Synergistic growth of two members from a mixed microbial consortium growing on biphenyl

Abstract A stable nine-membered aerobic bacterial consortium (BSEN-2) growing on biphenyl as the sole carbon and energy source was isolated from a polychlorinated biphenyl (PCB) contaminated soil. Characterisation of the members, strains BPSI-1 to 9, revealed three principal genera, Pseudomonas, Sphingomonas and Alcaligenes . Phenotypic analysis based on standard microbiological tests and Biolog identification, showed close relationship between community members with the exception of Sphingomonas paucimobilis strain BPSI-3. Some clusters revealed relationships unrelated to genus groupings. Strain BPSI-3 produced a bright yellow water soluble compound from biphenyl having absorption maxima at 412 and 337 nm at neutral pH. This is similar, but not identical, to those results reported for muconic semialdehydes, cleavage products of biphenyl and other aromatic compounds. Only four of the nine isolates, BPSI-2, 3, 4 and 7, were capable of growth on biphenyl as sole carbon and energy source. Two isolates, Alcaligenes faecalis type II strain BPSI-2 and S. paucimobilis strain BPSI-3, were isolated together and were difficult to separate into pure cultures. Growth studies in liquid culture showed that a co-culture of these two achieved a specific growth rate (μ) approximately twice as high as strain BPSI-2 and four times that of BPSI-3. Both strains grew equally well on benzoate with no significant difference in their specific growth rates. When compared to the original mixed culture, BSEN-2, the co-culture achieved 39% greater biomass and a specific growth rate twice as high. In the co-culture, the yellow colour seen with pure cultures of BPSI-3 was not observed. BPSI-2 was found to be able to utilise the yellow metabolites more effectively than BPSI-3. A model for the interaction of these two strains, based on the utilisation of biphenyl catabolites and degradation at the genetic level, has been proposed.     

Synergistic mineralization of biphenyl by Alcaligenes faecalis type II BPSI-2 and Sphingomonas paucimobilis BPSI-3

Polychlorinated biphenyls (PCBs) are important environmental pollutants and have been found to have adverse effects on a number of different organisms. Aerobic biodegradation of PCBs occurs through direct oxidation of the biphenyl nucleus. Biphenyl degraders are instrumental in the mineralization of PCBs to CO₂ and water. Here two bacteria, Alcaligenes faecalis type II strain BPSI-2 and Sphingomonas paucimobilis strain BPSI-3, are described that exhibit synergistic mineralization of biphenyl (using ¹⁴C-UL-biphenyl) when grown as a co-culture. Mineralization rates (23·7 and 9·1 nmol nmol⁻¹ h⁻¹, respectively) and extent of mineralization (38·1% and 24·4%, respectively) were significantly different between the strains as well as when compared to the co-culture (35·2 nmol nmol⁻¹ h⁻¹ and 45·2%). Both strains were originally isolated from an enrichment culture, BSEN-2. The co-culture of BPSI-2 and 3 showed a threefold increase in mineralization rate compared with the parent culture and a decrease in the time taken for ¹⁴CO₂ evolution to occur. There was no significant difference in the extent of mineralization between the co-culture and BSEN-2. Examination of enrichment cultures at the community level may play a role in optimizing bioremediation programmes.     

Polymer selection for biphenyl degradation in a solid-liquid two-phase partitioning bioreactor

The commercially available thermoplastic polymer Hytrel was selected as the delivery phase for the hydrophobic model compound biphenyl in a solid-liquid two-phase partitioning bioreactor (TPPB), and 2.9 g biphenyl could successfully be degraded in 1-L TPPBs by a pure culture of the biphenyl-degrading bacterium Burkholderia xenovorans LB400 in 50 h and by a mixed microbial consortium isolated from contaminated soil in 45 h. TPPBs consist of an aqueous cell-containing phase and an immiscible second phase that partitions toxic and/or poorly soluble substrates (in this case biphenyl) on the basis of maintaining a thermodynamic equilibrium. This paper illustrates a rational strategy for selecting a suitable solid polymeric substance for the delivery of the poorly water-soluble model compound biphenyl. The partitioning of biphenyl between the selected polymers and water was analogous to partitioning of solutes between two immiscible liquid phases. The partitioning coefficients varied between 180 for Nylon 6.6 and 11,000 for Desmopan, where the later numerical value is comparable to biphenyl partitioning coefficients between water and organic solvents. Employing a solid delivery phase enabled the utilization of a surfactant-producing microbial mixed culture, which could not be cultivated in liquid-liquid TPPBs and thereby extended the range of biocatalysts that can be employed in TPPBs.     

Degradation of biphenyl by methanogenic microbial consortium

Biphenyl was readily degraded and mineralized to CO₂ and CH₄ by a PCB-dechlorinating anaerobic microbial consortium. Degradation occurred when biphenyl was supplied as a sole source of carbon or as a co-metabolic substrate together with glucose and methanol. p-Cresol was detected and confirmed by mass spectroscopy as a transient intermediate. Production of ¹⁴ C-CO₂ and ¹⁴C-CH₄ from ¹⁴C-biphenyl was observed in the approximate ratio of 1:2. The results indicated the existence of novel pathways for biphenyl degradation in a natural anaerobic microbial community.     

高矿浆浓度下黄铜矿浸出过程中微生物群落结构变化规律

目的：为阐明微生物群落演替及功能与浸出效率之间关系奠定基础,以及如何提高黄铜矿生物浸出效率和铜回收率提供理 论依据。方法：通过连续传代培养进行驯化,使得复合菌群的矿浆浓度耐受能力达到25 %（w/v）。采用该复合菌群在25 %矿浆浓 度下浸出黄铜矿,同时利用变性梯度凝胶电泳和克隆文库技术分析浸出过程中的微生物多样性。最后,采用实时荧光定量PCR 对 浸出过程中微生物群落结构进行定量解析。结果：28天内黄铜矿浸出率能够达到95.1 %,而驯化前的浸出率只有51.5%。该复合 菌群主要由Acidithiobacillus caldus, Sulfobacillus acidophilus,和Fereoplasma theroplasma thermophilum组成,其中Acidithbacillus caldus是浸出前期和后期的优势种群,而Sulfobacillus acidophilus在浸出中期均有竞争优势, Ferroplasma thermophilum在整个浸出过程中占 据整个群落的比例均较低。结论：本研究获得的复合菌群具有较强的浸出黄铜矿能力, Acidithiobacillus caldus和Sulfobacillus acidophilus在浸出过程中起着重要的作用,pH 值和铜浸出率与群落结构相关性较高。     

A road map for the development of community systems (CoSy) biology

Microbial interactions are essential for all global geochemical cycles and have an important role in human health and disease. Although we possess general knowledge about the major processes within a microbial community, we are presently unable to decipher what role individual microorganisms have and how their individual actions influence others in the community. We also have limited knowledge with which to predict the effects of microbial interactions and community composition on the environment and vice versa. In this Opinion article, we describe how community systems (CoSy) biology will enable us to decode these complex relationships and will therefore improve our understanding of individual members of the community and the modes of interactions in which they engage.     

Comparison of autochthonous bacteria and commercially available cultures with respect to their effectiveness in fuel oil degradation

Summary This study examined the microbial degradation of fuel oil by nine highly adapted different commercially available mixed bacterial cultures (DBC-plus, Flow Laboratories, Meckenheim, F.R.G.) and a bacterial community from a domestic sewage sludge sample. All mixed cultures were cultivated under aerobic batch conditions shaking (110 rpm) at 20°C in a mineral base medium containing 1 or 5% (v/v) fuel oil as the sole carbon source. Percent degradation of fuel oil and the n-alkane fraction was recorded for the nine DBC-plus cultures and the mixed population of the activated sludge sample. The increase in colony counts, protein, and optical density was studied during a 31-day incubation period for DBC-plus culture A, DBC-plus culture A2 and the activated sludge sample. The activated sludge mixed culture was most effective in degrading fuel oil, but various isolated bacterial strains from this bacterial community were not able to grow on fuel oil as the sole carbon source. In contrast, the n-alkane degradation rates of the DBC-cultures were lower, but single strains from the commercially available mixed cultures were able to mineralize fuel oil hydrocarbons. Strains ofPseudomonas aeruginosa were isolated most frequently and these organisms were able to grow very rapidly on fuel oil as a complex sole carbon source. The results indicate that fuel oil degradation in domestic sewage sludge is performed by mixed populations of naturally occurring bacteria and does not depend on the application of highly adapted commercially available cultures.     

Anaerobic degradation of the aromatic hydrocarbon biphenyl by a sulfate-reducing enrichment culture

The aromatic hydrocarbon biphenyl is a widely distributed environmental pollutant. Whereas the aerobic degradation of biphenyl has been extensively studied, knowledge of the anaerobic biphenyl-oxidizing bacteria and their biochemical degradation pathway is scarce. Here, we report on an enrichment culture that oxidized biphenyl completely to carbon dioxide under sulfate-reducing conditions. The biphenyl-degrading culture was dominated by two distinct bacterial species distantly affiliated with the Gram-positive genus Desulfotomaculum . Moreover, the enrichment culture has the ability to grow with benzene and a mixture of anthracene and phenanthrene as the sole source of carbon, but here the microbial community composition differed substantially from the biphenyl-grown culture. Biphenyl-4-carboxylic acid was identified as an intermediate in the biphenyl-degrading culture. Moreover, 4-fluorobiphenyl was converted cometabolically with biphenyl because in addition to the biphenyl-4-carboxylic acid, a compound identified as its fluorinated analog was observed. These findings are consistent with the general pattern in the anaerobic catabolism of many aromatic hydrocarbons where carboxylic acids are found to be central metabolites.     

Leitbakteria of microbial biofilm communities causing occlusion of biliary stents

Biliary stents inserted to relieve obstructive jaundice caused by biliary or pancreatic malignancies inevitably become occluded by microbial growth in the form of diverse microbial community biofilms. The scarce information available on these communities is based on cultivation methods, but such methods usually provide distorted overviews of community composition, so commonalities and differences in biliary stent communities are uncertain. We extracted DNA and RNA from the microbial communities of 11 biliary stents explanted from nine patients in hospitals from two different countries, amplified 16S rRNA and rDNA sequences, analysed the amplicons by the single-strand conformation polymorphism (SSCP) method, and sequenced and deduced phylogenetic assignments of the major amplicons representing the major biofilm community members. We used a Modified Robbins Device (MRD) to study de novo development of a stent biofilm from a patient stent microbial community. Single-strand conformation polymorphism fingerprinting revealed the same six abundant bacterial species, here designated Leitbakteria, namely Klebsiella pneumoniae, Enterococcus faecalis, Pseudomonas aeruginosa, Enterobacter aerogenes, and two unculturable bacteria distantly related to E. coli and Shigella sonnei, in all of the stent biofilm communities. In the experimental biliary stent system, a sequential colonization of the stent surface was observed, with P. aeruginosa being the pioneer colonizer, followed by K. pneumoniae and one of the unculturable Leitbakteria, followed by the remainder of the community. The overview of microbial biofilm communities of biliary stents gained by the use of culture-independent methods revealed new unculturable bacteria as major members of biliary stent biofilms, and the diversity of the abundant members of the stent biofilms is considerably lower than suggested from earlier studies based on cultivation methods, and that communities from different stents from different patients in different countries are remarkably similar and have similar major members, the stent Leitbakteria.     

喀斯特地区三种人工林土壤微生物群落结构特征

为揭示不同人工植被修复模式对喀斯特土壤微生物群落的影响,采用氯仿熏蒸提取法和磷脂脂肪酸(phospholipid fatty acid, PLFA)法研究人工构建的降香黄檀(Dalbergia odorifera)纯林(PDOP)、顶果木(Acrocarpus fraxinifolius)纯林(PAFP)、顶果木×降香黄檀混交林(MADP)对土壤微生物生物量及土壤微生物群落结构的影响。结果表明:(1)PDOP的土壤微生物生物量碳(MBC)和微生物生物量氮(MBN)含量显著高于PAFP和MADP,PAFP显著高于MADP。(2)三种人工林土壤真菌、丛枝菌根真菌和总PLFA含量无显著差异,但PDOP土壤细菌、放线菌、丛枝菌根真菌和总PLFA含量均高于PAFP和MADP,PAFP高于MADP。PDOP的土壤细菌、革兰氏阳性菌、革兰氏阴性菌、放线菌的PLFA含量显著高于MADP。MADP的真菌细菌比显著高于PDOP,但与PAFP无显著差异。(3)冗余分析表明,土壤阳离子交换量、pH和C:N是影响土壤微生物群落组成的最主要影响因子。从三种人工林的土壤微生物生物量及微生物群落结构来看,在喀斯特地区MADP并未显示出酸性土地区混交林提高土壤微生物生物量、改善土壤微生物群落结构的优势,但混交林的真菌细菌比最高,更有利于提高土壤生态系统的稳定性。     

Effects of media composition on substrate removal by pure and mixed bacterial cultures

Continuous culture experiments with identical experimental designs were run with a mixed microbial community of activated sludge origin and an axenic bacterial culture derived from it. Each culture received 2-chlorophenol (2-CP) at a concentration of 160 mg/L as COD and L-lysine at a concentration of 65 mg/L as COD. A factorial experimental design was employed with dilution rate and media composition as the two controlled variables. Three dilution rates were studied: 0.015, 0.0325, and 0.05 h^–1. Media composition was changed by adding four biogenic compounds (butyric acid, thymine, glutamic acid and lactose) in equal COD proportions at total concentrations of 0, 34, 225, and 1462 mg/L as COD. The measured variables were the effluent concentrations of 2-CP as measured by the 4-aminoantipyrene test and lysine as measured by the o-diacetylbenzene procedure. The results suggest that community structure and substrate composition play important roles in the response of a microbial community to mixed substrates. The addition of more biogenic substrates to the axenic culture had a deleterious effect on the removal of both lysine and 2-CP, although the effect was much larger on lysine removal. In contrast, additional substrates had a positive effect on the removal of 2-CP by the mixed community and much less of a negative effect on the removal of lysine. The dilution rate at which the cultures were growing had relatively little impact on the responses to the additional substrates.Abbreviations COD chemical oxygen demand - 2-CP 2-chlorophenol - DOC dissolved organic carbon - MDL method detection limit - SS suspended solids     

Predator-specific enrichment of actinobacteria from a cosmopolitan freshwater clade in mixed continuous culture

We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyte Cryptomonas sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (Ochromonas sp.) always resulted in an immediate bloom of a single phylotype population of members of the class Actinobacteria (Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate Cyclidium glaucoma. However, when the Ochromonas sp. was added after the establishment of C. glaucoma, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an Aquabacterium sp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community.     

Simultaneous degradation of acetonitrile and biphenyl by Pseudomonas aeruginosa

A bacterium capable of utilizing either acetonitrile as the sole source of carbon and nitrogen or biphenyl as the sole source of carbon was isolated from soil and identified as Pseudomonas aeruginosa. The bacterium also utilized other nitriles, amides, and polychlorinated biphenyls (PCBs) as growth substrates. Acetonitrile- or biphenyl-grown cells oxidized these substrates without a lag. In studies with [14C]acetonitrile, nearly 74% of the carbon was recovered as 14CO2 and 8% was associated with the biomass. In studies with [14C]biphenyl, nearly 68% of the carbon was recovered as 14CO2 and nearly 6% was associated with the biomass. Although higher concentrations of acetonitrile as the sole sources of nitrogen inhibited the rates of [14C]biphenyl mineralization, lower concentrations (0.05%, w/v) gave a 77% stimulation in 14CO2 recovery. Pseudomonas aeruginosa metabolized acetonitrile to ammonia and acetic acid and biphenyl to benzoic acid. The bacterium also simultaneously utilized biphenyl as the sole carbon source and acetonitrile as the sole nitrogen source. However, biphenyl utilization increased only after the depletion of acetonitrile. Metabolites of the mixed substrate were ammonia and benzoic acid, which completely disappeared in the later stages of incubation. Nitrile hydratase and amidase were responsible for the transformation of acetonitrile to acetic acid and ammonia.     

用ERIC-PCR结合分子杂交监测焦化废水处理系统(A2/O)中微生物群落结构的变化

建立一种不依赖纯培养 ,可以在废水处理工业现场使用的监测微生物群落结构变化的分子技术。以处理焦化工业废水(A2 /O生物膜工艺 )不同构筑物中的悬浮污泥的微生物群落为研究对象 ,每周采样 1次 ,连续 4周。获得悬浮污泥总 DNA的ERIC- PCR指纹图谱 ,结合分子杂交进一步区分相同条带间的不同序列信息。结果表明 ,在缺氧池 (A2池 )和好氧池 (O池 )之间 ,各个采样点的 ERIC- PCR图谱差异不大 ,悬浮污泥在各构筑物之间交流充分 ;同一采样点的图谱在不同采样时期具有明显差异 ,显示了在此期间微生物群落的连续动态变化过程。通过对生物膜系统中悬浮污泥的微生物群落结构的指纹图谱分析 ,可开发出对该系统微生物群落结构动态变化进行检测的技术     

16S rDNA-RFLP分析六氯苯好氧降解菌群的结构及其多样性

从某化工厂排水沟底泥中取样,经2个月的富集驯化得到六氯苯好氧降解菌群。通过测定该微生物菌群在降解六氯苯过程中累积耗氧量、微生物生长曲线及Cl-浓度的变化,证明在好氧条件下该微生物菌群能够以六氯苯为唯一碳源和能源生长。当培养温度为30℃,pH为7.0时,该菌群能在18d内将无机盐培养基中浓度为4.5mg/L的六氯苯降解55%以上,降解速率达到137.5μg/(L.d)。对降解菌群提取总DNA,选择性扩增细菌16S rDNA片段,建立克隆文库。通过限制性内切酶(限制性内切酶HaeⅢ和RsaⅠ)分析,得到9种不同的谱型,其中3种谱型是主要谱型。对主要谱型的克隆子测序,结果表明,它们分别与Alcaligenes和Azospirillum菌属相似性最高。该菌群在去除环境中难降解的有机氯污染物方面具有应用前景。     

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.     

Increases in soil respiration following labile carbon additions linked to rapid shifts in soil microbial community composition

Organic matter decomposition and soil CO₂ efflux are both mediated by soil microorganisms, but the potential effects of temporal variations in microbial community composition are not considered in most analytical models of these two important processes. However, inconsistent relationships between rates of heterotrophic soil respiration and abiotic factors, including temperature and moisture, suggest that microbial community composition may be an important regulator of soil organic matter (SOM) decomposition and CO₂ efflux. We performed a short-term (12-h) laboratory incubation experiment using tropical rain forest soil amended with either water (as a control) or dissolved organic matter (DOM) leached from native plant litter, and analyzed the effects of the treatments on soil respiration and microbial community composition. The latter was determined by constructing clone libraries of small-subunit ribosomal RNA genes (SSU rRNA) extracted from the soil at the end of the incubation experiment. In contrast to the subtle effects of adding water alone, additions of DOM caused a rapid and large increase in soil CO₂ flux. DOM-stimulated CO₂ fluxes also coincided with profound shifts in the abundance of certain members of the soil microbial community. Our results suggest that natural DOM inputs may drive high rates of soil respiration by stimulating an opportunistic subset of the soil bacterial community, particularly members of the Gammaproteobacteria and Firmicutes groups. Our experiment indicates that variations in microbial community composition may influence SOM decomposition and soil respiration rates, and emphasizes the need for in situ studies of how natural variations in microbial community composition regulate soil biogeochemical processes.     

凋落物对土壤有机碳与微生物功能多样性的影响

森林凋落物是影响土壤微生物群落和有机碳含量的重要因素,但其作用的程度和机制尚不清楚,研究该问题对于分析森林生态系统碳循环和资源管理具有重要意义。研究凋落物去除与添加处理下土壤有机碳含量与土壤微生物对碳源利用的差异,明确凋落物去除与添加对土壤微生物群落代谢功能及其多样性的影响,探究不同处理下SOC含量变化的土壤微生物群落代谢机理。选取承德市雾灵山1405-1435 m海拔范围内核桃楸-蒙古栎混交林的表层土壤,采用室内培养结合Biolog-ECO方法,测定了培养第21天的土壤有机碳（soil organic carbon,SOC）含量及微生物群落的AWCD值、Shannon-Wiener多样性指数、Simpson优势度指数、McIntosh均匀度指数、Pielou丰富度指数,分析培养期内凋落物的不同处理下SOC含量与微生物功能多样性的变化特征。结果表明：1）不同凋落物处理对SOC含量与土壤微生物群落多样性具有显著影响（P<0.05）,DL > HL > NL > CK;2）不同凋落物处理下土壤微生物群落代谢活性和土壤微生物对碳源的利用程度具有显著差异（P<0.05）,碳水化合物类和氨基酸类是土壤微生物的主要碳源;3）不同处理的SOC含量与土壤微生物多样性具有正相关关系。双倍凋落物添加在短期内对土壤微生物多样性影响难以达到显著水平且在一定程度上对土壤微生物的代谢活性具有抑制作用,土壤微生物群落功能多样性对SOC含量具有重要影响。     

Structural and functional characterization of a stable, 4-chlorosalicylic-acid-degrading,bacterial community in a chemostat

A mixed, stable microbial community, obtained by continuous enrichment of a sediment core using 4-chlorosalicylic acid as sole source of carbon and energy, contained 10 different bacterial species, including Klebsiella pneumonia, Pseudomonas fluorescens, P. mendocina and P. cichorii. The members of the community were grown separately on various chlorinated compounds which were readily degraded.The author is with the Department of Environmental Sciences, College of Basic Sciences and Humanities, Gobind Ballav Pant University of Agriculture and Technology, Pantnagar, Naini-tal-263145, India