Diverse cellulolytic bacteria isolated from the high humus, alkaline-saline chinampa soils

Aiming at learning the functional bacterial community in the high humus content, saline-alkaline soils of chinampas, the cellulolytic bacteria were quantified and 100 bacterial isolates were isolated and characterized in the present study. Analysis of 16S-23S IGS (intergenic spacer) RFLP (restriction fragment length polymorphism) grouped the isolates into 48 IGS types and phylogenetic analysis of 16S rRNA genes identified them into 42 phylospecies within 29 genera and higher taxa belonging to the phyla Actinobacteria, Firmicutes and Proteobacteria, dominated by the genera Arthrobacter, Streptomyces, Bacillus, Pseudomonas, Pseudoxanthomonas and Stenotrophomonas. Among these bacteria, 63 isolates represent 26 novel putative species or higher taxa, while 37 were members of 17 defined species according to the phylogenetic relationships of 16S rRNA gene. Except for the novel species, the cellulolytic activity was not reported previously in 9 of the 17 species. They degraded cellulose in medium at pH?4.5–10.0 or supplied with NaCl up to 9 %. In addition, 84.8 and 71.7 % of them degraded xylan and Avicel, respectively. These results greatly improved the knowledge about the diversity of cellulolytic bacteria and demonstrated that the chinampa soils contain diverse and novel cellulolytic bacteria functioning at a wide range of pH and salinity levels, which might be a valuable biotechnological resource for biotransformation of cellulose.     

The microbial ecology of anaerobic cellulose degradation in municipal waste landfill sites: evidence of a role for fibrobacters

Cellulose is reputedly the most abundant organic polymer in the biosphere, yet despite the fundamental role of cellulolytic microorganisms in global carbon cycling and as potential sources of novel enzymes for biotechnology, their identity and ecology is not well established. Cellulose is a major component of landfill waste and its degradation is therefore a key feature of the anaerobic microbial decomposition process. Here, we targeted a number of taxa containing known cellulolytic anaerobes (members of the bacterial genus Fibrobacter, lineages of Clostridium clusters I, III, IV and XIV, and anaerobic fungi of the Neocallimastigales) in landfill leachate and colonized cellulose 'baits' via PCR and quantitative PCR (qPCR). Fibrobacter spp. and Clostridium clusters III, IV and XIV were detected in almost all leachate samples and cluster III and XIV clostridia were the most abundant (1-6% and 1-17% of total bacterial 16S rRNA gene copies respectively). Two landfill leachate microcosms were constructed to specifically assess those microbial communities that colonize and degrade cellulose substrates in situ. Scanning electron microscopy (SEM) of colonized cotton revealed extensive cellulose degradation in one microcosm, and Fibrobacter spp. and Clostridium cluster III represented 29% and 17%, respectively, of total bacterial 16S rRNA gene copies in the biofilm. Visible cellulose degradation was not observed in the second microcosm, and this correlated with negligible relative abundances of Clostridium cluster III and Fibrobacter spp. (≤ 0.1%), providing the first evidence that the novel fibrobacters recently detected in landfill sites and other non-gut environments colonize and degrade cellulose substrates in situ.     

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.     

Characterization of wheat straw-degrading anaerobic alkali-tolerant mixed cultures from soda lake sediments by molecular and cultivation techniques

Alkaline pretreatment has the potential to enhance the anaerobic digestion of lignocellulosic biomass to biogas. However, the elevated pH of the substrate may require alkalitolerant microbial communities for an effective digestion. Three mixed anaerobic lignocellulolytic cultures were enriched from sediments from two soda lakes with wheat straw as substrate under alkaline (pH 9) mesophilic (37°C) and thermophilic (55°C) conditions. The gas production of the three cultures ceased after 4 to 5 weeks, and the produced gas was composed of carbon dioxide and methane. The main liquid intermediates were acetate and propionate. The physiological behavior of the cultures was stable even after several transfers. The enrichment process was also followed by molecular fingerprinting (terminal restriction fragment length polymorphism) of the bacterial 16S rRNA gene and of the mcrA/mrtA functional gene for methanogens. The main shift in the microbial community composition occurred between the sediment samples and the first enrichment, whereas the structure was stable in the following transfers. The bacterial communities mainly consisted of Sphingobacteriales, Clostridiales and Spirochaeta, but differed at genus level. Methanothermobacter and Methanosarcina genera and the order Methanomicrobiales were predominant methanogenes in the obtained cultures. Additionally, single cellulolytic microorganisms were isolated from enrichment cultures and identified as members of the alkaliphilic or alkalitolerant genera. The results show that anaerobic alkaline habitats harbor diverse microbial communities, which can degrade lignocellulose effectively and are therefore a potential resource for improving anaerobic digestion.     

Characterization of Clostridium thermocellum Isolates Grown on Cellulose and Sugarcane Bagasse

Although plant cell walls may be degraded by microbial free enzymes, many bacteria degrade cellulose via enzyme complexes called cellulosomes. The study of the structures and mechanisms of these large macromolecular complexes is an active and ongoing research topic, with the goal of developing methods to improve lignocellulosic biomass conversion using cellulosomes. The aim of the present work was to evaluate and characterize the holocellulolytic activities produced by two new isolates (ISO1 and ISO2) of the spore-forming thermophilic anaerobic bacterium Clostridium thermocellum, during growth on crystalline cellulose and sugarcane bagasse, in comparison with activities obtained from the C. thermocellum strain CthJW. The pH and temperature values for optimal growth of the isolates were pH 7 and 60 °C, respectively. The isolates produced cellulolytic, xylanolytic, and pectinolytic activities when cultured on crystalline cellulose or sugarcane bagasse, which have never been used previously as the sole carbon source for these bacteria. The profiles of secreted proteins for these isolates, ISO1 and ISO2, were quite different from those obtained for the standard strain CthJW and from each other, as shown by 2D gel electrophoresis maps, and these profiles also depend on the carbon source used. Different protein isoforms were also detected in the maps for all growth conditions and bacterial strains. MALDI-TOF mass spectrometry was used to identify the differentially expressed proteins for ISO1 and ISO2 under growth in the presence of cellulose as carbon source. Twenty-five differentially expressed spots were identified and grouped into 8 functional categories: metabolism (20 %), motor function (20 %), protein synthesis (12 %), oxidative stress (16 %), secretory pathway (12 %), cellulose hydrolysis (4 %), protein folding (4 %), and defense (12 %). Spots 200 and 197, identified as a glycosyl hydrolase family member 9 and as a chaperone GroEL, respectively, were detected for all isolates and are potentially related to cellulosome architecture.     

嗜热厌氧纤维素分解菌的分离、鉴定及其酶学特性

【目的】分离高效降解纤维素的嗜热厌氧菌,通过与嗜热产乙醇菌株联合培养的方式,为生产纤维素乙醇提供微生物资源。【方法】利用厌氧分离技术从降解纤维素的马粪富集物中分离到一株嗜热厌氧细菌HCp。采用形态学观察、生理生化鉴定、结合16S rDNA序列的系统发育学分析确定该菌株的分类地位,利用DNS酶活分析方法测定此分离菌株的酶学性质。【结果】分离菌株HCp革兰氏染色阴性,直杆,细胞单个或成对出现,菌体大小为(0.35-0.50)μm×(2.42-6.40)μm,严格厌氧,形成芽胞,能运动,对新霉素有一定的抗性。此菌能利用滤纸纤维素、纤维素粉、微晶纤维素、脱脂棉和水稻秸秆、明胶等,还可以利用葡萄糖、纤维二糖、木糖、木聚糖、果糖、蔗糖、核糖、半乳糖、麦芽糖、山梨糖、海藻糖、蜜二糖、甘露糖等。该菌株在pH6.5-8.5、温度35-70℃、盐浓度0%-1.0%范围内利用纤维素生长,最适pH为6.85,最适温度为60℃,最适NaCl浓度为0.2%,最佳生长条件下,在10 d内滤纸纤维素降解率可达90.40%。在HCp的纤维小体中,滤纸酶、羧甲基纤维素酶、β-葡萄糖苷酶、木聚糖酶的最适作用温度分别为70℃、70℃、70℃、60℃,并且羧甲基纤维素酶具有较高的热稳定性。部分长度的16S rDNA序列分析表明,分离菌株HCp与Acetivibrio cellulolyticus、A.cellulosolvens相似性为97.5%。【结论】分离菌株HCp是从马粪富集物中分离到的一株嗜热厌氧细菌,该菌具有较强的降解纤维素能力,生长温度范围广,酶的热稳定性好,纤维素底物利用广泛等特性,为纤维素降解产乙醇提供了良好的材料。     

Diversity of Bacteria and Glycosyl Hydrolase Family 48 Genes in Cellulolytic Consortia Enriched from Thermophilic Biocompost

The enrichment from nature of novel microbial communities with high cellulolytic activity is useful in the identification of novel organisms and novel functions that enhance the fundamental understanding of microbial cellulose degradation. In this work we identify predominant organisms in three cellulolytic enrichment cultures with thermophilic compost as an inoculum. Community structure based on 16S rRNA gene clone libraries featured extensive representation of clostridia from cluster III, with minor representation of clostridial clusters I and XIV and a novel Lutispora species cluster. Our studies reveal different levels of 16S rRNA gene diversity, ranging from 3 to 18 operational taxonomic units (OTUs), as well as variability in community membership across the three enrichment cultures. By comparison, glycosyl hydrolase family 48 (GHF48) diversity analyses revealed a narrower breadth of novel clostridial genes associated with cultured and uncultured cellulose degraders. The novel GHF48 genes identified in this study were related to the novel clostridia Clostridium straminisolvens and Clostridium clariflavum, with one cluster sharing as little as 73% sequence similarity with the closest known relative. In all, 14 new GHF48 gene sequences were added to the known diversity of 35 genes from cultured species.The exploration and understanding of cellulose fermentation capabilities in nature could inform and enable industrial processes converting cellulosic biomass to fuels and other products. Enrichment of microbial communities that can utilize cellulose is useful in this context for the identification of novel organisms, novel metabolisms, and novel functions. Of particular interest are communities that can utilize cellulose at high temperatures and under anaerobic conditions, featuring high rates of solubilization under conditions where the energy and the reducing power of substrates are conserved in potentially useful fermentation products.Some evidence indicates that cocultures may be able to utilize cellulose more fully and produce higher concentrations of ethanol than pure cultures of model cellulolytic organisms such as Clostridium thermocellum and Clostridium straminisolvens (16, 20, 34). An initial step toward understanding the functional roles of community members in cooperative cellulose degradation is answering the question of what organisms are present in cellulolytic consortia obtained from nature. Currently, diversity estimation methods applied to cellulolytic communities range from traditional methods targeting the 16S rRNA gene (4, 12) to complex metagenomic analyses targeting the breadth of functional genes present in genomes of mixed cultures and the environment (3).From a functional gene standpoint, cellulase systems are complex assemblages of multifunctional glycosyl hydrolases. Even particularly relevant families, such as family 5 and family 9, tend to include hydrolases with multiple substrate specificities, deep evolutionary roots, and extensive sequence diversity within the same organism (19). However, family 48 glycosyl hydrolases include a select group of cellulosomal and unbound cellulases thought to play an essential role in cellulose solubilization by model cellulolytic clostridia (5, 7, 15), actinobacteria (6, 13), and anaerobic fungi (31). One key feature of this family of glycosyl hydrolases (mostly exoglucanases) is their ability to enhance cellulose solubilization in synergistic interactions with family 9 glycosyl hydrolases (2, 13). But unlike the latter, and with the notable exception of CelS and CelY in Clostridium thermocellum, family 48 hydrolases are present mostly in single copies in the genomes of cellulolytic microbes, making family 48 hydrolase genes a desirable target for primer design and molecular characterization.In this paper we describe the enrichment of microbial communities from a thermophilic compost pile and provide an assessment of diversity in stable cellulolytic enrichments by addressing total bacterial diversity using the 16S rRNA gene as well as introducing a novel method to assess functional diversity in cellulolytic consortia by targeting glycosyl hydrolase family 48 (GHF48) genes.     

Degradation of benzene, toluene, and xylene isomers by a bacterial consortium obtained from rhizosphere soil of Cyperus sp. grown in a petroleum-contaminated area

Increasing contamination of soil and groundwater with benzene, toluene, and xylene (BTX) due to activities of the chemical and oil refinery industry has caused serious environmental damage. Efficient methods are required to isolate and degrade them. Microorganisms associated with rhizosphere soil are considered efficient agents to remediate hydrocarbon contamination. In this study, we obtained a stabilized bacterial consortium from the rhizosphere soil of Cyperus sp. grown in a petroleum-contaminated field in Southern Mexico. This consortium was able to completely degrade BTX in 14 days. Bacteria isolated from the consortium were identified by 16S rRNA gene sequence analysis as Ralstonia insidiosa, Cellulomonas hominis, Burkholderia kururiensis, and Serratia marcescens. The BTX-degradation capacity of the bacterial consortium was confirmed by the detection of genes pheA, todC1, and xylM, which encoded phenol hydroxylase, toluene 1,2-dioxygenase, and xylene monooxygenase, respectively. Our results demonstrate feasibility of BTX biodegradation by indigenous bacteria that might be used for soil remediation in Southern Mexico.     

Assessment of microbial communities associated with fermentative–methanogenic biodegradation of aromatic hydrocarbons in groundwater contaminated with a biodiesel blend (B20)

A controlled field experiment was conducted to assess the potential for fermentative–methanogenic biostimulation (by ammonium-acetate injection) to enhance biodegradation of benzene, toluene, ethylbenzene and xylenes (BTEX) as well as polycyclic aromatic hydrocarbons (PAHs) in groundwater contaminated with biodiesel B20 (20:80 v/v soybean biodiesel and diesel). Changes in microbial community structure were assessed by pyrosequencing 16S rRNA analyses. BTEX and PAH removal began 0.7 year following the release, concomitantly with the increase in the relative abundance of Desulfitobacterium and Geobacter spp. (from 5 to 52.7 % and 15.8 to 37.3 % of total Bacteria 16S rRNA, respectively), which are known to anaerobically degrade hydrocarbons. The accumulation of anaerobic metabolites acetate and hydrogen that could hinder the thermodynamic feasibility of BTEX and PAH biotransformations under fermentative/methanogenic conditions was apparently alleviated by the growing predominance of Methanosarcina. This suggests the importance of microbial population shifts that enrich microorganisms capable of interacting syntrophically to enhance the feasibility of fermentative–methanogenic bioremediation of biodiesel blend releases.     

Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was established and analyzed. Results showed that a net 538 ??mol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes and a decrease in the alkanes profile was also observed. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the archaeal phylotypes were mostly related to members of the orders Methanobacteriales and Methanosarcinales. The bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. However, most of the bacterial clones retrieved from the enrichment cultures showed low similarity to 16S rRNA gene sequences of the cultured members, indicating that the enrichment cultures contained novel bacterial species. Though alkane-degrading methanogenic enrichment consortium has rarely been reported from petroleum reservoirs, our results indicated that oilfield production water harbors a microbial community capable of syntrophic conversion of n-alkanes to methane, which sheds light on the bio-utilization of marginal oil reservoirs for enhanced energy recovery.     

复合菌系降解纤维素过程中微生物群落结构的变化

为明确高效纤维素降解复合菌系降解过程中微生物群落结构的变化规律及关键的降解功能菌,利用该复合菌系对滤纸和稻秆进行生物处理,通过底物降解、微生物生长量、发酵液pH的变化情况,选择不同降解时期复合菌系提取的总DNA进行细菌16S rRNA基因扩增子高通量测序。通过分解特性试验确定在接种后培养第12、72、168 h分别作为降解初期、高峰期、末期。该复合菌系分别主要由1个门、2个纲、2个目、7个科、11个属组成。随着降解的进行,短芽胞杆菌属Brevibacillus、喜热菌属Caloramator的相对丰度逐渐降低;梭菌属Clostridium、芽胞杆菌属Bacillus、地芽胞杆菌属Geobacillus、柯恩氏菌属Cohnella的相对丰度逐渐升高;解脲芽胞杆菌属Ureibacillus、泰氏菌属Tissierella、刺尾鱼菌属Epulopiscium在降解高峰期时相对丰度最高;各时期类芽胞杆菌属Paenibacillus、瘤胃球菌属Ruminococcus的相对丰度无明显变化。上述11个主要菌属均属于厚壁菌门,具有嗜热、耐热、适应广泛pH、降解纤维素或半纤维素的特性。好氧型细菌是降解初期的主要优势功能菌,到中后期厌氧型细菌逐渐增多,并逐步取代好氧型细菌成为降解纤维素的主要细菌。     

Degradation of Cyanophycin by Sedimentibacter hongkongensis Strain KI and Citrobacter amalonaticus Strain G Isolated from an Anaerobic Bacterial Consortium

Using a combination of various enrichment techniques, the strictly anaerobic, gram-positive, endospore-forming bacterium Sedimentibacter hongkongensis strain KI as revealed by 16S rRNA analysis and the gram-negative enterobacterium Citrobacter amalonaticus strain G as revealed by physiological tests were isolated from an anaerobic cyanophycin (CGP)-degrading bacterial consortium. S. hongkongensis strain KI is the first anaerobic bacterium with the ability to hydrolyze CGP to β-Asp-Arg and β-Asp-Lys dipeptides, as revealed by electrospray ionization-mass spectrometry and reversed-phase high-performance liquid chromatography analysis. However, these primary accumulated hydrolysis products were only partially used by S. hongkongensis strain KI, and significant growth on CGP did not occur. On the other hand, C. amalonaticus strain G did not degrade CGP but grew on the β-linked iso-dipeptides formed in vitro by enzymatic CGP degradation or in vivo by metabolic activity of S. hongkongensis strain KI. Dipeptide utilization occurred at the highest rate if both strains were used in cocultivation experiments with CGP, indicating that cooperation between different bacteria occurs in anaerobic natural environments for complete CGP turnover. The amino acids obtained from the cleavage of dipeptides were fermented to ethanol, acetic acid, and succinic acid, as revealed by gas chromatographic analysis and by spectrophotometric enzyme assays.     

Comparative metagenomic analysis of microcosm structures and lignocellulolytic enzyme systems of symbiotic biomass-degrading consortia

Decomposition of lignocelluloses by cooperative microbial actions is an essential process of carbon cycling in nature and provides a basis for biomass conversion to fuels and chemicals in biorefineries. In this study, structurally stable symbiotic aero-tolerant lignocellulose-degrading microbial consortia were obtained from biodiversified microflora present in industrial sugarcane bagasse pile (BGC-1), cow rumen fluid (CRC-1), and pulp mill activated sludge (ASC-1) by successive subcultivation on rice straw under facultative anoxic conditions. Tagged 16S rRNA gene pyrosequencing revealed that all isolated consortia originated from highly diverse environmental microflora shared similar composite phylum profiles comprising mainly Firmicutes, reflecting convergent adaptation of microcosm structures, however, with substantial differences at refined genus level. BGC-1 comprising cellulolytic Clostridium and Acetanaerobacterium in stable coexistence with ligninolytic Ureibacillus showed the highest capability on degradation of agricultural residues and industrial pulp waste with CMCase, xylanase, and β-glucanase activities in the supernatant. Shotgun pyrosequencing of the BGC-1 metagenome indicated a markedly high relative abundance of genes encoding for glycosyl hydrolases, particularly for lignocellulytic enzymes in 26 families. The enzyme system comprised a unique composition of main-chain degrading and side-chain processing hydrolases, dominated by GH2, 3, 5, 9, 10, and 43, reflecting adaptation of enzyme profiles to the specific substrate. Gene mapping showed metabolic potential of BGC-1 for conversion of biomass sugars to various fermentation products of industrial importance. The symbiotic consortium is a promising simplified model for study of multispecies mechanisms on consolidated bioprocessing and a platform for discovering efficient synergistic enzyme systems for biotechnological application.     

The effect of heavy metals on microbial community structure of a sulfidogenic consortium in anaerobic semi-continuous stirred tank reactors

The effect of heavy metals on community structure of a heavy metal tolerant sulfidogenic consortium was evaluated by using a combination of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene and dissimilatory sulfite reductase (dsrB) gene fragments, 16S rRNA gene cloning analysis and fluorescence in situ hybridization (FISH). For this purpose, four anaerobic semi-continuous stirred tank reactors (referred as R1–R4) were run in parallel for 12 weeks at heavy metal loading rates of 1.5, 3, 4.5 and 7.5 mg l^?1 d^?1 each of Cu²⁺, Ni²⁺, Zn²⁺, and Cr⁶⁺, respectively. The abundance ratio of Desulfovibrio vulgaris detected by FISH to total cell counts was consistent with the obtained results of cloning and DGGE. This indicated that D. vulgaris was dominant in all analyzed samples and played a key role in heavy metal removal in R1, R2, and R3. In contrast, after 4 weeks of operation of R4, a distinct biomass loss was observed and no positive hybridized cells were detected by specific probes for the domain Bacteria, sulfate-reducing bacteria and D. vulgris. High removal efficiencies of heavy metals were achieved in R1, R2 and R3 after 12 weeks, whereas the precipitation of heavy metals in R4 was significantly decreased after 4 weeks and almost not observed after 6 weeks of operation. In addition, the anaerobic bacteria, such as Pertrimonas sulfuriphila, Clostridium sp., Citrobacter amalonaticus, and Klebsiella sp., identified from DGGE bands and clone library were hypothesized as heavy metal resistant bacteria at a loading rate of 1.5 mg l^?1 d^?1 of Cu²⁺, Ni²⁺, Zn²⁺, and Cr^6+.     

Characterization of three plant biomass-degrading microbial consortia by metagenomics- and metasecretomics-based approaches

The selection of microbes by enrichment on plant biomass has been proposed as an efficient way to develop new strategies for lignocellulose saccharification. Here, we report an in-depth analysis of soil-derived microbial consortia that were trained to degrade once-used wheat straw (WS1-M), switchgrass (SG-M) and corn stover (CS-M) under aerobic and mesophilic conditions. Molecular fingerprintings, bacterial 16S ribosomal RNA (rRNA) gene amplicon sequencing and metagenomic analyses showed that the three microbial consortia were taxonomically distinct. Based on the taxonomic affiliation of protein-encoding sequences, members of the Bacteroidetes (e.g. Chryseobacterium, Weeksella, Flavobacterium and Sphingobacterium) were preferentially selected on WS1-M, whereas SG-M and CS-M favoured members of the Proteobacteria (e.g. Caulobacter, Brevundimonas, Stenotrophomonas and Xanthomonas). The highest degradation rates of lignin (~59 %) were observed with SG-M, whereas CS-M showed a high consumption of cellulose and hemicellulose. Analyses of the carbohydrate-active enzymes in the three microbial consortia showed the dominance of glycosyl hydrolases (e.g. of families GH3, GH43, GH13, GH10, GH29, GH28, GH16, GH4 and GH92). In addition, proteins of families AA6, AA10 and AA2 were detected. Analysis of secreted protein fractions (metasecretome) for each selected microbial consortium mainly showed the presence of enzymes able to degrade arabinan, arabinoxylan, xylan, β-glucan, galactomannan and rhamnogalacturonan. Notably, these metasecretomes contain enzymes that enable us to produce oligosaccharides directly from wheat straw, sugarcane bagasse and willow. Thus, the underlying microbial consortia constitute valuable resources for the production of enzyme cocktails for the efficient saccharification of plant biomass.     

Naturally Occurring DNA Transfer System Associated with Membrane Vesicles in Cellulolytic Ruminococcus spp. of Ruminal Origin

A genetic transformation system with similarities to those reported for gram-negative bacteria was found to be associated with membrane vesicles of the ruminal cellulolytic genus Ruminococcus. Double-stranded DNA was recovered from the subcellular particulate fraction of all the cellulolytic ruminococci examined. Electron microscopy revealed that the only particles present resembled membrane vesicles. The likelihood that the DNA was associated with membrane vesicles (also known to contain cellulosomes) was further supported by the adherence of the particles associated with the subcellular DNA to cellulose powder added to culture filtrates. The particle-associated DNA comprised a population of linear molecules ranging in size from <20 kb to 49 kb (Ruminococcus sp. strain YE73) and from 23 kb to 90 kb (Ruminococcus albus AR67). Particle-associated DNA from R. albus AR67 represented DNA derived from genomic DNA of the host bacterium having an almost identical HindIII digestion pattern and an identical 16S rRNA gene. Paradoxically, particle-associated DNA was refractory to digestion with EcoRI, while the genomic DNA was susceptible to extensive digestion, suggesting that there is differential restriction modification of genomic DNA and DNA exported from the cell. Transformation using the vesicle-containing fraction of culture supernatant of Ruminococcus sp. strain YE71 was able to restore the ability to degrade crystalline cellulose to two mutants that were otherwise unable to do so. The ability was heritable and transferred to subsequent generations. It appears that membrane-associated transformation plays a role in lateral gene transfer in complex microbial ecosystems, such as the rumen.     

Erratum to: Two novel species Enterococcus lemanii sp. nov. and Enterococcus eurekensis sp. nov., isolated from a swine-manure storage pit

A polyphasic taxonomic study using morphological, biochemical, chemotaxonomic and molecular genetic methods was performed on six strains of an unknown Gram-positive, nonspore-forming, facultative anaerobic coccus-shaped bacterium isolated from a swine-manure storage pit. On the basis of 16S rRNA, RNA polymerase-subunit (rpoA), and the 60-kilodalton chaperonin (cpn60) gene sequence analyses, it was shown that all the isolates were enterococci but formed two separate lines of descent. Pairwise 16S rRNA sequence comparisons demonstrated that the two novel organisms were most closely related to each other (97.9 %) and to Enterococcus aquimarinus (97.8 %). Both organisms contained major amounts of C_16:0, C_16:1 ω7c, and C_18:1 ω7c/12t/9t as the major cellular fatty acids. Based on biochemical, chemotaxonomic, and phylogenetic evidence, the names Enterococcus lemanii sp. nov. (type strain PC32^T = CCUG 61260^T = NRRL B-59661^T) and Enterococcus eurekensis sp. nov. (type strain PC4B^T = CCUG 61259^T = NRRL B-59662^T) are proposed for the hitherto undescribed species.     

Effect of herbicide adjuvants on the biodegradation rate of the methylthiotriazine herbicide prometryn

A microbial community, selected by its ability to degrade triazinic herbicides was acclimatized by successive transfers in batch cultures. Initially, its ability to degrade prometryn, was evaluated using free cells or cells attached to fragments of a porous support. As carbon, nitrogen and sulfur sources, prometryn, (98.8 % purity), or Gesagard, a herbicide formulation containing 44.5 % prometryn and 65.5 % of adjuvants, were used. In batch cultures, a considerable delay in the degradation of prometryn, presumptively caused by the elevated concentration of inhibitory adjuvants, occurred. When pure prometryn was used, volumetric removal rates remarkably higher than those obtained with the herbicide formulation were estimated by fitting the raw experimental data to sigmoidal decay models, and differentiating them. When the microbial consortium was immobilized in a continuously operated biofilm reactor, the negative effect of adjuvants on the rate and removal efficiency of prometryn could not be detected. Using the herbicide formulation, the consortium showed volumetric removal rates greater than 20 g m^?3 h^?1, with prometryn removal efficiencies of 100 %. The predominant bacterial strains isolated from the microbial consortium were Microbacterium sp., Enterobacter sp., Acinetobacter sp., and Flavobacterium sp. Finally, by comparison of the prometryn removal rates with others reported in the literature, it can be concluded that the use of microbial consortia immobilized in a biofilm reactor operated in continuous regime offer better results than batch cultures of pure microbial strains.     

Evaluating Models of Cellulose Degradation by Fibrobacter succinogenes S85

Fibrobacter succinogenes S85 is an anaerobic non-cellulosome utilizing cellulolytic bacterium originally isolated from the cow rumen microbial community. Efforts to elucidate its cellulolytic machinery have resulted in the proposal of numerous models which involve cell-surface attachment via a combination of cellulose-binding fibro-slime proteins and pili, the production of cellulolytic vesicles, and the entry of cellulose fibers into the periplasmic space. Here, we used a combination of RNA-sequencing, proteomics, and transmission electron microscopy (TEM) to further clarify the cellulolytic mechanism of F. succinogenes. Our RNA-sequence analysis shows that genes encoding type II and III secretion systems, fibro-slime proteins, and pili are differentially expressed on cellulose, relative to glucose. A subcellular fractionation of cells grown on cellulose revealed that carbohydrate active enzymes associated with cellulose deconstruction and fibro-slime proteins were greater in the extracellular medium, as compared to the periplasm and outer membrane fractions. TEMs of samples harvested at mid-exponential and stationary phases of growth on cellulose and glucose showed the presence of grooves in the cellulose between the bacterial cells and substrate, suggesting enzymes work extracellularly for cellulose degradation. Membrane vesicles were only observed in stationary phase cultures grown on cellulose. These results provide evidence that F. succinogenes attaches to cellulose fibers using fibro-slime and pili, produces cellulases, such as endoglucanases, that are secreted extracellularly using type II and III secretion systems, and degrades the cellulose into cellodextrins that are then imported back into the periplasm for further digestion by β-glucanases and other cellulases.     

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.