Absence of microbial mineralization of lignin in anaerobic enrichment cultures

The existence of anaerobic biodegradation of lignin was examined in mixed microflora. Egyptian soil samples, in which rapid mineralization of organic matter takes place in the presence of an important anaerobic microflora, were used to obtain the anaerobic enrichment cultures for this study. Specifically, 14CO2 or [14C]lignin wood was used to investigate the release of labeled gaseous or soluble degradation products of lignin in microbial cultures. No conversion of 14C-labeled lignin to 14CO2 or 14CH4 was observed after 6 months of incubation at 30 degrees C in anaerobic conditions with or without NO3-. A small increase in soluble radioactivity was observed in certain cultures, but it could not be related to the release of catabolic products during the anaerobic biodegradation of lignin.     

Characteristics of anaerobic oxalate-degrading enrichment cultures from the rumen.

Enrichment cultures of rumen bacteria degraded oxalate within 3 to 7 days in a medium containing 10% rumen fluid and an initial level of 45 mM sodium oxalate. This capability was maintained in serially transferred cultures. One mole of methane was produced per 3.8 mol of oxalate degraded. Molecular hydrogen and formate inhibited oxalate degradation but not methanogenesis; benzyl viologen and chloroform inhibited both oxalate degradation and methanogenesis. Attempts to isolate oxalate-degrading bacteria from these cultures were not successful. Oxalate degradation was uncoupled from methane production when enrichments were grown in continuous culture at dilution rates greater than or equal to 0.078 h-1. Growth of the uncoupled population (lacking methanogens) in batch culture was accompanied by degradation of 45 mM oxalate within 24 h and production of 0.93 mol of formate per mol of oxalate degraded. Oxalate degradation by the uncoupled population was not inhibited by molecular hydrogen or formate. Cell yields (grams [dry weight]) per mole of oxalate degraded by the primary enrichment and the uncoupled populations were 1.7 and 1.0, respectively.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.     

Metabolic by-products of anaerobic toluene degradation by sulfate-reducing enrichment cultures.

Two dead-end metabolites of anaerobic toluene transformation, benzylsuccinic acid and benzylfumaric acid, accumulated in sulfate-reducing enrichment cultures that were fed toluene as the sole carbon source. Stable isotope-labeled toluene and gas chromatography-mass spectrometry were used to confirm that the compounds resulted from toluene metabolism. The two metabolites constituted less than 10% of the toluene carbon (over 80% was mineralized to carbon dioxide, according to a previous study). This study demonstrates that the novel nonproductive pathway proposed by Evans and coworkers (P. J. Evans, W. Ling, B. Goldschmidt, E. R. Ritter, and L. Y. Young, Appl. Environ. Microbiol. 58:496-501, 1992) for a denitrifying pure culture applies to disparate anaerobic bacteria.     

Microbial dynamics in anaerobic enrichment cultures degrading di-n-butyl phthalic acid ester

Although anaerobic biodegradation of di-n-butyl phthalic acid ester (DBP) has been studied over the past decade, only little is known about the microorganisms involved in the biological anaerobic degradation pathways. The aim of this work is to characterize the microbial community dynamics in enrichment cultures degrading phthalic acid esters under methanogenic conditions. A selection pressure was applied by adding DBP at 10 and 200 mg L(-1) in semi-continuous anaerobic reactors. The microbial dynamics were monitored using single strand conformation polymorphism (SSCP). While only limited abiotic losses were observed in the sterile controls (20-22%), substantial DBP biodegradation was found in the enrichment cultures (90-99%). In addition, significant population changes were observed. The dominant bacterial species in the DBP-degrading cultures was affiliated to Soehngenia saccharolytica, a microorganism described previously as an anaerobic benzaldehyde degrader. Within the archaeal community, there was a shift between two different species of the genus Methanosaeta sp., indicating a highly specific impact of DBP or degradation products on archaeal species. RNA-directed probes were designed from SSCP sequences, and FISH observations confirmed the dominance of S. saccharolytica, and indicated floccular microstructures, likely providing favourable conditions for DBP degradation.     

The role of iron in enhancing anaerobic toluene degradation in sulfate-reducing enrichment cultures

Ferrous iron enhanced the toluene degradation rate of sulfidogenic enrichment cultures inoculated with contaminated subsurface soil from an aviation fuel storage facility near the Patuxent River (Md.). Ferrous iron had an analogous effect on the degradation rate of benzoic acid, a transient metabolite of anaerobic toluene degradation in these cultures, when benzoic acid was used as a sole carbon and energy source. Two hypotheses were proposed to explain iron's effect: (a) Iron may have prevented sulfide toxicity via precipitation of sulfide as FeS, and (b) iron might have been a limiting nutrient required for degradation (i.e., amendments of iron could have compensated for iron removed from solution by precipitation as FeS). To test these hypotheses, substrate degradation rates were compared in the presence of FeSO₄ (a sulfate source that both precipitates sulfide species and precludes iron limitation) versus ZnSO₄ (a sulfate source that precipitates sulfide species but does not preclude iron limitation) versus MgSO₄ (a sulfate source that neither precipitates sulfide nor precludes iron limitation). For both toluene and benzoic acid, FeSO₄ and ZnSO₄ were comparable in their enhancement of substrate degradation rates and were superior to MgSO₄ in that respect. Thus, iron appears to ameliorate sulfide toxicity, not nutritional iron limitation, in these cultures. The observation that ethylenediaminetetraacetic acid, a chelating agent capable of retaining iron in solution in the presence of sulfide, did not stimulate the cultures is consistent with this conclusion. The implications of these results for bioremediation of fuel-contaminated aquifers that contain sulfate-reducing bacteria are discussed. Correspondence to: H.R. Beller.     

Selecting for lactic acid producing and utilising bacteria in anaerobic enrichment cultures

Lactic acid-producing bacteria are important in many fermentations, such as the production of biobased plastics. Insight in the competitive advantage of lactic acid bacteria over other fermentative bacteria in a mixed culture enables ecology-based process design and can aid the development of sustainable and energy-efficient bioprocesses. Here we demonstrate the enrichment of lactic acid bacteria in a controlled sequencing batch bioreactor environment using a glucose-based medium supplemented with peptides and B vitamins. A mineral medium enrichment operated in parallel was dominated by Ethanoligenens species and fermented glucose to acetate, butyrate and hydrogen. The complex medium enrichment was populated by Lactococcus, Lactobacillus and Megasphaera species and showed a product spectrum of acetate, ethanol, propionate, butyrate and valerate. An intermediate peak of lactate was observed, showing the simultaneous production and consumption of lactate, which is of concern for lactic acid production purposes. This study underlines that the competitive advantage for lactic acid-producing bacteria primarily lies in their ability to attain a high biomass specific uptake rate of glucose, which was two times higher for the complex medium enrichment when compared to the mineral medium enrichment. The competitive advantage of lactic acid production in rich media can be explained using a resource allocation theory for microbial growth processes.     

Batch culture enrichment of ANAMMOX populations from anaerobic and aerobic seed cultures

Discharge of nitrate and ammonia rich wastewaters into the natural waters encourage eutrophication, and contribute to aquatic toxicity. Anaerobic ammonium oxidation process (ANAMMOX) is a novel biological nitrogen removal alternative to nitrification-denitrification, that removes ammonia using nitrite as the electron acceptor. The feasibility of enriching the ANAMMOX bacteria from the anaerobic digester sludge of a biomethanation plant treating vegetable waste and aerobic sludge from an activated sludge process treating domestic sewage is reported in this paper. ANAMMOX bacterial activity was monitored and established in terms of nitrogen transformations to ammonia, nitrite and nitrate along with formation of hydrazine and hydroxylamine.     

Complete degradation of tetrachloroethene by combining anaerobic dechlorinating and aerobic methanotrophic enrichment cultures

 Degradation of tetrachloroethene (perchloroethylene, PCE) was investigated by combining the metabolic abilities of anaerobic bacteria, capable of reductive dechlorination of PCE, with those of aerobic methanotrophic bacteria, capable of co-metabolic degradation of the less-chlorinated ethenes formed by reductive dechlorination of PCE. Anaerobic communities reductively dechlorinating PCE, trichloroethene (TCE) and dichloroethenes were enriched from various sources. The maximum rates of dechlorination observed for various chloroethenes in these batch enrichments were: PCE to TCE (341 μmol l^-1 day^-1), TCE to cis-dichloroethene (159 μmol l^-1 day^-1), cis-dichloroethene to chloroethene (99 μmol l^-1 day^-1) and trans-dichloroethene to chloroethene (22 μmol l^-1 day^-1). A mixture of these enrichments was inoculated into an anoxic fixed-bed upflow column. In this column PCE was converted mainly into cis-1, 2-dichloroethene, small amounts of TCE and chloroethene, and chloride. Enrichments of aerobic methanotrophic bacteria were grown in an oxic fixed-bed downflow column. Less-chlorinated ethenes, formed in the anoxic column, were further metabolized in this oxic methanotrophic column. On the basis of analysis of chloride production and the disappearance of chlorinated ethenes it was demonstrated that complete degradation of PCE was possible by combining these two columns. Operation of the two-column system under various process conditions indicated that the sensitivity of the methanotrophic bacteria to chlorinated intermediates represented the bottle-neck in the sequential anoxic/oxic degradation process of PCE. Received: 24 October 1994 / Received revision: 20 January 1995 / Accepted: 23 January 1995     

Urea production by Thiosphaera pantotropha and by anaerobic enrichment cultures from marine sediments

Abstract The production of urea by Thiosphaera pantotropha was studied. Batch cultures were grown on acetate as energy source and with NO₃⁻ or O₂ as terminal electron acceptor. Urea accumulated in the media during exponential growth in aerobic and anaerobic cultures of T. pantotropha . Urea production continued after the cells had entered the stationary growth phase. Bacterial ability to produce urea was supported by studies of cultures enriched for denitrifying, sulphate-reducing and fermenting bacteria. The results implied that urea production was common among bacteria normally considered to be important in marine sediments.     

An improved gas distribution system for anaerobic screening of multiple microbial cultures

A cultivation set-up for multiple cultures has been designed that can be used for anaerobic screening for quantitative changes in growth rate or other analyses, e.g. protein composition of different strains. The developed gas distribution system provides a reproducible level of anaerobicity in 30 cultivation flasks and resembles the open system of a high-performance bioreactor in that it ensures cultivation at atmospheric pressure and avoids supersaturation of carbon dioxide. The system is cheap and user-friendly and allows rapid screenings of many strains simultaneously.     

The microbial diversity in picoplankton enrichment cultures: a molecular screening of marine isolates

Picoplankton bacteria from a North Sea water sample were cultured under a variety of different conditions (nutrients, temperature, light, agitation, adhesion). Fluorescent in situ hybridization (FISH) analysis of the enrichments showed complex communities which were dominated by gamma-Proteobacteria or beta-Proteobacteria, followed by alpha-Proteobacteria and bacteria from the Cytophaga/Flavobacterium/Bacteroides (CFB) cluster. Among 410 isolates, a high degree of diversity was found, both with respect to colony color and morphology and with respect to genetic diversity. Isolated bacteria were classified into the main taxa by a special PCR approach, termed signature PCR (SIG-PCR). It was based on an oligo primer mixture targeting 16S rDNA which yielded PCR products of taxon-specific lengths. Again, gamma-Proteobacteria dominated (48%), followed by alpha-Proteobacteria (20%). beta-Proteobacteria were rarely isolated (eight strains of 410). The CFB cluster comprised the second largest phylum (14%), and 7.5% of all isolates belonged to the high-GC Gram-positives. Thus, isolated bacteria were representative of enrichment communities with the exception of the beta-Proteobacteria, which were detected in high abundance in certain enrichments by FISH but not isolated, and the high-GC Gram-positives, which were cultivated but not detected by FISH. A genomic fingerprinting technique, randomly amplified polymorphic DNA, showed that among 58 CFB isolates only 18 identical genotypes were found, and among the 84 alpha-Proteobacteria only eight identical genotypes were present. The data show the enormous diversity of cultivated bacteria from picoplankton enrichment cultures of one North Sea water sample, which is only a small fraction of the total picoplankton community.     

Development and characterization of stable sediment-free anaerobic bacterial enrichment cultures that dechlorinate aroclor 1260

We have developed sediment-free anaerobic enrichment cultures that dechlorinate a broad spectrum of highly chlorinated polychlorinated biphenyls (PCBs). The cultures were developed from Aroclor 1260-contaminated sediment from the Housatonic River in Lenox, MA. Sediment slurries were primed with 2,6-dibromobiphenyl to stimulate Process N dechlorination (primarily meta dechlorination), and sediment was gradually removed by successive transfers (10%) to minimal medium. The cultures grow on pyruvate, butyrate, or acetate plus H(2). Gas chromatography-electron capture detector analysis demonstrated that the cultures extensively dechlorinate 50 to 500 mug/ml of Aroclor 1260 at 22 to 24 degrees C by Dechlorination Process N. Triplicate cultures of the eighth transfer without sediment dechlorinated 76% of the hexa- through nonachlorobiphenyls in Aroclor 1260 (250 mug/ml) to tri- through pentachlorobiphenyls in 110 days. At least 64 PCB congeners, all of which are chlorinated on both rings and 47 of which have six or more chlorines, were substrates for this dechlorination. To characterize the bacterial diversity in the enrichments, we used eubacterial primers to amplify and clone 16S rRNA genes from DNA extracted from cultures grown on acetate plus H(2). Restriction fragment length polymorphism analysis of 107 clones demonstrated the presence of Thauera-like Betaproteobacteria, Geobacter-like Deltaproteobacteria, Pseudomonas species, various Clostridiales, Bacteroidetes, Dehalococcoides of the Chloroflexi group, and unclassified Eubacteria. Our development of highly enriched, robust, stable, sediment-free cultures that extensively dechlorinate a highly chlorinated commercial PCB mixture is a major and unprecedented breakthrough in the field. It will enable intensive study of the organisms and genes responsible for a major PCB dechlorination process that occurs in the environment and could also lead to effective remediation applications.     

Biodiversity within hot spring microbial mat communities: molecular monitoring of enrichment cultures

We have begun to examine the basis for incongruence between hot spring microbial mat populations detected by cultivation or by 16S rRNA methods. We used denaturing gradient gel electrophoresis (DGGE) to monitor enrichments and isolates plated therefrom. At near extincting inoculum dilutions we observed Chloroflexus-like and cyanobacterial populations whose 16S rRNA sequences have been detected in the New Pit Spring Chloroflexus mat and the Octopus Spring cyanobacterial mat. Cyanobacterial populations enriched from 44 to 54°C and 56 to 63°C samples at near habitat temperatures were similar to those previously detected in mat samples of comparable temperatures. However, a lower temperature enrichment from the higher temperature sample selected for the populations found in the lower temperature sample. Three Thermus populations detected by both DGGE and isolation exemplify even more how enrichment may bias our view of community structure. The most abundant population was adap ted to the habitat temperature (50°C), while populations adapted to 65°C and 70°C were 102- and 104-fold less abundant, respectively. However, enrichment at 70°C favored the least abundant strain. Inoculum dilution and incubation at the habitat temperature favored the more numerically relevant populations. We enriched many other aerobic chemoorganotropic populations at various inoculum dilutions and substrate concentrations, most of whose 16S rRNA sequences have not been detected in mats. A common feature of numerically relevant cyanobacterial, Chloroflexus-like and aerobic chemorganotrophic populations, is that they grow poorly and resist cultivation on solidified medium, suggesting plating bias, and that the medium composition and incubation conditions may not reflect the natural microenvironments these populations inhabit.     

Effect of carbon source during enrichment on BTEX degradation by anaerobic mixed bacterial cultures

A comprehensive study on the effects of different carbon sources during the bacterial enrichment on the removal performances of benzene, toluene, ethylbenzene, and xylenes (BTEX) compounds when present as a mixture was conducted. Batch BTEX removal kinetic experiments were performed using cultures enriched with individual BTEX compounds or BTEX as a mixture or benzoate alone or benzoate–BTEX mixture. An integrated Monod-type non-linear model was developed and a ratio between maximum growth rate (μ _max) and half saturation constant (K_s) was used to fit the non-linear model. A higher μ _max/K_s indicates a higher affinity to degrade BTEX compounds. Complete removal of BTEX mixture was observed by all the enriched cultures; however, the removal rates for individual compounds varied. Degradation rate and the type of removal kinetics were found to be dependent on the type of carbon source during the enrichment. Cultures enriched on toluene and those enriched on BTEX mixture were found to have the greatest μ _max/K_s and cultures enriched on benzoate had the least μ _max/K_s. Removal performances of the cultures enriched on all different carbon sources, including the ones enriched on benzoate or benzoate–BTEX mixture were also improved during a second exposure to BTEX. A molecular analysis showed that after each exposure to the BTEX mixture, the cultures enriched on benzoate and those enriched on benzoate–BTEX mixture had increased similarities to the culture enriched on BTEX mixture.     

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.     

Molecular characterization of surfactant-driven microbial community changes in anaerobic phenanthrene-degrading cultures under methanogenic conditions

SDS and Triton X-100 added at their critical micelle concentrations (CMCs), increased phenanthrene solubility in the presence of sediments and inhibited phenanthrene biodegradation. Triton X-100 caused more inhibition than SDS. 16S rDNA analyses revealed that both surfactants changed the microbial communities of phenanthrene-degrading cultures. Further, after the surfactant additions, parts of the microbial populations were not detected and methane production decreased. Surfactant applications, necessary to achieve actual CMCs, alter microbial community structure and diminish methanogenic activity under anaerobic conditions. We propose that this change may be related to the inhibitory effects of SDS and Triton X-100 on phenanthrene biodegradation under methanogenic conditions.     

The fate of lignin and lignin‐derived compounds in anaerobic environments

During ODP Leg 201 microbial communities in Eastern Equatorial Pacific Ocean and Peru Margin sediments were investigated. The sediment layers sampled extended down to 420 m below the sea floor, with estimated ages of up to 40 million years. Contamination-free anoxic slurries were inoculated into media containing different substrate combinations, all at micromolar concentration. These culture media were designed for a broad spectrum of physiological groups. A total of 162 pure cultures were isolated that could be grouped into 19 different phylotypes based on 16S rRNA gene analysis. The isolates belonged to the Alpha-, Gamma- and Deltaproteobacteria, the Firmicutes, Actinobacteria, and Bacteroidetes. The genera most frequently isolated were Bacillus (68 isolates) and Rhizobium (40 isolates). Comparison of strains with the same phylotypes by enterobacterial repetitive intergenic consensus (ERIC-PCR) analysis revealed the presence of several subgroups that did not correlate with medium, sediment depth or sampling site. The majority of the isolates, although obtained from anoxic environments and isolated under strictly anoxic conditions, turned out to be facultativly aerobic. Physiologically, the isolates were characterized as generalists, able to utilize a broad variety of electron donors with either oxygen, nitrate and in some cases manganese oxides as electron acceptors. The diversity inferred from physiological tests was even higher than that on the phylogenetic or genomic level. The outcome of the contamination tests, the isolation of close relatives of already known subsurface bacteria, the repeated finding of the same phylotype from different sites and the level of diversity present in the culture collection strongly suggest that indigenous deep-biosphere bacteria had been isolated.