Bacterial ecology of abattoir wastewater treated by an anaerobic digestor

Linda Jabari Hana Gannoun Eltaief Khelifi Jean-Luc Cayol Jean-Jacques Godon Moktar Hamdi Marie-Laure Fardeau About the authors

Abstract

Wastewater from an anaerobic treatment plant at a slaughterhouse was analysed to determine the bacterial biodiversity present. Molecular analysis of the anaerobic sludge obtained from the treatment plant showed significant diversity, as 27 different phyla were identified. Firmicutes, Proteobacteria, Bacteroidetes, Thermotogae, Euryarchaeota (methanogens), and msbl6 (candidate division) were the dominant phyla of the anaerobic treatment plant and represented 21.7%, 18.5%, 11.5%, 9.4%, 8.9%, and 8.8% of the total bacteria identified, respectively. The dominant bacteria isolated were Clostridium, Bacteroides, Desulfobulbus, Desulfomicrobium, Desulfovibrio and Desulfotomaculum. Our results revealed the presence of new species, genera and families of microorganisms. The most interesting strains were characterised. Three new bacteria involved in anaerobic digestion of abattoir wastewater were published.

Keywords
Bacterial ecology; Biodiversity; Bacterial culture; SSCP; PCR

Introduction

For hygienic reasons, abattoirs use copious amounts of water in their processing operations (slaughtering and cleaning), which creates significant wastewater. In addition, the increased use of automated machines to process carcasses, along with the incorporation of washing at every stage, has increased water consumption in slaughterhouse facilities. The high fat and protein content of slaughterhouse waste makes wastewater a good substrate for anaerobic digestion, due to its expected high methane yield.11 Chasteen TG, Fuentes DE, Tantaleán JC, Vásquez CC. Tellurite: history, oxidative stress, and molecular mechanisms of resistance. FEMS Microbiol Rev. 2009;33:820-832; Chen YT, Chang HY, Lai YC, Pan CC, Tsai SF, Peng HL. Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43. Gene. 2004;337:189-198; Scherr KE, Lundaa T, Klose V Bochmann G, Loibner AP Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation. J Biotechnol. 2012;157:564-572; Palatsi J, Vinas M, Guivernau M, Fernandez B, Flotats X. Anaerobic digestion of slaughterhouse waste: main process limitations and microbial community interactions. Bioresour Technol. 2011;102:2219-2227. Numerous microorganisms are involved in the anaerobic degradation of slaughterhouse waste, any step of which may be rate-limiting depending on the waste being treated as well as the process involved.

The microorganisms involved in anaerobic digestion have not been fully identified; however, at least four groups of microorganisms are involved in this process.22 Hassan AN, Nelson BK. Invited review: anaerobic fermentation of dairy food wastewater. J Dairy Sci. 2012;95:6188-6203. The first group are the hydrolytic bacteria that degrade complex compounds (protein, carbohydrates, and fat) into simpler compounds, such as organic acids, alcohols, carbon dioxide (CO2) and hydrogen. The second group are the hydrogen producing acetogenic bacteria that use organic acids and alcohols to produce acetate and hydrogen. The third group contains homoacetogenic bacteria that can only form acetate from hydrogen, CO2, organic acids, alcohols, and carbohydrates. The fourth group comprises methanogens that form methane from acetate, CO2, and hydrogen. Hydrolytic, acetogenic, and methanogenic microorganisms play an equally important role in anaerobic digestion and methane production. Optimal methane production is only achieved via the interaction of multiple microorganisms,33 Chartrain M, Bhatnagar L, Zeikus JG. Microbial ecophysiology of whey biomethanation: comparison of carbon transformation parameters, species composition, and starter culture performance in continuous culture. Appl Environ Microbiol. 1987;53:1147-1156. and therefore, biodegradation of molecules in wastewater depends on the activity of all microbial groups involved.

Common fermentative bacteria include Lactobacillus, Eubacterium, Clostridium, Escherichia coli, Fusobacterium, Bacteroides, Leuconostoc, and Klebsiella. Acetogenic bacteria include Acetobacterium, Clostridium, and Desulfovibrio.22 Hassan AN, Nelson BK. Invited review: anaerobic fermentation of dairy food wastewater. J Dairy Sci. 2012;95:6188-6203. Methane producing organisms are classified under domain Archaea and phylum Euryarchaeota.44 Boone DR, Castenholz RW. Bergey's Manual of Systematic Bacteriology. vol. 1, 2nd ed. New York, NY: Springer-Verlag; 2001.

In order to better understand the function of a bacterial population, a detailed description of the microbial ecosystem is necessary. One method is via molecular biology techniques.55 Khelifi E, Bouallagui H, Fardeau ML, et al. Fermentative and sulphate-reducing bacteria associated with treatment of an industrial dye effluent in an up-flow anaerobic fixed bed bioreactor. Biochem Eng J. 2009;45:136-144. Recent advances in the molecular analysis of bacterial ecosystems allow a better understanding of the specific microorganisms involved in wastewater treatment. There are only a few studies focused on microbial populations, diversity and evolution in reactors fed with complex organic wastes.11 Chasteen TG, Fuentes DE, Tantaleán JC, Vásquez CC. Tellurite: history, oxidative stress, and molecular mechanisms of resistance. FEMS Microbiol Rev. 2009;33:820-832; Chen YT, Chang HY, Lai YC, Pan CC, Tsai SF, Peng HL. Sequencing and analysis of the large virulence plasmid pLVPK of Klebsiella pneumoniae CG43. Gene. 2004;337:189-198; Scherr KE, Lundaa T, Klose V Bochmann G, Loibner AP Changes in bacterial communities from anaerobic digesters during petroleum hydrocarbon degradation. J Biotechnol. 2012;157:564-572; Palatsi J, Vinas M, Guivernau M, Fernandez B, Flotats X. Anaerobic digestion of slaughterhouse waste: main process limitations and microbial community interactions. Bioresour Technol. 2011;102:2219-2227. Therefore, little is known about the composition of these reactors. The development of advanced molecular biology techniques has contributed to the detection, quantification, and identification of the bacterial populations involved in the treatment of abattoir wastewater. For example, cloning and sequencing of 16S rRNA gene fragments provide information about the phylogeny of the microorganisms. Additionally, single stranded conformation polymorphism (SSCP) offers a simple, inexpensive and sensitive method for detecting whether or not DNA fragments are identical in sequence, and can greatly reduce the amount of sequencing necessary.66 Gannoun H, Khelifi E, Omri I, et al. Microbial monitoring by molecular tools of an upflow anaerobic filter treating abattoir wastewaters. Bioresour Technol. 2013;142:269-277.

This work aimed to study the bacterial ecology of an anaerobic digestor through both bacterial culture and molecular biological techniques. The bacteria involved in the anaerobic digestion of abattoir wastewater were identified using classic microbiology techniques and molecular tools (sequencing of 16S rRNA and SSCP). Additionally, our results were compared with those of Gannoun et al.66 Gannoun H, Khelifi E, Omri I, et al. Microbial monitoring by molecular tools of an upflow anaerobic filter treating abattoir wastewaters. Bioresour Technol. 2013;142:269-277. to evaluate the effect of storage at 4 °C on the bacterial diversity of the sludge.

Material and methods

Origin of the sludge

Anaerobic sludge samples were collected from an upflow anaerobic filter that treats abattoir wastewater in Tunisia.77 Gannoun H, Bouallagui H, Okbi A, Sayadi S, Hamdi M. Mesophilic and thermophilic anaerobic digestion of biologically pretreated abattoir wastewaters in an upflow anaerobic filter. J Hazard Mater. 2009;170:263-271. The digestor operated under both mesophilic (37 °C) and thermophilic (55 °C) conditions. Samples were taken at the end of thermophilic phase and stored at 4 °C. The sludge was then analysed to determine the bacterial diversity present, first via bacterial culture.

DNA extraction, PCR and SSCP analysis of the digestor sludge

Four milliliters of the anaerobic sludge sample were centrifuged at 6000 rpm for 10 min. Pellets were re-suspended in 4 mL of 4 M guanidine thiocyanate–0.1 M Tris pH 7.5 and 600 µL of N-lauroyl sarcosine 10%. Two hundred and fifty microlitres of treated samples were transferred in 2 mL tubes and stored at −20 °C.

Extraction and purification of total genomic DNA was implemented according to the protocol developed by Godon et al.88 Godon JJ, Zumstein E, Dabert P, Habouzit F. Moletta R. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol. 1997;63:2802-2813.

Highly variable V3 regions of bacterial 16S rRNA genes were amplified by PCR using bacterial (w49–w34) primers (Table 1). Samples were treated according to the protocol previously described by Delbès et al.99 Delbès C, Moletta R, Godon J. Bacterial and archaeal 16S rDNA and 16S rRNA dynamics during an acetate crisis in an anaerobic digestor ecosystem. FEMS Microbiol Ecol. 2001;35:19-26.

Table 1
Sequences and target positions of primers used in this study.

For electrophoresis, PCR–SSCP products were diluted in water before mixing with 18.75 µL formamide (Genescan-Applied Biosystems) and 0.25 µL internal standards (ROX, Genescan-Applied Biosystems) according to the protocol of SSCP described by Delbès et al.99 Delbès C, Moletta R, Godon J. Bacterial and archaeal 16S rDNA and 16S rRNA dynamics during an acetate crisis in an anaerobic digestor ecosystem. FEMS Microbiol Ecol. 2001;35:19-26.

SSCP analyses were performed on an automatic sequencer abi310 (Applied Biosystems). RNA fragment detection was done on the fluorescent w34 primer. The results obtained were analysed by GeneScan Analysis 2.0.2 Software (Applied Biosystems) as specified by Gannoun et al.66 Gannoun H, Khelifi E, Omri I, et al. Microbial monitoring by molecular tools of an upflow anaerobic filter treating abattoir wastewaters. Bioresour Technol. 2013;142:269-277. For bacterial identification, pyrosequencing of the DNA samples using a 454 protocol was performed (Research and Testing Laboratory, Lubbock, USA).

Methods of analysis for pyrosequencing data used herein have been described previously.1010 Wolcott RD, Gontcharova V, Sun YZischakau A, Dowd SE. Bacterial diversity in surgical site infections: not just aerobic cocci any more. J Wound Care. 2009;18:317-323.1414 Pitta DW, Pinchak E, Dowd SE, et al. Rumen bacterial diversity dynamics associated with changing from bermudagrass hay to grazed winter wheat diets. Microb Ecol. 2010;59:511-522. Sequences are first depleted of barcodes and primers. Then, short sequences under 200 bp are removed, sequences with ambiguous base calls are removed, and sequences with homopolymer runs exceeding 6 bp are removed. Sequences are then denoised and chimeras removed. Operational taxonomic units were defined after removal of singleton sequences, clustering at 3% divergence (97% similarity).1515 Dowd SE, Callaway TR, Wolcott RD, et al. Evaluation of the bacterial diversity in the feces of cattle using 16S rDNA bacterial tag-encoded FLX amplicon pyrosequencing (bTEFAP). BMC Microbiol. 2008;8:125.2121 Swanson KS, Dowd SE, Suchodolski JS, et al. Phylogenetic and gene-centric metagenomics of the canine intestinal microbiome reveals similarities with humans and mice. ISME J. 2011;5:639-649. Operational taxonomic units were then taxonomically classified using BLASTn against a curated GreenGenes database2222 DeSantis TZ, Hugenholtz P, Larsen N, et al. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microbiol. 2006;72:5069-5072. and compiled into each taxonomic level into both “counts” and “percentage” files. Counts files contain the actual number of sequences, while the percentage files contain the percentage of sequences within each sample that map to the designated taxonomic classification.

Enrichment and isolation procedures of fermentative and SRB bacteria

The Hungate technique2323 Hungate RE. A roll tube method for cultivation of strict anaerobes In: Norris JR, Ribbons DW, eds. Methods in Microbiology. vol. 3B. New York, USA: Academic Press; 1969:117-132. was used throughout this study. Inoculations were done with 10% of culture. Samples were collected from the anaerobic filter at the end of thermophilic phase. A 0.5 mL aliquot of sample was inoculated into Hungate tubes containing 5 mL of basal medium.

For both SRB and fermentative bacteria, enrichment and isolation was done according to the protocol described previously by Hungate2323 Hungate RE. A roll tube method for cultivation of strict anaerobes In: Norris JR, Ribbons DW, eds. Methods in Microbiology. vol. 3B. New York, USA: Academic Press; 1969:117-132. and Khelifi et al.55 Khelifi E, Bouallagui H, Fardeau ML, et al. Fermentative and sulphate-reducing bacteria associated with treatment of an industrial dye effluent in an up-flow anaerobic fixed bed bioreactor. Biochem Eng J. 2009;45:136-144.

Purification of the DNA, PCR amplification and sequencing of the 16S rRNA gene of isolated bacteria

Purification of the DNA, PCR amplification and sequencing of the 16S rRNA gene were performed as described previously.2424 Thabet OB, Fardeau ML, Joulian C, et al. Clostridium tunisiense sp. nov., a new proteolytic, sulfur-reducing bacterium isolated from an olive mill wastewater contaminated by phosphogypse. Anaerobe. 2004;10:185-190. The partial sequences generated were assembled using BioEdit version 5.0.92525 Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp Ser. 1999;41:95-98. and the consensus sequence of 1495 nucleotides was corrected manually for errors. The most closely related sequences in GenBank (version 178), the Ribosomal Database Project (release 10) identified using BLAST,2626 Altschul SF, Gish WMiller WMyers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215:403-410. and the Sequence Match program,2727 Cole JR, Wang Q, Cardenas E, et al. The ribosomal database project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009;37(Database issue):D141-D145. were extracted and aligned. The consensus sequence was then adjusted manually to conform to the 16S rRNA secondary structure model.2828 Winker S, Woese CR. A definition of the domains Archaea, Bacteria and Eucarya in terms of small subunit ribosomal RNA characteristics. Syst Appl Microbiol. 1991;14:305-310. Nucleotide ambiguities were omitted and evolutionary distances were calculated using the Jukes and Cantor option.2929 Jukes TH, Cantor CR. Evolution of protein molecules. In: Munro HN, ed. Mammalian Protein Metabolism. New York, USA: Academic Press; 1969:21-132. Dendrograms were constructed with the TREECON program using the neighbor-joining method.3030 Saitou N, Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987;4:406-425. Tree topology was re-examined by the bootstrap method (1000 replications) of re-sampling.3131 Felsenstein J. Confidence limits on phylogenies: an approach using the bootstrap. Evolution. 1985;39:783-791. Its topology was also supported using the maximum-parsimony and maximum-likelihood algorithms.

Data analyses

The two main factors taken into account when measuring diversity are richness and evenness. Richness is a measure of the number of different kinds of organisms present in a particular sample. Evenness (Es) compares the similarity of population sizes between each of the species present. The reciprocal of Simpson's index (diversity richness) (1/D), which is widely used for ecological studies, was also used as a measure of diversity. Richness and evenness were calculated and interpreted as described previously by Simpson3232 Simpson EH. Measurement of diversity. Nature. 1949;163:688. and Gannoun et al.66 Gannoun H, Khelifi E, Omri I, et al. Microbial monitoring by molecular tools of an upflow anaerobic filter treating abattoir wastewaters. Bioresour Technol. 2013;142:269-277.

Results and discussion

Diversity and abundance of the bacterial communities in the bioreactor sludge using SSCP and DNA sequencing

There was significant microbial diversity of the upflow anaerobic filter, which operated under both mesophilic (37 °C) and thermophilic (55 °C) conditions. Twenty-seven different phyla were identified, and the six most common phyla (Firmicutes, Proteobacteria, Bacteroidetes, Thermotogae, Euryarchaeota (the methanogenic bacteria), and the msbl6 (candidate division)) represented 78.8% of the total (Table 2). The 21 less common phyla represented 21% of the total.

Table 2
The different phyla found in the digestor.

Gram-positive bacteria, including Firmicutes (low G + C), were the most common type of bacteria in the anaerobic digestor, comprising 21.7% of the total. Both Gram-positive low G + C bacteria and the Bacteroidetes phylum are known for their fermentative properties. Furthermore Bacteroidetes play an important role in the degradation of complex polymers. Firmicutes and Bacteroidetes are also the two main groups encountered in the study by Godon et al.88 Godon JJ, Zumstein E, Dabert P, Habouzit F. Moletta R. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol. 1997;63:2802-2813. of a fluidised bed anaerobic digestor fed with vinasse. These two groups of bacteria hydrolyse the polymer substrates which are not degraded during the previous stages of anaerobic digestion (such as polysaccharides, proteins and lipids) into acetate, long chain fatty acids, CO2, formate and hydrogen.

Bacteria within the Proteobacteria phylum were also commonly found in the digestor. These Gram-negative bacteria are considered to be some of the most cultivable microorganisms.3333 Giovannoni SJ, Rappé M. Evolution, diversity, and molecular ecology of marine prokaryotes. In: Kirchman DL, ed. Microbial Ecology ofthe Oceans. New York, NY: John Wiley & Sons, Inc.; 2000:47-84.,3434 Zwart G, Crump BC, Kamst-Van Agterveld MP, Hagen F, Han SK. Typical freshwater bacteria: an analysis of available 16S rRNA gene sequences from plankton of lakes and rivers. Aquat Microb Ecol. 2002;28:141-155. The Proteobacteria have an important role in the hydrolysis and acetogenesis steps of anaerobic digestion, and include delta, gamma and beta varieties. Deltaproteobacteria contains many syntrophic anaerobic bacteria, which participate in sulphate reduction. Among the Gammaproteobacteria, there are many denitrifying bacteria or bacteria that accumulate phosphates.3535 Crocetti GR, Hugenholtz P Bond PL, et al. Identification of polyphosphate-accumulating organisms and design of 16S rRNA-directed probes for their detection and quantitation. Appl Environ Microbiol. 2000;66:1175-1182. The Betaproteobacteria are involved in nitrification, and are potentially also involved in denitrification.

Phylogenetic analysis of the domain Bacteria also helped to highlight the existence of a poorly known order, Thermotogales, which was relatively abundant within the digestor at 9.4%. Thermotogales contains anaerobic bacteria that are heterotrophic with a fermentative metabolism.3636 Huber R, Langworthy TA. Thermotoga maritima sp. Nov. represents a new genu of unique extremely thermophilic eubacteria growing up to 90 °C. Arch Microbiol. 1986;144:324-333. These bacteria are also found in other anaerobic digestors.88 Godon JJ, Zumstein E, Dabert P, Habouzit F. Moletta R. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol. 1997;63:2802-2813.

The Planctomycetales group represented 1.9% of the bacteria within the digestor. Bacteria within Planctomycetales are limited to five kinds and only eight species are described. Aerobic heterotrophic Planctomycetes have been successfully isolated from brackish marine sediments, freshwater sediments, soil, hot springs, salt pits and tissues from giant tiger prawn postlarvae.3737 Neef A, Amann R, Schlesner H, Schleifer KH. Monitoring a widespread bacterial group: in situ detection of planctomycetes with 16S rRNA-targeted probes. Microbiology. 1998;144(Pt 12):3257-3266.,3838 Chouari R, Le Paslier D, Daegelen PGinestet P, Weissenbach J, Sghir A. Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant. Appl Environ Microbiol. 2003;69:7354-7363. In addition, a special group of Planctomycetes were implicated in the oxidation of ammonia under anaerobic conditions in wastewater plants, coastal marine sediments, and oceanic and freshwater oxygen minimum zones.3939 Elshahed MS, Youssef NH, Luo Q, et al. Phylogenetic and metabolic diversity of Planctomycetes from anaerobic, sulfide-and sulfur-rich Zodletone Spring, Oklahoma. Appl Environ Microbiol. 2007;73:4707-4716. Furthermore, a wide variety of Planctomycetales were found during analysis of samples from aquatic anaerobic environments, a sulphide- and sulphur-rich spring, activated sludge wastewater treatment plants and in anaerobic digestors.88 Godon JJ, Zumstein E, Dabert P, Habouzit F. Moletta R. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol. 1997;63:2802-2813.,3838 Chouari R, Le Paslier D, Daegelen PGinestet P, Weissenbach J, Sghir A. Molecular evidence for novel planctomycete diversity in a municipal wastewater treatment plant. Appl Environ Microbiol. 2003;69:7354-7363.,3939 Elshahed MS, Youssef NH, Luo Q, et al. Phylogenetic and metabolic diversity of Planctomycetes from anaerobic, sulfide-and sulfur-rich Zodletone Spring, Oklahoma. Appl Environ Microbiol. 2007;73:4707-4716.

The Chloroflexi represented 3.2% of the digestor's bacteria. Chloroflexi have been identified from many environments through 16S rRNA gene profiling, including marine and freshwater sediments. Despite this, the Chloroflexi remain a relatively understudied bacterial lineage. At present, there are 19 complete genomes available for the Chloroflexi.4040 Hug LA, Castelle CJ, Wrighton KC, et al. Community genomic analyses constrain the distribution of metabolic traits across the Chloroflexi phylum and indicate roles in sediment carbon cycling. Microbiome. 2013;1:22.

Tang et al.4141 Tang Y,Shigematsu T, Morimura S, Kida K. Microbial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation. J Biosci Bioeng. 2005;99:150-164. showed that three phyla were involved in the mesophilic anaerobic treatment of synthetic rejection containing bovine serum albumin: Bacteroidetes, followed by Firmicutes and Proteobacteria. In another study by Fang et al.4242 Fang HHP, Liang DW, Zhang T, Liu Y.Anaerobic treatment of phenol in wastewater under thermophilic condition. Water Res. 2006;40:427-434. that evaluated the anaerobic degradation of phenol rich rejection in an upflow anaerobic sludge blanket (UASB) reactor, eight phylogenetic groups were detected, namely Thermotogales (38.9% of clones), Firmicutes (27.8%), Chloroflexi (11.1%), candidate division OP8 (9.3%), candidate division OP 5 (5.5%), Proteobacteria (3.7%), Bacteroidetes (1.9%) and Nitrospirae (1.9%). These results are comparable with the results of our study.

The Euryarchaeota, which consist mainly of methanogenic bacteria, represented 8.9% of bacteria within the anaerobic digestor. The Crenarchaeota, extreme thermoacidophiles, were also detected in the digestor with an abundance of 1.4%. The digestor seems to have limited archaeal diversity. These results are similar to the results of another study on the bacterial diversity of an anaerobic digestor fed with vinasse, which contained only 4% of Archaea Bacteria sequences.88 Godon JJ, Zumstein E, Dabert P, Habouzit F. Moletta R. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol. 1997;63:2802-2813.

Overall, there was significant bacterial diversity within the digestor. There were 23 species each consisting of greater than 1%, and the most abundant species was Kosmotoga spp. with a percentage of 9.37% (Table 3).

Table 3
The main genus and species of the digestor.

SSCP analysis of the effect of storage on the diversity and abundance of bacterial communities within the bioreactor sludge

SSCP analyses (Fig. 1) show the results of two samples of sludge collected from the same digestor at the end of the thermophilic phase.66 Gannoun H, Khelifi E, Omri I, et al. Microbial monitoring by molecular tools of an upflow anaerobic filter treating abattoir wastewaters. Bioresour Technol. 2013;142:269-277. The second sample was stored at 4 °C for two months and showed different SSCP patterns. The analysis of the two SSCP patterns showed significant change in the bacterial community over time, which can be explained by the fact the sludge is not stable over time.

Fig. 1
Effect of storage on the dynamics of single strand conformation polymorphism patterns of bacterial 16S rRNA gene amplification products of the anaerobic digestor.

The dynamics of bacterial communities were monitored by PCR-SSCP methods. The profile obtained for the domain Bacteria is shown in Fig. 1. The SSCP pattern revealed the high diversity of bacteria, with at least 48 distinguishable peaks and about 23 prominent peaks. The bacterial diversity richness (1/D) and species evenness (Es) were used as a measure of diversity and abundance. The obtained values were 38.97 (which offers toward the number of species S = 48) (indicated maximum diversity)) and 0.811 (which offers toward to 1: species in the sample are quite evenly distributed), respectively. This result is in agreement with other molecular studies based on the PCR-SSCP methods.

Several research groups confirmed our results and demonstrated that diversity in bacterial digestors varies depending on several factors. However, Zumstein et al.,4343 Zumstein E, Moletta R, Godon JJ. Examination of two years of community dynamics in an anaerobic bioreactor using fluorescence polymerase chain reaction (PCR) single-strand conformation polymorphism analysis. Environ Microbiol. 2000;2:69-78. who studied the community dynamics in an anaerobic bioreactor using fluorescence PCR single-strand conformation polymorphism analysis, indicated that throughout the period of the study, rapid significant shifts in the species composition of the bacterial community occurred. In fact, the bacterial community was followed for two years through the analysis of 13 SSCP patterns, with one SSCP pattern every two months. The analysis of the SSCP patterns showed a continuous change in the bacterial community during that time. Typically, a microorganism initially present at low levels in the community grew, peaked and decreased. Moreover, some microorganisms seemed to fluctuate simultaneously.

Keskes et al.4444 Keskes S, Hmaied F. Gannoun H, Bouallagui H, Godon JJ, Hamdi M. Performance of a submerged membrane bioreactor for the aerobic treatment of abattoir wastewater. Bioresour Technol. 2012;103:28-34. studied the effect of the prolonged sludge retention time on bacterial communities involved in the aerobic treatment of abattoir wastewater by a submerged membrane bioreactor. Their results showed that the biodiversity varied significantly in relation with the environmental conditions, particularly TSS.

The sludge microorganisms and associations of microorganisms may occupy the same ecological niche successively. They could correspond to the ecological unit called an ecotype.4545 Begon M, Harper JL, Townsend CR. Ecology:Individuals, Populations and Communities. 2nd ed. Boston, MA: Blackwell Scientific Publications; 1990:945.

Isolation and identification of bacteria

Fermentative bacteria and SRB involved in the anaerobic digestion of organic matter in abattoir effluents were investigated by two approaches: classical microbiology and molecular taxonomy.

The isolation of bacteria is preceded by an enrichment phase which favors the growth of a given microorganism (fermentative bacteria or SRB), selected according to the physical and nutritional conditions of the medium category. For this purpose, two different culture media were used, as described in the Materials and Methods section. The first culture medium was specially designed for the detection of fermentative bacteria and the second for SRB. Both culture media were tested at 37 °C and 55 °C. Glucose was used as an energy source to isolate fermentative bacteria, whereas lactate, acetate and H2CO2 were reserved for the isolation of SRB.

Isolation and culture of bacteria allowed us to study the biodiversity of these bioreactors as well as highlighted the dominant bacteria in the different conditions tested. The isolation of the different strains was carried out on an agar medium. This step helped to isolate strains that are identified based on their morphological differences (size, shape, and color).

Following this step, molecular analysis of the isolated strains was performed. After extraction of the bacterial DNA, the 16S rDNA was amplified by PCR. The quality of the amplified DNA was visualised with ultra-violet light after migration on gel agarose at 1%. The PCR product was subsequently sequenced to identify the different strains. The genes coding for 16S rRNA have been chosen as taxonomic markers, as they have a universal distribution and conserved function. These genes possess both highly conserved areas, providing information on the evolution of distant species, and variable areas, to differentiate organisms belonging to the same genus, and eventually the same species.4646 Woese CR. Bacterial evolution. Microbiol Rev. 1987;51:221-271.

Each microorganism was then identified by comparing the 16S rDNA sequences with those of known microorganisms, and each was cataloged in computer databases. Given the large number of isolated bacteria in recent underwent restriction analysis by ARDRA technique to eliminate identical strains restriction profiles to keep only the amplifias of different strains priori which will be the object of phylogenetic studies. Phylogenetic affiliations of the 16S rRNA of bacteria sequences are presented in Table 4 with the closest relatives of isolated mesophilic and thermophilic fermentative bacteria and SRB.

Table 4
Phylogenetic affiliation of the 16S rRNA bacteria sequences of isolated strains.

Fermentative bacterial communities in the bioreactor sludge

Strains LINBA, LINBL1, LINBA2 are closest phylogenetically to Clostridium sp. D3RC-2 with a percentage sequence similarity of 99%. This bacterium was firstly detected in the rumen of a yak in China4747 Zhang K. Clostridium sp. from yak rumen in China. Unpublished. 2006.,4848 Zhang K, Song L, Dong X. Proteiniclasticum ruminis gen. nov., sp. nov., a strictly anaerobic proteolytic bacterium isolated from yak rumen. Int J Syst Evol Microbiol. 2010;60(Pt 9):2221-2225. but it is not yet described. The strain LIND8A shares 96% of sequences with Clostridium sp. D3RC-2. This strain seems to be a new species and differs from the latter. The strain LIND8L2 has also been recently affiliated with Clostridia species. LIND8L2 is a strain similar to Clostridium novyi with 96% sequence similarity. C. novyi is a pathogenic bacterium phylogenetically close to C. botulinum and C. haemolyticum.4949 Sasaki Y,Takikawa N, Kojima A, Norimatsu M, Suzuki S, Tamura Y.Phylogenetic positions of Clostridium novyi and Clostridium haemolyticum based on 16S rDNA sequences. Int J Syst Evol Microbiol. 2001;51(Pt 3):901-904. The nearest strain to LIND8A is Clostridium sp 13A1, previously isolated from an anaerobic bioreactor treating cellulose wastes,5050 Burrell PC. The isolation of novel anaerobic cellulolytic bacteria from a landfill leachate bioreactor. Unpublished. 2004. with 99% sequence similarity. The closest phylogenetic relatives of the strain LIND7HT are Parabacteroides merdae,5151 Johnson JL, Moore WEC, Moore LVH. Bacteroides caccae sp. nov., Bacteroides merdae sp. nov., and Bacteroides stercoris sp. nov. isolated from human feces. Int J Syst Bacteriol. 1986;36:499-501.P. goldsteinii5252 Song Y,Liu C, Lee J, Bolanos M, Vaisanen ML, Finegold SM. Bacteroides goldsteinii sp. nov. isolated from clinical specimens of human intestinal origin. J Clin Microbiol. 2005;43:4522-4527.,5353 Sakamoto M, Benno Y.Reclassification of Bacteroides distasonis, Bacteroides goldsteinii and Bacteroides merdae as Parabacteroides distasonis gen. nov., comb. nov., Parabacteroides goldsteinii comb. nov. and Parabacteroides merdae comb. nov. Int J Syst Evol Microbiol. 2006;56(Pt 7):1599-1605. and P. gordonii,5454 Sakamoto M, Suzuki N, Matsunaga N, et al. Parabacteroides gordonii sp. nov., isolated from human blood cultures. IntJ Syst Evol Microbiol. 2009;59(Pt 11):2843-2847. with 91.4%, 91.3% and 91.2% sequence similarity, respectively. This novel strain was identified and characterised by Jabari et al.5555 Jabari L, Gannoun H, Cayol JL, et al. Characterization of Defluviitalea saccharophila gen. nov., sp. nov., a thermophilic bacterium isolated from an upflow anaerobic filter treating abattoir wastewaters, and proposal of Defluviitaleaceae fam. nov. IntJ Syst Evol Microbiol. 2012;62(Pt 3):550-555. On the basis of phylogenetic inference and phenotypic properties, LIND7HT is proposed as the type strain of a novel genus and species within the family Porphyromonadaceae, Macellibacteroides fermentans gen. nov., sp. nov.

Strains isolated in mesophilic conditions were determined to belong to Firmicutes and Bacteroidetes, which are known for their fermentative activity and which are the two groups mainly encountered in the study by Godon et al.88 Godon JJ, Zumstein E, Dabert P, Habouzit F. Moletta R. Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis. Appl Environ Microbiol. 1997;63:2802-2813. on an anaerobic digestor. They hydrolyse the polymer substrates not degraded during the stages of remediation (such as polysaccharides, proteins and lipids) to acetate, long chain fatty acids, CO2, formate and hydrogen.

The isolation of fermentative, thermophilic bacteria obtained some strains classified as “uncultured bacterium” indicating they cannot be grown on synthetic material in a laboratory. These strains are LIND6LT2, LIND8AT, LINBAT1 and LINBLT2. The strain LIND6LT2 was detected in an anaerobic digestor treating solid waste in thermophilic conditions.5656 Li T. Microbial functional groups in a thermophilic anaerobic solid waste digestor revealed by stable isotope probing. Unpublished. 2007. The closest phylogenetic relative to this strain is Parasporobacterium paucivorans with 87.17% sequence similarity.5757 Lomans BP, Leijdekkers P Wesselink JJ, et al. HJ. Obligate sulfide-dependent degradation of methoxylated aromatic compounds and formation of methanethiol and dimethyl sulfide by a freshwater sediment isolate, Parasporobacterium paucivorans gen. nov., sp. nov. Appl Environ Microbiol. 2001;67:4017-4023. This novel strain was initially identified and characterised by Jabari et al.5858 Jabari L, Gannoun H, Cayol JL, et al. Macellibacteroides fermentans gen. nov., sp. nov., a member of the family Porphyromonadaceae isolated from an upflow anaerobic filter treating abattoir wastewaters. IntJ Syst Evol Microbiol. 2012;62(Pt 10):2522-2527. On the basis of phylogenetical and physiological properties, the strain LIND6LT2T is proposed as the strain type of Defluviitalea saccharophila gen. nov., sp. nov., placed in Defluviitaleaceae fam. nov., within the phylum Firmicutes, class Clostridia, order Clostridiales. The strain LIND8AT has 97% similarity with a strain classified as uncultivable bacterium, probably involved in metal reduction.5959 Brodie EL, Desantis TZ, Joyner DC, et al. Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation. Appl Environ Microbiol. 2006;72:6288-6298.

According to the phylogenetic tree (Fig. 2), the strains LINBAT1 and LINBLT2 are phylogenetically close. These sequences were also identified within an anaerobic digestor treating pharmaceutical wastes.6060 LaPara TM, Nakatsu CH, Pantea L, Alleman JE. Phylogenetic analysis of bacterial communities in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Appl Environ Microbiol. 2000;66:3951-3959. The closest relative to strain LIND4FT1 is Caloramator coolhaasii, with 96% sequence similarity which suggests that this strain is a new species. C. coolhaasii has been isolated from an anaerobic granular sludge bioreactor that degrades glutamate. It is moderately thermophilic and strictly anaerobic.6161 Plugge CM, Zoetendal EG, Stams AJ. Caloramator coolhaasii sp. nov., a glutamate-degrading, moderately thermophilic anaerobe. Int J Syst Evol Microbiol. 2000;50(Pt 3):1155-1162. LIND8HT strain is close to Lutispora thermophile,6262 Shiratori H, Ohiwa H, Ikeno H, et al. Lutispora thermophila gen. nov., sp. nov., a thermophilic, spore-forming bacterium isolated from a thermophilic methanogenic bioreactor digesting municipal solid wastes. Int J Syst Evol Microbiol. 2008;58(Pt 4):964-969. with 99% sequence similarity which is firstly isolated from an enrichment culture derived from an anaerobic thermophilic bioreactor treating artificial solid wastes. Strains LINBLT and LINBHT2 were affiliated with C. tertium and C. thermosuccinogenes, respectively, each with 98% sequence similarity.

Fig. 2
Phylogenetic tree based on the gene encoding the 16S RNA showing the positioning of fermentative mesophilic and thermophilic bacteria isolated from the anaerobic digestor.

The mesophilic and thermophilic fermentative strains which were isolated were also designated to the group Firmicutes, which seems to be the most abundant group in our anaerobic digestor. Almost all isolates were represented by Clostridium species. This is not surprising, because during the hydrolysis phase in bioreactors, macromolecules such as polysaccharides, lipids, proteins and nucleic acids are cleaved, typically by specific extracellular enzymes, producing monomers (monosaccharides, fatty acids, amino acids and nitrogen bases) which are transported into the cell where they are fermented. The bacteria involved in this stage have a strictly anaerobic or facultative metabolism. During the acidogenesis phase, these monomers are metabolised by fermentative microorganisms to primarily produce volatile fatty acids (acetate, propionate, butyrate, isobutyrate, valerate and isovalerate), but also alcohols, sulphide (H2S), CO2 and hydrogen. Acidogenesis leads to simplified products of fermentation, and the bacteria involved in this step may be facultative or strictly anaerobic. Strictly anaerobic bacteria of the genus Clostridium are often a large fraction of the population participating in the anaerobic step of acid formation.

Fermentative bacteria were abundant, particularly the proteolytic Clostridium species. These species hydrolyse proteins to polypeptides and amino acids, while lipids are hydrolysed via oxidation to long-chain fatty acids, and glycerol and polycarbohydrates are hydrolysed to sugars and alcohols. After that, fermentative bacteria convert the intermediates to volatile fatty acids, hydrogen and CO2.66 Gannoun H, Khelifi E, Omri I, et al. Microbial monitoring by molecular tools of an upflow anaerobic filter treating abattoir wastewaters. Bioresour Technol. 2013;142:269-277.

Sulphate-reducing bacterial communities in the sludge bioreactor

Sulphate-reducing bacteria were isolated in mesophilic and thermophilic conditions on various substrates. In anaerobic digestion, the acidogenesis products are converted into acetate and hydrogen in the acetogenesis phase. The hydrogen is normally used by the microbial community's methanogenic hydrogenophiles to reduce CO2 to methane (CH4) while acetate is converted by methanogenic acetoclastes to CH4.

The presence of sulphate in the medium may change the flow of the substrate available for methanogens. In fact, the SRB may oxidise a portion of the substrate (mainly via the hydrogen) using sulphate as an electron acceptor. In such situations, the substrate is converted to sulphur if the pH of the medium is acidic. These methanogenic bacteria can therefore compete with other bacterial groups such as sulphate-reducing bacteria.6363 Garcia JL, Patel BK, Ollivier B. Taxonomic, phylogenetic, and ecological diversity of methanogenic Archaea. Anaerobe. 2000;6:205-226. The SRB may also be involved in the hydrolysis6464 McInerney MJ. Anaerobic hydrolis and fermentatio of fats and proteins. In: Zehnde AJ, ed. Biology of Anaerobic Microorganisms. New York, USA: John Wiley and Sons, Inc.; 1988:373-415. and acetogenesis steps.6565 DolfingJ. Acetogenesis. In: Zehnder AJB, ed. Biology of anaerobic microorganisms. New York, USA: John Wiley and Sons, Inc.; 1988:417-468. In addition, the SRB are known to play a key role in the biodegradation of a number of environmental pollutants.6666 Ensley BD, Suflita JM. Metabolism of environmental contaminants by mixed and pure cultures of sulfate reducing bacteria. In: Barton LL, ed. Sulfate Reducing Bacteria. vol. 8. New York, USA: Peplum Press; 1995:293-332.

The strain LINDBL, isolated at 37 °C (Table 4) in the presence of 20 mM lactate, is most closely related to Desulfobulbus propionicus with a 99% sequence similarity. D. propionicus was first isolated from a fluidised bed bioreactor treating effluent containing acid and metals.6767 Kaksonen AH. Diversity and community fatty acid profiling of sulfate-reducing fluidized-bed reactors treating acidic, metal-containing wastewater. Geomicrobiol J. 2004;21: 469-480. Strains LINDBH and LINDBH2 were affiliated phylogenetically to Desulfovibrio vulgaris with 99% sequence similarity. Both strains were isolated at 37 °C on basal medium supplemented with H2CO2 (2 bars) as a substrate. D. vulgaris is used as a model for the study of SRB to analyse the mechanisms of metal corrosion and to treat toxic metal ions from the environment.6868 Heidelberg JF, Seshadri R, Haveman SA, et al. The genome sequence of the anaerobic, sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough. Nat Biotechnol. 2004;22:554-559.

LINDBA1 and LINDBHT1 are two mesophilic strains that were isolated on basal medium for SRB using two different substrates, the first one in the presence of 20 mM acetate and the second one in the presence of H2CO2 (at 2 bars). These strains have Desulfomicrobium baculatum as their closest relative, with 99% sequence similarity. Phylogenetic analysis demonstrated that the strain LINDBHT1T belonged within the genus Desulfotomaculum. This strain (LINDBHT1T) had Desulfotomaculum halophilum6969 Tardy-Jacquenod C, Magot M, Patel BKC, Matheron R, Caumette P. Desulfotomaculum halophilum sp. nov., a halophilic sulfate-reducing bacterium isolated from oil production facilities. Int J Syst Bacteriol. 1998;48(Pt 2):333-338. and Desulfotomaculum alkaliphilum7070 Pikuta E, Lysenko A, Suzina N, et al. Desulfotomaculum alkaliphilum sp. nov., a new alkaliphilic, moderately thermophilic, sulfate-reducing bacterium. Int J Syst Evol Microbiol. 2000;50(Pt 1):25-33. as its closest phylogenetic relatives with approximately 89% sequence similarity. LINDBHT1T is a novel anaerobic thermophilic sulphate-reducing bacterium. This bacterium constitutes a new species of the genus Desulfotomaculum, D. peckii7171 Jabari L, Gannoun H, Cayol JL, et al. Desulfotomaculum peckii sp. nov., a moderately thermophilic member of the genus Desulfotomaculum, isolated from an upflow anaerobic filter treating abattoir wastewaters. IntJ Syst Evol Microbiol. 2013;63(Pt 6):2082-2087. (Fig. 3).

Fig. 3
Phylogenetic tree based on the gene encoding the 16S RNA showing the positioning of SRB isolated from the anaerobic digestor.

Various SRB were isolated from the anaerobic digestor which shows that they are involved in the degradation process. In order to gain an overall idea of the cultivable bacterial diversity of the digestor, we grouped all of the bacteria isolated on to the same phylogenetic tree (Fig. 4).

Fig. 4
Phylogenetic tree based on the 16S RNA gene of bacteria isolated from digestor treating abattoir wastewaters.

Analysis of the microbial populations obtained from the anaerobic sludge samples in both mesophilic and thermophilic conditions led to the isolation of many morphologically distinct bacteria. Molecular and microbial analyses showed that fermentative bacteria (primarily Clostridium spp. and Parabacteroides spp.), Desulfobulbus spp., Desulfomicrobium spp., Desulfovibrio ssp. and Desulfotomaculum ssp. were the prominent members of the bacterial community in the bioreactor (Fig. 4). The diversity of the microbial community within the digestor may reflect the metabolic diversity of microorganisms involved in anaerobic digestion. The interactions of this complex microbial community allows for complete degradation of natural polymers such as polysaccharides, proteins, lipids and nucleic acids into methane and carbon dioxide.

Conclusion

In conclusion, the use of both bacterial culture and molecular techniques enabled us to establish a picture of the existing microbial biodiversity in an anaerobic digestor. The culture approach was essential, especially with regard to culture and/or isolation of microorganisms with no known cultivable representative. Further research in this area can only improve our knowledge of microbial anaerobic digestors, including the role of different microbial populations involved in anaerobic degradation of waste, which will improve control of these treatment processes. A comprehensive molecular inventory would also support and complement our study of the microbial diversity of anaerobic cultures as it would link information on the diversity and function of microbial communities in their environment.

  • Associate Editor: Welington Luiz de Araújo

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Publication Dates

  • Publication in this collection
    Jan-Mar 2016

History

  • Received
    19 Sept 2014
  • Accepted
    6 July 2015
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