Microbial Consortia for Hydrogen Production Enhancement

Ten efficient hydrogen-producing strains affiliated to the Clostridium genus were used to develop consortia for hydrogen production. In order to determine their saccharolytic and proteolytic activities, glucose and meat extract were tested as fermentation substrates, and the best hydrogen-producing strains were selected. The C. roseum H5 (glucose-consuming) and C. butyricum R4 (protein-degrading) co-culture was the best hydrogen-producing co-culture. The end-fermentation products for the axenic cultures and co-cultures were analyzed. In all cases, organic acids, mainly butyrate and acetate, were produced lowering the pH and thus inhibiting further hydrogen production. In order to replace the need for reducing agents for the anaerobic growth of clostridia, a microbial consortium including Clostridium spp. and an oxygen-consuming microorganism able to form dense granules (Streptomyces sp.) was created. Increased yields of hydrogen were achieved. The effect of adding a butyrate-degrading bacteria and an acetate-consuming archaea to the consortia was also studied.     

Biodegradation of Oil Tank Bottom Sludge using Microbial Consortia

We present a rationale for the selection of a microbial consortia specifically adapted to degrade toxic components of oil refinery tank bottom sludge (OTBS). Sources such as polluted soils, petrochemical waste, sludge from refinery-wastewater plants, and others were used to obtain a collection of eight microorganisms, which were individually tested and characterized to analyze their degradative capabilities on different hydrocarbon families. After initial experiments using mixtures of these strains, we developed a consortium consisting of four microorganisms (three bacteria and one yeast) selected in the basis of their cometabolic effects, emulsification properties, colonization of oil components, and degradative capabilities. Although the specific contribution each of the former parameters makes is not clearly understood, the activity of the four-member consortium had a strong impact not only on linear alkane degradation (100%), but also on the degradation of cycloalkanes (85%), branched alkanes (44%), and aromatic and sulphur–aromatic compounds (31–55%). The effectiveness of this consortium was significantly superior to that obtained by individual strains, commercial inocula or an undefined mixture of culturable and non-culturable microorganisms obtained from OTBS-polluted soil. However, results were similar when another consortium of four microorganisms, previously isolated in the same OTBS-polluted soil, was assayed.     

Cr(VI) Reduction by Sulfidogenic and Nonsulfidogenic Microbial Consortia

In time course experiments, bacterial community compositions were compared between a sulfidogenic and two nonsulfidogenic Cr(VI)-reducing consortia enriched from metal-contaminated sediments. The consortia were subjected to 0 and 0.85 mM or 1.35 mM Cr(VI), and Cr(VI) reduction, growth, and denaturing gradient gel electrophoresis profiles of PCR products of small-subunit (16S) ribosomal genes were compared. Results showed that although Cr(VI) was completely reduced by the three consortia, Cr(VI) inhibited cell growth, with sulfate-reducing bacteria being particularly sensitive to Cr(VI) toxicity relative to other bacteria in the consortia.     

Temporal Stability and Biodiversity of Two Complex Antilisterial Cheese-Ripening Microbial Consortia

The temporal stability and diversity of bacterial species composition as well as the antilisterial potential of two different, complex, and undefined microbial consortia from red-smear soft cheeses were investigated. Samples were collected twice, at 6-month intervals, from each of two food producers, and a total of 400 bacterial isolates were identified by Fourier-transform infrared spectroscopy and 16S ribosomal DNA sequence analysis. Coryneform bacteria represented the majority of the isolates, with certain species being predominant. In addition, Marinolactobacillus psychrotolerans, Halomonas venusta, Halomonas variabilis, Halomonas sp. (10⁶ to 10⁷ CFU per g of smear), and an unknown, gram-positive bacterium (10⁷ to 10⁸ CFU per g of smear) are described for the first time in such a consortium. The species composition of one consortium was quite stable over 6 months, but the other consortium revealed less diversity of coryneform species as well as less stability. While the first consortium had a stable, extraordinarily high antilisterial potential in situ, the antilisterial activity of the second consortium was lower and decreased with time. The cause for the antilisterial activity of the two consortia remained unknown but is not due to the secretion of soluble, inhibitory substances by the individual components of the consortium. Our data indicate that the stability over time and a potential antilisterial activity are individual characteristics of the ripening consortia which can be monitored and used for safe food production without artificial preservatives.     

Metagenomic DNA Extraction of Natural Cellulose-Degrading Consortia

A natural cellulose-degrading consortium is able to efficiently convert various types of lignocellulose to organic products, which is considered a potential method for the production of biofuels and biochemicals from lignocellulose materials. Metagenomics is an efficient method for determining the complex microbiota structure and performing metabolic mechanism analysis. However, natural cellulose-degrading consortia not only have complicated microbiome compositions and metabolites but also have tight adsorption between cells and cellulose fibers, which could have a significant influence on metagenomic DNA extraction, leading to a reduction of the DNA concentration, integrity, and diversity. Here, we found that bacterial cells that bind to cellulose fibers were efficiently washed off by a high-salt solution treatment and that the wall-breaking ratios of the biological enzyme and mechanical homogenization method were significantly higher than those of grinding and the freeze-thaw method. These results helped to establish a new method for extraction of the consortia metagenome that efficiently decreased absorption between cells and cellulose fibers, reduced the adverse impact of metabolites on DNA extraction, and improved the quality of DNA extraction.     

Biofuel Cells Select for Microbial Consortia That Self-Mediate Electron Transfer

Microbial fuel cells hold great promise as a sustainable biotechnological solution to future energy needs. Current efforts to improve the efficiency of such fuel cells are limited by the lack of knowledge about the microbial ecology of these systems. The purposes of this study were (i) to elucidate whether a bacterial community, either suspended or attached to an electrode, can evolve in a microbial fuel cell to bring about higher power output, and (ii) to identify species responsible for the electricity generation. Enrichment by repeated transfer of a bacterial consortium harvested from the anode compartment of a biofuel cell in which glucose was used increased the output from an initial level of 0.6 W m⁻² of electrode surface to a maximal level of 4.31 W m⁻² (664 mV, 30.9 mA) when plain graphite electrodes were used. This result was obtained with an average loading rate of 1 g of glucose liter⁻¹ day⁻¹ and corresponded to 81% efficiency for electron transfer from glucose to electricity. Cyclic voltammetry indicated that the enhanced microbial consortium had either membrane-bound or excreted redox components that were not initially detected in the community. Dominant species of the enhanced culture were identified by denaturing gradient gel electrophoresis and culturing. The community consisted mainly of facultative anaerobic bacteria, such as Alcaligenes faecalis and Enterococcus gallinarum, which are capable of hydrogen production. Pseudomonas aeruginosa and other Pseudomonas species were also isolated. For several isolates, electrochemical activity was mainly due to excreted redox mediators, and one of these mediators, pyocyanin produced by P. aeruginosa, could be characterized. Overall, the enrichment procedure, irrespective of whether only attached or suspended bacteria were examined, selected for organisms capable of mediating the electron transfer either by direct bacterial transfer or by excretion of redox components.     

Quantitative Molecular Assay for Fingerprinting Microbial Communities of Wastewater and Estrogen-Degrading Consortia

A quantitative fingerprinting method, called the real-time terminal restriction fragment length polymorphism (real-time-t-RFLP) assay, was developed for simultaneous determination of microbial diversity and abundance within a complex community. The real-time-t-RFLP assay was developed by incorporating the quantitative feature of real-time PCR and the fingerprinting feature of t-RFLP analysis. The assay was validated by using a model microbial community containing three pure strains, an Escherichia coli strain (gram negative), a Pseudomonas fluorescens strain (gram negative), and a Bacillus thuringiensis strain (gram positive). Subsequently, the real-time-t-RFLP assay was applied to and proven to be useful for environmental samples; the richness and abundance of species in microbial communities (expressed as the number of 16S rRNA gene copies of each ribotype per milliliter) of wastewater and estrogen-degrading consortia (enriched with 17α-estradiol, 17β-estradiol, or estrone) were successfully characterized. The results of this study strongly suggested that the real-time-t-RFLP assay can be a powerful molecular tool for gaining insight into microbial communities in various engineered systems and natural habitats.     

Characterization of Microbial Consortia in Paddy Rice Soil by Phospholipid Analysis

Abstract Microbial biomass and community structure in paddy rice soil during the vegetation period of rice were estimated by analysis of their phospholipid fatty acids (PLFA), hydroxy fatty acids of lipopolysaccharides (LPS-HYFA), and phospholipid ether lipids (PLEL) directly extracted from the soil. A clear change in the composition of the community structure at different sampling periods was observed, indicated by the principal component analysis of the PLFA. A dramatic decline of ester-linked PLFA was observed in the soil samples taken at the second sampling time. In contrast to the ester-linked PLFA, the non-ester-linked PLFA composition did not change. The hydroxy fatty acids of lipopolysaccharides as well as ether lipids decreased consecutively during the observation period. Total microbial abundance was estimated to be (4.1–7.3) × 10⁹ cells g^-1 soil (dry weight). About 44% account for aerobic and 32% for facultative anaerobic bacteria, and 24% for archaea, on average. According to the profile and patterns of PLFA in the soil sample, it may be suggested that the paddy soil at the August sampling period contained more abundant facultative anaerobic bacteria (ca. 36%) and archaea (ca. 37%), but the total microbial biomass was significantly lower than in the remaining sampling periods. As the plant approached maturity, the microbial community structure in the soil changed to contain more abundant Gram-negative bacteria and methanotrophs. Received: 23 September 1999; Accepted: 28 February 2000; Online Publication: 12 May 2000     

Degradation of Nickel Protoporphyrin Disodium and Vanadium Oxide Octaethylporphyrin by Philippine Microbial Consortia

Microbial degradation of nickel and vanadium porphyrins is an economically important and environment-friendly alternative to physicochemical processes currently used in refining crude oil. This study involved the screening of 23 microbial isolates from crude oil–contaminated soils in the Philippines. Two microbial consortia concocted out of four bacteria and three fungi from Guimaras Island Province degraded significantly higher amounts of nickel protoporphyrin disodium (NiPPDS) and vanadium oxide octaethyl porphyrin (VOOEP) than their corresponding member components. Culture parameters were varied and then optimized by the Taguchi method in assays in minimal salt medium supplemented with metalloporphyrins. Optimal degradations by consortium GI-2,3 (Bacterium megaterium–Enterobacter cloacae) were 79 ± 1.5% for NiPPDS at 40 mg/L, pH 7, 30°C and 89 ± 1.7% for VOOEP at 20 mg/L, pH 6, 30°C. For consortium As-2,P (Aspergillus unguis–Penicillium griseofulvum), optimal degradations w`ere 71 ± 1.3% for NiPPDS at 20 mg/L, pH 5.5, 30°C and 90 ± 2.8% for VOOEP at 20 mg/L, pH 4.5, 40°C.     

Development of Steady-State Diffusion Gradients for the Cultivation of Degradative Microbial Consortia

A diffusion gradient plate was constructed and evaluated for its potential use in the isolation of degradative microbial consortia from natural habitats. In this model, a steady-state concentration gradient of diclofop methyl, established by diffusion through an agarose gel, provided the carbon for microbial growth. Colonization of the gel surface was observed with epifluorescence and scanning confocal laser microscopy to determine microbial responses to the diclofop gradient. A detectable gradient developed over a narrow band (<10 mm). Consequently, quantitative analyses of the microbial response to the gradient were difficult to obtain. A two-dimensional, finite-element numerical transport model for advective-diffusive transport was used to simulate concentration and flux profiles in the physical model. The simulated profiles were correlated with the measured concentration gradient (R² = 0.89) and the cell numbers on the gel surface (R² = 0.85). The numerical model was subsequently used to redesign the physical model. The detectable concentration gradient in the modified physical model extended over the length of the gel (38 mm). The simulated profile again showed a good correlation with the measured profile (R² = 0.96) and the microbial responses to the concentration gradient (R² = 0.99). It was concluded that these gradients provide the steady-state environments needed to sustain steady-state consortia. They also provide a physical pathway for the development of degradative biofilms from low to high concentrations of toxicants and simulate conditions under which low concentrations of toxicant are supplied at a constant flux over long periods of time, such as the conditions that could occur in natural environments.     

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.     

利用高通量测序技术对水稻秸秆中、低温降解菌系的比较分析

采用Illumina MiSeq高通量测序技术,对中、低温（中温25 ℃、低温10 ℃）富集驯化所得水稻秸秆降解菌系进行测序;采用生物信息学方法对中、低温秸秆降解菌系群落生物多样性进行分析。结果表明,测序质控后获得601 489条16S rDNA序列,平均长度273 bp,经Silval 132数据库比对,中温降解菌系包括18个门,172个属,302个OTU,其中优势菌属为弓形杆菌属（Arcobacter）、拟杆菌属（Bacteroides）、屠场杆菌属（Macellibacteroides）、假单胞菌属（Pseudomonas）和梭形杆菌属（Lysinibacillus）;低温降解菌系包括16个门,169个属,280个OTU,其中优势菌属为弓形杆菌属、屠场杆菌属、拟杆菌属、丛毛单胞属（Comamonas）和假单胞菌属。温度对降解菌系中优势菌门的相对丰度无显著影响,中、低温秸秆降解菌系主要菌属相对丰度差异显著。     

Novel Microbial Populations in Ambient and Mesophilic Biogas-Producing and Phenol-Degrading Consortia Unraveled by High-Throughput Sequencing

Methanogenesis from wastewater-borne organics and organic solid wastes (e.g., food residues) can be severely suppressed by the presence of toxic phenols. In this work, ambient (20 °C) and mesophilic (37 °C) methane-producing and phenol-degrading consortia were enriched and characterized using high-throughput sequencing (HTS). 454 Pyrosequencing indicated novel W22 (25.0 % of bacterial sequences) in the WWE1 and Sulfurovum-resembled species (32.0 %) in the family Campylobacterales were the most abundant in mesophilic and ambient reactors, respectively, which challenges previous knowledge that Syntrophorhabdus was the most predominant. Previous findings may underestimate bacterial diversity and low-abundance bacteria, but overestimate abundance of Syntrophorhabdus. Illumina HTS revealed that archaeal populations were doubled in ambient reactor and tripled in mesophilic reactor, respectively, compared to the ～4.9 % (of the bacteria and archaea sequences) in the seed sludge. Moreover, unlike the dominance of Methanosarcina in seed sludge, acetotrophic Methanosaeta predominated both (71.4–76.5 % of archaeal sequences) ambient and mesophilic enrichments. Noteworthy, this study, for the first time, discovered the co-occurrence of green sulfur bacteria Chlorobia, sulfur-reducing Desulfovibrio, and Sulfurovum-resembling species under ambient condition, which could presumably establish mutualistic relationships to compete with syntrophic bacteria and methanogens, leading to the deterioration of methanogenic activity. Taken together, this HTS-based study unravels the high microbial diversity and complicated bacterial interactions within the biogas-producing and phenol-degrading bioreactors, and the identification of novel bacterial species and dominant methanogens involved in the phenol degradation provides novel insights into the operation of full-scale bioreactors for maximizing biogas generation.     

Genomic Mining for Novel FADH2-Dependent Halogenases in Marine Sponge-Associated Microbial Consortia

Many marine sponges (Porifera) are known to contain large amounts of phylogenetically diverse microorganisms. Sponges are also known for their large arsenal of natural products, many of which are halogenated. In this study, 36 different FADH₂-dependent halogenase gene fragments were amplified from various Caribbean and Mediterranean sponges using newly designed degenerate PCR primers. Four unique halogenase-positive fosmid clones, all containing the highly conserved amino acid motif “GxGxxG”, were identified in the microbial metagenome of Aplysina aerophoba. Sequence analysis of one halogenase-bearing fosmid revealed notably two open reading frames with high homologies to efflux and multidrug resistance proteins. Single cell genomic analysis allowed for a taxonomic assignment of the halogenase genes to specific symbiotic lineages. Specifically, the halogenase cluster S1 is predicted to be produced by a deltaproteobacterial symbiont and halogenase cluster S2 by a poribacterial sponge symbiont. An additional halogenase gene is possibly produced by an actinobacterial symbiont of marine sponges. The identification of three novel, phylogenetically, and possibly also functionally distinct halogenase gene clusters indicates that the microbial consortia of sponges are a valuable resource for novel enzymes involved in halogenation reactions.     

Comparison of Microbial Consortia in Refuse-Derived Fuel (RDF) Preparations between Japan and Germany

Refuse-derived fuels (RDF) pellets manufactured in Japan have been reported to contain a relatively high number of viable bacterial cells, and these bacteria generated a large amount of hydrogen gas during fermentation under wet conditions. In this study, we compared hydrogen gas generation from RDF pellets manufactured in Japan and in Germany and found that a large amount of hydrogen gas was generated from the Japanese RDF pellets but not from the German ones. This difference can be explained by the absence and presence of a biodegradation process before molding of raw garbage into RDF pellets. That is, the German process includes a biodegradation (or biological drying) process with forced aeration for a week, and this appears to reduce BOD in the garbage. Denaturing gradient gel electrophoresis analysis of 16S rRNA gene followed by DNA sequencing indicated that microbiotas of the RDF pellets manufactured in Japan and in Germany were very different.     

DNA末端构型调控RecJ核酸外切酶活性

目的 核酸酶介导的DNA双链末端切割对同源重组修复至关重要。然而,DNA末端构型对RecJ 5’-3’核酸外切酶活性的调控尚不清楚。本研究旨在探究DNA3’端和5’端构型对RecJ核酸外切酶活性的影响及其机制。方法 为探究DNA3’端构型对RecJ核酸外切酶活性的影响,使用含有Mg²⁺的体系,对具有不同3’突出末端长度（9 nt与18 nt）和3’突出末端修饰（磷酸化和硫代磷酸酯修饰）的单链DNA分别进行RecJ核酸酶活性检测。为揭示DNA 3’端构型对RecJ外切酶活性的调控机制,在Mg²⁺缺失的体系中,使RecJ与底物结合后进行凝胶迁移实验（EMSA）。为探索其他调控因子与DNA3’端构型对RecJ的协同作用,分别检测5’端磷酸化修饰和单链DNA结合蛋白（SSB）对DNA3’突出末端修饰的影响。结果 DNA3’端构型包括突出末端的长度和修饰（磷酸化和硫代磷酸酯修饰）均会抑制RecJ外切酶活性。DNA 3’端磷酸化和硫代磷酸酯修饰通过重塑RecJ-DNA的结合模式抑制RecJ外切酶活性。DNA 5’端磷酸化修饰可增强RecJ对具有不同3’端...