Time (min) Fig. 1. Biological sulfide removal from swine waste biogas using a photobioreactor seeded with an anaerobic bacterial consortium enriched from swine waste. The control reactor photobioreactor was seeded with a purple non-sulfur oxidizing bacterium (Rhodopseudomonas sp.).
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et al., 2006). The estimated removal rates of y350e390 mg L1 h1 were, however, significantly higher than previously reported values for continuous stirred tank reactors (14.6e109 mg L1 h1) (Syed et al., 2006). The absence of measurable sulfide removal in the biological negative control reactor inoculated with non-sulfur Rhodopseudomonas indicated that abiotic losses (e.g., volatilization) were negligible. After 180 min of reaction, the experimental reactor was sparged a second time with biogas to examine the robustness of the biofiltering process. Re-spiking biogas into reactor did not affect the sulfide removal by the enriched anaerobic consortium. Therefore, the robustness of this anaerobic consortium ultimately reflects on its potential applicability as inoculum for biofilters systems designed to treat sulfide-rich biogas stream. Analysis of the 16S rRNA gene sequencing obtained from the sulfur oxidizing-enriched anaerobic consortium grown in batch reactor revealed the presence of dominant bacteria (55% of the total 20 sequenced clones) most closely (>99% similarity) associated with Azospirillum sp. 5c, (Fig. 2) belonging to the Rhodospirillaceae family of the a-proteobacteria class (Yoon et al., 2007). Whereas no reports exist to support sulfide-oxidation by Azospirillum sp. C5, several members of Rhodospirillaceae are known to phototrophically oxidize sulfide to elemental sulfur (Imhoff et al., 1981). The next highest percentage of the clone library was represented by sequences most similar to Azoarcus communis, representing 25% of the clones recovered. Azoarcus spp., members of the b-subclass of the proteobacteria are best known for nitrogen fixation. The findings do not indicate that the Azospirillum spp. were solely responsible for sulfide removal in the reactor. However, enrichment of populations closely related to Azospirillum and the concomitant sulfide consumption suggests that it plays an important role in sulfide removal in these bioreactors. Isolation of Azospirillum was unsuccessful even after several serial dilutions of the enrichment. This may indicate a syntrophic relationship between the dominant microorganism and others in the community. The photomicrograph of the enriched consortium shown in Fig. 3 revealed the presence of structures that resemble the intracellular globules observed for sulfur storage by phototrophic, sulfide-oxidizing Azospirillum thiophilum isolated from a sulfide spring (Lavrinenko et al., 2010). These clusters of elemental sulfur are common among other sulfur-oxidizing bacteria such as Thiocystis sp. (Vasconcelos et al., 2006) and further indicate a role for Azospirillum-like populations in sulfide removal. Whereas the accumulation of exceeding biomass produced by field scale biofiltering process could serve as a source of sulfur-rich fertilizer remains unclear and warrants further investigation. Efficient, cost-effective sulfide removal from biogas streams will be an important component for ensuring the economic sustainability of methane production from livestock wastes. The findings presented here show that a phototrophic microbial community,
Fig. 3. Optical micrographs (1000 magnification) of the dominant bacterial cells present in the sulfide-enriched anaerobic consortium. Arrows indicate the presence of putative sulfur inclusions within cells.
enriched from a swine waste lagoon is capable of rapid and efficient sulfide removal. Such a consortium may be utilized in a biofilter for sulfide oxidation and may offer a more cost-effective approach for treating biogas. Further studies with this consortium are needed to identify which organisms are responsible for sulfide removal and optimize the design of a farm-scale application for evaluation. 4. Conclusions This work presented here demonstrates the ability of an anaerobic, phototrophic microbial consortium, enriched form a swine-waste lagoon to remove sulfide from a biogas reactor treating swine waste. The consortium was strongly enriched in species that were most similar to known Azospirillum spp. that may oxidize sulfide. This finding is important because native microbial species from the swine waste source will be better adapted to local environmental conditions and produce a more stable bioreactor for sufide removal. Such a community could more easily be utilized as a seed culture for innoculating biofilters to treat biogas streams. There are significant advantages to biofiltration using native species, including, overcoming technical limitations associated with maintenance of cultures against detrimental abiotic conditions and system costs and complexity associated with the delivery of oxygen for aerobic sulfide-oxidizing bacteria. Acknowledgments This research was funded by a Brazilian national electrical energy agency e ANEEL grant N 014/2012. The authors thank the Ministry of Education of Brazil for the CAPES-EMBRAPA postdoctoral scholarship (N 001/2011). The authors acknowledge Dr. Raquel Rech for the microscopy image analysis.
Fig. 2. Phylogenetic tree based on comparative analysis of 16S rRNA gene sequences recovered in a clone library from the phototrophic, sulfide-oxidizing enrichment. The distance matrix was generated using a Jukes-Cantor corrected distance model. The tree was created using weighted, neighbor joining and shows the relationship to known type cultures. The green text shows the clone name and the blue text, the percent of the clones recovered with >98% sequence similarity. Bootstrap values were obtained from 100 maximum likelihood replicates. Methanocaldococcus jannashii was used as the outgroup. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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