Comparative metagenomics of three Dehalococcoides-containing enrichment cultures: the role of the non-dechlorinating community

ABSTRACT: BACKGROUND: The Dehalococcoides are strictly anaerobic bacteria that gain metabolic energy via the oxidation of H2 coupled to the reduction of halogenated organic compounds. Dehalococcoides spp. grow best in mixed microbial consortia, relying on non-dechlorinating members to provide essential nutrients and maintain anaerobic conditions. A metagenome sequence was generated for the dechlorinating mixed microbial consortium KB-1. A comparative metagenomic study utilizing two additional metagenome sequences for Dehalococcoides-containing dechlorinating microbial consortia was undertaken to identify common features that are provided by the non-dechlorinating community and are potentially essential to Dehalococcoides growth. RESULTS: The KB-1 metagenome contained eighteen novel homologs to reductive dehalogenase genes. The metagenomes obtained from the three consortia were automatically annotated using the MG-RAST server, from which statistically significant differences in community composition and metabolic profiles were determined. Examination of specific metabolic pathways, including corrinoid synthesis, methionine synthesis, oxygen scavenging, and electron-donor metabolism identified the Firmicutes, methanogenic Archaea, and the delta-Proteobacteria as key organisms encoding these pathways, and thus potentially producing metabolites required for Dehalococcoides growth. CONCLUSIONS: Comparative metagenomics of the three Dehalococcoides-containing consortia identified that similarities across the three consortia are more apparent at the functional level than at the taxonomic level, indicating the non-dechlorinating organisms' identities can vary provided they fill the same niche within a consortium. Functional redundancy was identified in each metabolic pathway of interest, with key processes encoded by multiple taxonomic groups. This redundancy likely contributes to the robust growth and dechlorination rates in dechlorinating enrichment cultures.     

Biotechnological potential of microbial consortia and future perspectives

Design of a microbial consortium is a newly emerging field that enables researchers to extend the frontiers of biotechnology from a pure culture to mixed cultures. A microbial consortium enables microbes to use a broad range of carbon sources. It provides microbes with robustness in response to environmental stress factors. Microbes in a consortium can perform complex functions that are impossible for a single organism. With advancement of technology, it is now possible to understand microbial interaction mechanism and construct consortia. Microbial consortia can be classified in terms of their construction, modes of interaction, and functions. Here we discuss different trends in the study of microbial functions and interactions, including single-cell genomics (SCG), microfluidics, fluorescent imaging, and membrane separation. Community profile studies using polymerase chain-reaction denaturing gradient gel electrophoresis (PCR-DGGE), amplified ribosomal DNA restriction analysis (ARDRA), and terminal restriction fragment-length polymorphism (T-RFLP) are also reviewed. We also provide a few examples of their possible applications in areas of biopolymers, bioenergy, biochemicals, and bioremediation.     

Pesticide relevance and their microbial degradation: a-state-of-art

The extensive use of pesticide causes imbalance in properties of soil, water and air environments due to having problem of natural degradation. Such chemicals create diverse environmental problem via biomagnifications. Currently, microbial degradation is one of the important techniques for amputation and degradation of pesticide from agricultural soils. Some studies have reported that the genetically modified microorganism has ability to degrade specific pesticide but problem is that they cannot introduce in the field because they cause some other environmental problems. Only combined microbial consortia of indigenous and naturally occurring microbes isolated from particular contaminated environment have ability to degrade pesticides at faster rate. The bioaugumentation processes like addition of necessary nutrients or organic matter are required to speed up the rate of degradation of a contaminant by the indigenous microbes. The use of indigenous microbial strains having plant growth activities is ecologically superior over the chemical methods. In this review, we have attempted to discuss the recent challenge of pesticide problem in soil environment and their biodegradation with the help of effective indigenous pesticides degrading microorganisms. Further, we highlighted and explored the molecular mechanism for the pesticide degradation in soil with effective indigenous microbial consortium. This review suggests that the use of pesticide degrading microbial consortia which is an eco-friendly technology may be suitable for the sustainable agriculture production.     

Isolation of Paenibacillus sp. and Variovorax sp. strains from decaying woods and characterization of their potential for cellulose deconstruction

Prospection of cellulose-degrading bacteria in natural environments allows the identification of novel cellulases and hemicellulases that could be useful in second-generation bioethanol production. In this work, cellulolytic bacteria were isolated from decaying native forest soils by enrichment on cellulose as sole carbon source. There was a predominance of Gram positive isolates that belonged to the phyla Proteobacteria and Firmicutes. Many primary isolates with cellulolytic activity were not pure cultures. From these consortia, isolation of pure constituents was attempted in order to test the hypothesis whether microbial consortia are needed for full degradation of complex substrates. Two isolates, CB1-2-A-5 and VG-4-A-2, were obtained as the pure constituents of CB1-2 and VG-4 consortia, respectively. Based on 16S RNA sequence, they could be classified as Variovorax paradoxus and Paenibacillus alvei. Noteworthy, only VG-4 consortium showed measurable xylan degrading capacity and signs of filter paper degradation. However, no xylan or filter paper degrading capacities were observed for the pure cultures isolated from it, suggesting that other members of this consortium were necessary for these hydrolyzing activities. Our results indicated that Paenibacillus sp. and Variovorax sp. as well as VG-4 consortium, might be a useful source of hydrolytic enzymes. Moreover, although Variovorax sp. had been previously identified in metagenomic studies of cellulolytic communities, this is the first report on the isolation and characterization of this microorganism as a cellulolytic genus.     

Self-organization, layered structure, and aggregation enhance persistence of a synthetic biofilm consortium

Microbial consortia constitute a majority of the earth's biomass, but little is known about how these cooperating communities persist despite competition among community members. Theory suggests that non-random spatial structures contribute to the persistence of mixed communities; when particular structures form, they may provide associated community members with a growth advantage over unassociated members. If true, this has implications for the rise and persistence of multi-cellular organisms. However, this theory is difficult to study because we rarely observe initial instances of non-random physical structure in natural populations. Using two engineered strains of Escherichia coli that constitute a synthetic symbiotic microbial consortium, we fortuitously observed such spatial self-organization. This consortium forms a biofilm and, after several days, adopts a defined layered structure that is associated with two unexpected, measurable growth advantages. First, the consortium cannot successfully colonize a new, downstream environment until it self-organizes in the initial environment; in other words, the structure enhances the ability of the consortium to survive environmental disruptions. Second, when the layered structure forms in downstream environments the consortium accumulates significantly more biomass than it did in the initial environment; in other words, the structure enhances the global productivity of the consortium. We also observed that the layered structure only assembles in downstream environments that are colonized by aggregates from a previous, structured community. These results demonstrate roles for self-organization and aggregation in persistence of multi-cellular communities, and also illustrate a role for the techniques of synthetic biology in elucidating fundamental biological principles.     

Molecular biology-based methods for quantification of bacteria in mixed culture: perspectives and limitations

Species-specific enumeration of mixed community is invaluable as it facilitates a better understanding of the significance of the individual strains, their interactions, and the underlying mechanisms of community dynamics. Mixed microbial community has been characterized by microbiological, biochemical, or molecular biology-based methods. While microbiological and biochemical techniques do not provide adequate quantitative information of the members of the consortia and require additional techniques for a more comprehensive analysis, molecular biology-based methods analyze the microbial consortium based on specific DNA sequences and do not require isolation and culturing of bacteria for quantitative analysis. These methods outshine conventional culture-based techniques in terms of better sensitivity, reproducibility, and reliability. Quantitative molecular biology methods have been classified as PCR-based and probe hybridization methods. The PCR-based methods includes quantitative real-time PCR and terminal restriction fragment length polymorphism, while fluorescent in situ hybridization and DNA microarrays fall under probe hybridization methods. The workflow, the quantification methods, and their potential applications are discussed in this review by highlighting their advantages and possible limitations.     

The microbial degradation of azimsulfuron and its effect on the soil bacterial community

AIMS: Azimsulfuron is a recently introduced sulfonylurea herbicide useful in controlling weeds in paddy fields. To date very little information is available on the biodegradation of this pesticide and on its effect on the soil microbial community. The aim of this work was to study its biodegradation both in slurry soil microcosms and in batch tests with mixed and pure cultures. METHODS AND RESULTS: Azimsulfuron was applied to forest bulk soil in order to study its effect on the structure of the bacterial soil community, as detectable by denaturant gradient gel electrophoresis (DGGE) analyses. Biodegradation and abiotic processes were investigated by HPLC analyses. In addition, a microbial consortium was selected, that was able to use azimsulfuron as the sole energy and carbon source. One of the metabolites produced by the consortium was isolated and identified through LC-MS analyses. Cultivable bacteria of the consortium were isolated and identified by 16S rDNA sequencing (1400 bp). CONCLUSIONS: Azimsulfuron treatment seems to have the ability to cause changes in the bacterial community structure that are detectable by DGGE analyses. It is easily biodegraded both in microcosms and in batch tests, with the formation of an intermediate that was identified as 2-methyl-4-(2-methyl-2H-tetrazol-5-yl)-2H-pyrazole-3-sulfonamide. SIGNIFICANCE AND IMPACT OF THE STUDY: The study increases the knowledge on the biodegradation of azimsulfuron and its effects on the soil microbiota.     

Studies on composition and stability of a large membered bacterial consortium degrading phenol

A ten member microbial consortium (AS) consisting of eight phenol-degrading and two non-phenol-degrading strains of bacteria was developed and maintained in a fed-batch reactor by feeding 500 mg l⁻¹ phenol for four years at 28 ± 3 °C. The consortium could degrade 99% of 500 mg l⁻¹ phenol after 24 hours incubation with a biomass increase of 2.6 × 10⁷ to 4 × 10¹² CFU ml⁻¹. Characterization of the members revealed that it consisted of 4 principal genera, Bacillus, Pseudomonas, Rhodococcus, Streptomyces and an unidentified bacterium. Phenol degradation by the mixed culture and Bacillus subtilis, an isolate from the consortium was compared using a range of phenol concentrations (400 to 700 mg l⁻¹) and by mixing with either 160 mg l⁻¹ glucose or 50 mg l⁻¹ of 2,4-dichlorophenol in the medium. Simultaneous utilization of unrelated mixed substrates (glucose/2,4-dichlorophenol) by the consortium and Bacillus subtilis, indicated the diauxic growth pattern of the organisms. A unique characteristic of the members of the consortia was their ability to oxidize chloro aromatic compounds via meta pathway and methyl aromatic compounds via ortho cleavage pathway. The ability of a large membered microbial consortia to maintain its stability with respect to its composition and effectiveness in phenol degradation indicated its suitability for bioremediation applications.     

Genomic and mechanistic insights into the biodegradation of organic pollutants

Several new methodologies have enabled recent studies on the microbial biodegradation mechanisms of organic pollutants. Culture-independent techniques for analysis of the genetic and metabolic potential of natural and model microbial communities that degrade organic pollutants have identified new metabolic pathways and enzymes for aerobic and anaerobic degradation. Furthermore, structural studies of the enzymes involved have revealed the specificities and activities of key catabolic enzymes, such as dioxygenases. Genome sequencing of several biodegradation-relevant microorganisms have provided the first whole-genome insights into the genetic background of the metabolic capability and biodegradation versatility of these organisms. Systems biology approaches are still in their infancy, but are becoming increasingly helpful to unravel, predict and quantify metabolic abilities within particular organisms or microbial consortia.     

Growth of Chlorinated Solvent‐Degrading Consortia in Fed‐Batch Bioreactors and Development of a Double‐Substrate High‐Performing Microbial Inoculum

The long‐term growth process of two microbial consortia effective in the aerobic cometabolic biodegradation of a mixture of 6‐chlorinated aliphatic hydrocarbons (CAHs), the effectiveness of these consortia as inocula for the bio‐augmentation of different types of microcosms and the development of a double‐substrate, high‐performing consortium is presented. The propane‐utilizing consortium generally proved to be the most effective one, being able to biodegrade vinyl chloride, cis‐ and trans‐1,2‐dichloroethylene, trichloroethylene, 1,1,2‐trichloroethane and 1,1,2,2‐tetrachloroethane at all the CAH concentrations tested (0–4 μM). Both consortia maintained unaltered CAH degradation capacities over a 300‐day growth period in the absence of the CAHs and were effective in inducing the rapid onset of CAH depletion upon inoculation in slurry microcosms set up with five types of aquifer materials. A consortium supplied with both methane and propane combined the best degradation capacities of the two single‐substrate consortia, and maintained stable performances for 150 days under slurry conditions. The degree of conversion of the organic Cl to chloride ions was equal to 90 %.     

Degradation of Di- Through Hepta-Chlorobiphenyls in Clophen Oil Using Microorganisms Isolated from Long Term PCBs Contaminated Soil

Present work describes microbial degradation of selected polychlorinated biphenyls (PCBs) congeners in Clophen oil which is used as transformer oil and contains high concentration of PCBs. Indigenous PCBs degrading bacteria were isolated from Clophen oil contaminated soil using enrichment culture technique. A 15 days study was carried out to assess the biodegradation potential of two bacterial cultures and their consortium for Clophen oil with a final PCBs concentration of 100 mg kg⁻¹. The degradation capability of the individual bacterium and the consortium towards the varying range of PCBs congeners (di- through hepta-chlorobiphenyls) was determined using GCMS. Also, dehydrogenase enzyme was estimated to assess the microbial activity. Maximum degradation was observed in treatment containing consortium that resulted in up to 97 % degradation of PCB-44 which is a tetra chlorinated biphenyl whereas, hexa chlorinated biphenyl congener (PCB-153) was degraded up to 90 % by the consortium. This indicates that the degradation capability of microbial consortium was significantly higher than that of individual cultures. Furthermore, the results suggest that for degradation of lower as well as higher chlorinated PCB congeners; a microbial consortium is required rather than individual cultures.     

Aerobic MTBE biodegradation in the presence of BTEX by two consortia under batch and semi-batch conditions

This study explores the effect of microbial consortium composition and reactor configuration on methyl tert-butyl ether (MTBE) biodegradation in the presence of benzene, toluene, ethylbenzene and p-xylenes(BTEX). MTBE biodegradation was monitored in the presence and absence of BTEX in duplicate batch reactors inoculated with distinct enrichment cultures: MTBE only (MO—originally enriched on MTBE) and/or MTBE BTEX (MB—originally enriched on MTBE and BTEX). The MO culture was also applied in a semi-batch reactor which received both MTBE and BTEX periodically in fresh medium after allowing cells to settle. The composition of the microbial consortia was explored using a combination of 16S rRNA gene cloning and quantitative polymerase chain reaction targeting the known MTBE-degrading strain PM1^T. MTBE biodegradation was completely inhibited by BTEX in the batch reactors inoculated with the MB culture, and severely retarded in those inoculated with the MO culture (0.18 ± 0.04 mg/L-day). In the semi-batch reactor, however, the MTBE biodegradation rate in the presence of BTEX was almost three times as high as in the batch reactors (0.48 ± 0.2 mg/L-day), but still slower than MTBE biodegradation in the absence of BTEX in the MO-inoculated batch reactors (1.47 ± 0.47 mg/L-day). A long lag phase in MTBE biodegradation was observed in batch reactors inoculated with the MB culture (20 days), but the ultimate rate was comparable to the MO culture (0.95 ± 0.44 mg/L-day). Analysis of the cultures revealed that strain PM1^T concentrations were lower in cultures that successfully biodegraded MTBE in the presence of BTEX. Also, other MTBE degraders, such as Leptothrix sp. and Hydrogenophaga sp. were found in these cultures. These results demonstrate that MTBE bioremediation in the presence of BTEX is feasible, and that culture composition and reactor configuration are key factors.     

Chlorinated biphenyl mineralization by individual populations and consortia of freshwater bacteria

Comparative studies were performed to investigate the contribution of microbial consortia, individual microbial populations, and specific plasmids to chlorinated biphenyl biodegradation among microbial communities from a polychlorinated biphenyl-contaminated freshwater environment. A bacterial consortium, designated LPS10, was shown to mineralize 4-chlorobiphenyl (4CB) and dehalogenate 4,4'-dichlorobiphenyl. The LPS10 consortium involved three isolates: Pseudomonas testosteroni (LPS10A), which mediated the breakdown of 4CB and 4,4'-dichlorobiphenyl to 4-chlorobenzoic acid; an isolate tentatively identified as an Arthrobacter sp. (LPS10B), which mediated 4-chlorobenzoic acid degradation; and Pseudomonas putida bv. A (LPS10C), whose role in the consortium has not been determined. None of these isolates contained detectable plasmids or sequences homologous to the 4CB-degradative plasmid pSS50. A freshwater isolate, designated LBS1C1, was found to harbor a 41-megadalton plasmid that was related to the 35-megadalton plasmid pSS50, and this isolate was shown to mineralize 4CB. In chemostat enrichments with biphenyl and 4CB as primary carbon sources, the LPS10 consortium was found to outcomplete bacterial populations harboring plasmids homologous to pSS50. These results demonstrate that an understanding of the biodegradative capacity of individual bacterial populations as well as interacting populations of bacteria must be considered in order to gain a better understanding of polychlorinated biphenyl biodegradation in the environment.     

Chlorinated biphenyl mineralization by individual populations and consortia of freshwater bacteria.

Comparative studies were performed to investigate the contribution of microbial consortia, individual microbial populations, and specific plasmids to chlorinated biphenyl biodegradation among microbial communities from a polychlorinated biphenyl-contaminated freshwater environment. A bacterial consortium, designated LPS10, was shown to mineralize 4-chlorobiphenyl (4CB) and dehalogenate 4,4'-dichlorobiphenyl. The LPS10 consortium involved three isolates: Pseudomonas testosteroni (LPS10A), which mediated the breakdown of 4CB and 4,4'-dichlorobiphenyl to 4-chlorobenzoic acid; an isolate tentatively identified as an Arthrobacter sp. (LPS10B), which mediated 4-chlorobenzoic acid degradation; and Pseudomonas putida bv. A (LPS10C), whose role in the consortium has not been determined. None of these isolates contained detectable plasmids or sequences homologous to the 4CB-degradative plasmid pSS50. A freshwater isolate, designated LBS1C1, was found to harbor a 41-megadalton plasmid that was related to the 35-megadalton plasmid pSS50, and this isolate was shown to mineralize 4CB. In chemostat enrichments with biphenyl and 4CB as primary carbon sources, the LPS10 consortium was found to outcomplete bacterial populations harboring plasmids homologous to pSS50. These results demonstrate that an understanding of the biodegradative capacity of individual bacterial populations as well as interacting populations of bacteria must be considered in order to gain a better understanding of polychlorinated biphenyl biodegradation in the environment.     

The microbial degradation of benzothiazoles

The biodegradation of benzothiazoles by pure and mixed microbial cultures derived from activated sludge has been studied. The degradation of 2-aminobenzothiazole (ABT) by both pure and mixed bacterial cultures has been demonstrated for the first time. ABT is degraded to give high yields of ammonia and sulphate (87 and 100 %, respectively of the theoretical yield). We also report for the first time the isolation of a pure bacterial culture PA, thought to be a strain of Rhodococcus , capable of growing on benzothiazole (BTH) itself as a sole carbon, nitrogen and energy source. Evidence is presented to suggest that this organism degrades BTH via the meta-cleavage pathway. The Rhodococcus PA degrades BTH but only releases a small proportion (5%) of the sulphur as sulphate. Mixed cultures containing this organism released ca 100% of the sulphur as sulphate, suggesting that other members of the consortium catalyse conversion of a sulphur-containing intermediate to sulphate. 2-Mercaptobenzothiazole (MBT) could not act as a growth substrate for any of the cultures studied but some could cause biotransformation of MBT to some extent. Attempts to obtain cultures degrading ABT and BTH from polluted river water were unsuccessful.     

Effect of Fenton reagent shock and recovery periods on anaerobic microbial community structure and degradation of chlorinated aliphatics

This study investigates the effect of Fenton reagent on the structure and function of a microbial consortium during the anaerobic degradation of hexachloroethane (HCA) and tetrachloroethene (PCE). Anaerobic biodegradation tests of HCA and PCE were performed in batch reactors using an anaerobic microbial consortium that had been exposed to Fenton reagent for durations of 0, 0.04, and 2 days and then allowed to recover for periods of 0, 3, and 7 days. The bacterial community structure was determined using culture-independent methods of 16S rRNA gene sequencing and automated ribosomal intergenic spacer analysis. Larger recovery periods partially restored the microbial community structure; however, the recovery periods did not restore the loss of ability to degrade HCA and PCE in cultures shocked for 0.04 days, and PCE in cultures shocked for 2 days. Overall the exposure to Fenton reagent had an impact on bacterial community structure with downstream effects on HCA and PCE degradation. This study highlights that the impacts of short- and long-term shocks on microbial community structure and function can be correlated using a combination of biodegradation tests and community structure analysis tools.     

Microbial degradation of the biocide polyhexamethylene biguanide: isolation and characterization of enrichment consortia and determination of degradation by measurement of stable isotope incorporation into DNA

AIMS: To isolate micro-organisms capable of utilizing polyhexamethylene biguanide (PHMB) as a sole source of nitrogen, and to demonstrate biodegradation of the biocide. METHODS AND RESULTS: Two consortia of bacteria were successfully enriched at the expense of PHMB, using sand from PHMB-treated swimming pools as inoculum. Both consortia were shown to contain bacteria belonging to the genera Sphingomonas, Azospirillum and Mesorhizobium. It was shown that the presence of both Sphingomonas and Azospirillum spp. was required for extensive growth of the consortia. In addition, the Sphingomonads were the only isolates capable of growth in axenic cultures dosed with PHMB. Using a stable isotope (15N)-labelled PHMB, metabolism of the biocide by both consortia was demonstrated. By comparing the level of 15N atom incorporation into bacterial DNA after growth on either 15N-PHMB or 15N-labelled NH4Cl, it was possible to estimate the percentage of PHMB biodegradation. CONCLUSIONS: The microbial metabolism of nitrogen from the biguanide moiety of PHMB has been demonstrated. It was revealed that Sphingomonas and Azospirillum spp. are the principal organisms responsible for growth at the expense of PHMB. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate the microbial metabolism of PHMB.     

Stimulation of Diesel Fuel Biodegradation by Indigenous Nitrogen Fixing Bacterial Consortia

Abstract Successful stimulation of N₂ fixation and petroleum hydrocarbon degradation in indigenous microbial consortia may decrease exogenous N requirements and reduce environmental impacts of bioremediation following petroleum pollution. This study explored the biodegradation of petroleum pollution by indigenous N₂ fixing marine microbial consortia. Particulate organic carbon (POC) in the form of ground, sterile corn-slash (post-harvest leaves and stems) was added to diesel fuel amended coastal water samples to stimulate biodegradation of petroleum hydrocarbons by native microorganisms capable of supplying a portion of their own N. It was hypothesized that addition of POC to petroleum amended water samples from N-limited coastal waters would promote the growth of N₂ fixing consortia and enhance biodegradation of petroleum. Manipulative experiments were conducted using samples from coastal waters (marinas and less polluted control site) to determine the effects of POC amendment on biodegradation of petroleum pollution by native microbial consortia. Structure and function of the microbial consortia were determined by measurement of N₂ fixation (acetylene reduction), hydrocarbon biodegradation (¹⁴C hexadecane mineralization), bacterial biomass (AODC), number of hydrocarbon degrading bacteria (MPN), and bacterial productivity (³H-thymidine incorporation). Throughout this study there was a consistent enhancement of petroleum hydrocarbon degradation in response to the addition of POC. Stimulation of diesel fuel biodegradation following the addition of POC was likely attributable to increases in bacterial N₂ fixation, diesel fuel bioavailability, bacterial biomass, and metabolic activity. Toxicity of the bulk phase water did not appear to be a factor affecting biodegradation of diesel fuel following POC addition. These results indicate that the addition of POC to diesel-fuel-polluted systems stimulated indigenous N₂ fixing microbial consortia to degrade petroleum hydrocarbons. Received: 29 December 1998; Accepted: 6 April 1999