Community and Proteomic Analysis of Methanogenic Consortia Degrading Terephthalate

Degradation of terephthalate (TA) through microbial syntrophy under moderately thermophilic (46 to 50°C) methanogenic conditions was characterized by using a metagenomic approach (A. Lykidis et al., ISME J. 5:122–130, 2011). To further study the activities of key microorganisms responsible for the TA degradation, community analysis and shotgun proteomics were used. The results of hierarchical oligonucleotide primer extension analysis of PCR-amplified 16S rRNA genes indicated that Pelotomaculum, Methanosaeta, and Methanolinea were predominant in the TA-degrading biofilms. Metaproteomic analysis identified a total of 482 proteins and revealed a distinctive distribution pattern of microbial functions expressed in situ. The results confirmed that TA was degraded by Pelotomaculum spp. via the proposed decarboxylation and benzoyl-coenzyme A-dependent pathway. The intermediate by-products, including acetate, H₂/CO₂, and butyrate, were produced to support the growth of methanogens, as well as other microbial populations that could further degrade butyrate. Proteins related to energy production and conservation, and signal transduction mechanisms (that is, chemotaxis, PAS/GGDEF regulators, and stress proteins) were highly expressed, and these mechanisms were important for growth in energy-limited syntrophic ecosystems.     

Simulating the Contribution of Coaggregation to Interspecies Hydrogen Fluxes in Syntrophic Methanogenic Consortia

A simple model (termed the syntrophy model) for simulating the contribution of coaggregation to interspecies hydrogen fluxes between syntrophic bacteria and methanogenic archaea is described. We applied it to analyzing partially aggregated syntrophic cocultures with various substrates, revealing that large fractions of hydrogen molecules were fluxed in aggregates.     

酸性条件下耐酸产甲烷颗粒污泥的培养及特性*

分别采用中性颗粒污泥和河底沉积物接种运行两个颗粒污泥膨胀床(EGSB)反应器,通过逐级降低pH的运行策略,驯化和培养了耐酸产甲烷颗粒污泥,两个EGSB反应器均能在pH5．8-6．2条件下稳定运行,容积负荷可达5．5-7．5kg COD,(m^3／d),COD去除率约90％;两种颗粒污泥在低pH值下均能保持较高的产甲烷活性,pH5．5时,仍能保持pH7．0时活性的51．8％和55．6％;还对耐酸颗粒污泥的粒径分布、沉降性能、金属元素含量、微观结构及细菌在颗粒表面和内部的分布等进行了研究。     

Biomethanation of low pH petrochemical wastewater using up-flow fixed-film anaerobic bioreactors

Anaerobic digestion of wastewater from a petrochemical plant, manufacturing Nylon-6 was studied in continuously fed up-flow fixed-film column reactors using different biomass support materials such as bonechar, charcoal, bricks, plastic beads and polyurethane foam under varying hydraulic and organic loading rates. Experimental results showed bonechar as the best support material with high biomass-retaining capacity because of its high specific surface area (53.35m²g^–1 of bedding material) and pore specific volume (0.244cm³g^–1 of bedding material). This system could treat waste water at hydraulic retention times (HRT) as low as 2.5 days with organic loading rates as high as 21.76kg COD m^–3 day^–1 using acidic feed of pH 2.5 resulting in a 95% COD reduction with biogas production of 11.76m³ m^–3 of reactor volume day^–1. Total alkalinity of 1700mg CaCO₃ l^–1 and pH of 7.5 of the treated wastewater were observed at 2.5 days HRT, indicating that methanogenesis appear to be alkalizing step and wastewater with pH as low as 2.5 can be treated as such without neutralization with retention of methanogenic biomass on bonechar.     

Fermentation Enhancement of Methanogenic Archaea Consortia from an Illinois Basin Coalbed via DOL Emulsion Nutrition

Microbially enhanced coalbed methane technology must be used to increase the methane content in mining and generate secondary biogenic gas. In this technology, the metabolic processes of methanogenic consortia are the basis for the production of biomethane from some of the organic compounds in coal. Thus, culture nutrition plays an important role in remediating the nutritional deficiency of a coal seam. To enhance the methane production rates for microorganism consortia, different types of nutrition solutions were examined in this study. Emulsion nutrition solutions containing a novel nutritional supplement, called dystrophy optional modification latex, increased the methane yield for methanogenic consortia. This new nutritional supplement can help methanogenic consortia form an enhanced anaerobic environment, optimize the microbial balance in the consortia, and improve the methane biosynthesis rate.     

A Formalized Design Process for Bacterial Consortia That Perform Logic Computing

The concept of microbial consortia is of great attractiveness in synthetic biology. Despite of all its benefits, however, there are still problems remaining for large-scaled multicellular gene circuits, for example, how to reliably design and distribute the circuits in microbial consortia with limited number of well-behaved genetic modules and wiring quorum-sensing molecules. To manage such problem, here we propose a formalized design process: (i) determine the basic logic units (AND, OR and NOT gates) based on mathematical and biological considerations; (ii) establish rules to search and distribute simplest logic design; (iii) assemble assigned basic logic units in each logic operating cell; and (iv) fine-tune the circuiting interface between logic operators. We in silico analyzed gene circuits with inputs ranging from two to four, comparing our method with the pre-existing ones. Results showed that this formalized design process is more feasible concerning numbers of cells required. Furthermore, as a proof of principle, an Escherichia coli consortium that performs XOR function, a typical complex computing operation, was designed. The construction and characterization of logic operators is independent of “wiring” and provides predictive information for fine-tuning. This formalized design process provides guidance for the design of microbial consortia that perform distributed biological computation.     

Effect of pH on Anaerobic Mild Steel Corrosion by Methanogenic Bacteria

Methanogens can use H₂ produced by cathodic depolarization-mediated oxidation of elemental iron to produce methane. Thermodynamic consideration of the cathodic depolarization mechanism predicts more oxidation of Fe⁰ at lower pH. Methanogenic responses to pH by Methanococcus deltae, Methanococcus thermolithotrophicus, and Methanosarcina barkeri were examined. When grown on H₂-CO₂, these bacteria had pH optima from 6.2 to 7.0, but when all H₂ was supplied from Fe⁰, methanogenic pH optima were lower, 5.4 to 6.5. Corrosion was monitored with and without cultures and at various pHs; more corrosion occurred when cultures were present, biologically induced corrosion was greatest at the pH optima for methanogenesis from Fe⁰, and corrosion without cultures increased with a drop in pH.     

Heterogeneous Distribution of Microbial Activity in Methanogenic Aggregates: pH and Glucose Microprofiles

Methanogenic aggregates, harvested from an upflow anaerobic sludge blanket reactor treating potato starch wastewater, were acclimatized to either glucose or a mixture of sugars and organic nitrogen compounds (i.e., diluted molasses). Both types of granules exhibited internal pH and substrate concentration gradients in mineral medium (pH 7.0, 30°C) as was measured with microelectrodes. Glucose-acclimatized granules suspended in a mineral medium lacking glucose exhibited a distinct internal pH decrease of about 1 U within the granule, suggesting strong metabolism by the acidogenic bacteria. Molasses-acclimatized and aged granules suspended in mineral medium did not exhibit such a pH decrease, suggesting the importance of the metabolic state of these acidogens. The pH gradient did not occur in deactivated granules and was not observable in strongly buffered media (mineral medium containing 33 mM phosphate or reactor liquid). When glucose (0.5 to 5.0 mM) was added to the mineral medium, granules exhibited a convex pH profile. Glucose consumption was located exclusively in the outer 200 to 300 μm of the aggregates (mean diameter = 1.5 mm). The addition of 20 mM 2-bromoethanesulfonic acid to the mineral medium indicated that the higher pH levels in the centre of the granule appeared to be related to the activity of methanogens. It is suggested that acidogenic activity occurs predominantly in the outer 200 to 300 μm of the aggregate and methanogenic activity occurs predominantly in the center of the investigated granules.     

Biomethanation of industrial effluents for energy recovery in China

Summary China has large amounts of effluents containing high organic loads. It is paying special attention to intensifying the process of biomethanation to increase the biogas production per unit of reactor volume and to reduce pollution. At present, a few plants have been realized such as two plants treating urban wastewater sludge, one plant working on effluents from the citric acid industry and three plants working with effluent from three alcohol distilleries. Biomethanation of waste liquor from pulp processing by lime has been studied on the laboratory scale and 79% COD removal has been obtained. An Upflow Anaerobic Sludge Blanket (UASB) pilot plant of 24 m³ fed with filtered residual liquor from a distillery produces 5 m³/m³ digester per day with a COD removal of 93%. Until now the extension of biomethanation of industrial effluents has been limited because the COD content of the effluents is still beyond the effluent discharge standards.

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Resumen China tiene una gran cantidad de efluentes con un nivel de polución orgánica elevado. Actualmente se está prestando una atención especial a los procesos de biometanación para poder incrementar la producción de biogas por unidad de volumen del reactor y aumentar la reducción del nivel de polución. Hasta ahora tan solo existen 2 plantas de tratemiento de aguas residuales urbanas, 1 planta que trabajan con efluentes de una fábrica de ácido ctrico y 3 plantas que trabajan con efluentes de 3 destilerias de alcool. Se ha estudiado a nivel de laboratorio la biometanación del licor de desecho del tratamiento de la pulpa con cal obteniéndose una disminución del 79% del COD. Una planta piloto UASB de 24 m³ alimentada con licor residual de destilerias filtrado produce 5 m³ y por día con una disminución del COD del 93%. Hasta el momento la expansión de la biometanación se ha visto frenada porque el contenido en COD de los efluentes no está aún contemplado en la normativa sobre vertidos líquidos.

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Résumé La Chine produit de grandes quantités d'effluents à fortes concentrations en matières organiques. Elle attache donc une importance particulière à l'intensification des procédés de biométhanisation afin d'en augmenter la productivité et l'effet dépolluant. A l'heure actuelle il existe quelques réalisations telles que deux installations de traitement des boues d'eaux résiduaires urbaines, un installation alimentées par les effluents d'industries de production d'acide citrique, et trois installations traitant des effluents de distillerie d'alcool. La biométhanisation d'eaux résiduaires résultant du processing de la pâte à papier par la chaux a été étudiée à l'échelle laboratoire et une réduction de 79% de la DCO a été obtenue. Une installation pilote (Upflow Anaerobic Sludge Blanket, UASB) de 24 m³ alimentée par les eaux résiduaires filtrées d'une distillerie produit 5 m³ par m³ et par jour avec une élimination de 93% de la DCO. Jusqu'à présent le développement de la biométhanisation d'effluents industriels a été limitée par un contenu en DCO des effluents méthanisés encore supérieur aux normes de décharge.

     

Biomethanation of rice and wheat straw

When rice or wheat straw was added to cattle dung slurry and digested anaerobically, daily gas production increased from 176 to 331 l/kg total solids with 100% rice straw and to 194 l/kg total solids with 40% wheat straw. Not only was methane production enhanced by adding chopped crop residues but a greater biodegradability of organic matter in the straws was achieved.The authors are with the Microbiology and Molecular Genetics Unit, Tata Energy Research Institute, 158 Jor Bagh, New Delhi 110 003, India     

Engineering of Phytase for Improved Activity at Low pH

For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.     

Use of Silicate Minerals for pH Control during Reductive Dechlorination of Chloroethenes in Batch Cultures of Different Microbial Consortia

In chloroethene-contaminated sites undergoing in situ bioremediation, groundwater acidification is a frequent problem in the source zone, and buffering strategies have to be implemented to maintain the pH in the neutral range. An alternative to conventional soluble buffers is silicate mineral particles as a long-term source of alkalinity. In previous studies, the buffering potentials of these minerals have been evaluated based on abiotic dissolution tests and geochemical modeling. In the present study, the buffering potentials of four silicate minerals (andradite, diopside, fayalite, and forsterite) were tested in batch cultures amended with tetrachloroethene (PCE) and inoculated with different organohalide-respiring consortia. Another objective of this study was to determine the influence of pH on the different steps of PCE dechlorination. The consortia showed significant differences in sensitivities toward acidic pH for the different dechlorination steps. Molecular analysis indicated that Dehalococcoides spp. that were present in all consortia were the most pH-sensitive organohalide-respiring guild members compared to Sulfurospirillum spp. and Dehalobacter spp. In batch cultures with silicate mineral particles as pH-buffering agents, all four minerals tested were able to maintain the pH in the appropriate range for reductive dechlorination of chloroethenes. However, complete dechlorination to ethene was observed only with forsterite, diopside, and fayalite. Dissolution of andradite increased the redox potential and did not allow dechlorination. With forsterite, diopside, and fayalite, dechlorination to ethene was observed but at much lower rates for the last two dechlorination steps than with the positive control. This indicated an inhibition effect of silicate minerals and/or their dissolution products on reductive dechlorination of cis-dichloroethene and vinyl chloride. Hence, despite the proven pH-buffering potential of silicate minerals, compatibility with the bacterial community involved in in situ bioremediation has to be carefully evaluated prior to their use for pH control at a specific site.     

Low pH myoglobin photoproducts.

Recently, there has been interest in determining the conditions under which the iron-histidine bond ruptures in myoglobin at low pH, so that the effect of proximal heme ligation can be studied. A 220-cm-1 Raman mode, assigned to iron-histidine stretching, is clearly visible after photolysis of aqueous MbCO samples below pH4 at room temperature (Sage et al. Biochemistry. 30:1237-1247). In contrast, Iben et al. (Biophys. J. 59:908-919) do not observe this mode upon photolysis of a pH3 MbCO sample in a glycerol/water glass at low temperature. In order to account for both the low temperature and the room temperature experiments, Iben et al. suggest a scheme involving an unusual protonation state of the proximal histidine. Here, we discuss some inconsistencies in their explanation of the room temperature results and offer instead a simple modification of an earlier model. In addition, circular dichroism data are presented that indicate partial unfolding of MbCO in aqueous solution below pH4, and raise questions about the claim of Iben et al. that MbCO remains folded in 75% glycerol at pH3.     

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.     

Effects of pH, Temperature, and Nutrients on Propionate Degradation by a Methanogenic Enrichment Culture

Enrichment cultures were used to determine the conditions promoting fastest methanogenic propionate degradation and growth by adapting the cultures to various physical and chemical conditions and measuring the specific growth rate. We found that the fastest growth of propionate oxidizers occurred at pH 6.8 to 8.5 and 32 to 45°C. Acetate-degrading populations showed narrower optima for fastest growth (pH 6.8 to 7.2 and 37 to 43°C). Enrichment cultures grew as well in minimal medium as in complex medium, although individual microbial populations appeared to require growth factors which could be met by cross-feeding.     

Mixed-Culture Fermentor for Simulating Methanogenic Digestors

Propionate degradation in an anaerobic digestor degrading animal waste (10-day retention time, 5.75 g liter⁻¹ day⁻¹ volatile solids loading rate, 40°C) was 0.304 mM h⁻¹, measured with [2-¹⁴C]propionate; this value indicated that CH₄ produced from propionate accounted for 14.8% of the CH₄ produced in the digestor (34.5%, including acetate produced from propionate). The mean propionate concentration was 0.67 mM, giving a propionate turnover rate of 0.46 h⁻¹. A continuous-, mixed-culture fermentor was developed to mimic the digestor. When degradation rates of methanogenic precursors (H₂, CO₂, and acetate) equalled those measured in the digestor, propionate degradation was inhibited. When the H₂ turnover rate was lowered by decreasing addition of H₂-generating substrates or by allowing a portion of the H₂ degradation to occur in an isolated compartment, propionate degradation in the fermentor resumed. The possibility is discussed that in digestors, much of the H₂ is produced and degraded within microenvironments associated with particles. Thus, the gross turnover rate of H₂ measured in digestors is an average, and specific microenvironments within the digestor may have different rates of turnover.     

Biomethanation of white rotted and brown rotted rice straw

Biomethanation of white rotted and brown rotted rice straw was taken for the present investigation and their efficiency on biomethanation has been tested. Rice straw was treated with white rot fungus Phanerochaete chrysosporium (P_C) and brown rot fungus Polyporus ostreiformis (P_O). Biogas and methane production was increased by about 34.73% and 46.19% in P_C-treated straw and 21.12% and 31.94% in P_O-treated straw respectively. VFA production has also been increased in P_C and P_O treated straw compared to control straw which were 76.73% and 30.69% respectively. Reduction of COD has also been found during biomethanation. The rate of reduction of COD during the initial period of digestion was 59.01%, 55.55% and 26.00% in P_C-treated, P_O-treated and control straw respectively after 21 days of digestion.     

Liquid-to-Gas Mass Transfer in Anaerobic Processes: Inevitable Transfer Limitations of Methane and Hydrogen in the Biomethanation Process

Liquid-to-gas mass transfer in anaerobic processes was investigated theoretically and experimentally. By using the classical definition of k(L)a, the global volumetric mass transfer coefficient, theoretical development of mass balances in such processes demonstrates that the mass transfer of highly soluble gases is not limited in the usual conditions occurring in anaerobic fermentors (low-intensity mixing). Conversely, the limitation is important for poorly soluble gases, such as methane and hydrogen. The latter could be overconcentrated to as much as 80 times the value at thermodynamic equilibrium. Such overconcentrations bring into question the biological interpretations that have been deduced solely from gaseous measurements. Experimental results obtained in three different methanogenic reactors for a wide range of conditions of mixing and gas production confirmed the general existence of low mass transfer coefficients and consequently of large overconcentrations of dissolved methane and hydrogen (up to 12 and 70 times the equilibrium values, respectively). Hydrogen mass transfer coefficients were obtained from the direct measurements of dissolved and gaseous concentrations, while carbon dioxide coefficients were calculated from gas phase composition and calculation of related dissolved concentration. Methane transfer coefficients were based on calculations from the carbon dioxide coefficients. From mass balances performed on a gas bubble during its simulated growth and ascent to the surface of the liquid, the methane and carbon dioxide contents in the gas bubble appeared to be controlled by the bubble growth process, while the bubble ascent was largely responsible for a slight enrichment in hydrogen.     

A Versatile Medium for Cultivating Methanogenic Archaea

Background

Methanobrevibacter smithii, Methanobrevibacter oralis, Methanosphaera stadtmanae, Methanomassilicoccus luminyensis and Methanobrevibacter arboriphilicus have been cultured from human digestive microbiota. Each one of these fastidious methanogenic archaea requires a specific medium for its growth, hampering their routine isolation and the culture.

Methodology/Principal Findings

A new culture medium here referred as SAB medium was optimized and tested to cultivate methanogens associated with human microbiota, as well as two mesophile methanogens Methanobacterium beijingense and Methanosaeta concilii. It was further tested for the isolation of archaea from 20 human stool specimens including 10 specimens testing positive for PCR detection of M. smithii. After inoculating 10⁵ colony-forming-unit archaea/mL or 1 g stool specimen in parallel in SAB medium and reference DSMZ medium in the presence of negative controls, growth of archaea was determined by optical microscopy and the measurement of methane production by gas chromatography. While the negative controls remained sterile, all tested archaea grew significantly more rapidly in SAB medium than in reference medium in 1–3 days (P<0.05, Student test). Among PCR-positive stool specimens, 10/10 grew in the SAB medium, 6/10 in DSMZ 119 medium, 5/10 in DSMZ 322 medium and 3/10 in DSMZ 334 c medium. Four out of ten PCR-negative stool specimens grew after a 3-week incubation in the SAB-medium whereas no growth was detected in any of the reference media. 16S rRNA gene sequencing yielded 99–100% sequence similarity with reference M. smithii except for one specimen that yielded 99–100% sequence similarity with reference Methanobrevibacter millerae.

Conclusions/Significance

SAB medium allows for the versatile isolation and growth of methanogenic archaea associated with human gut microbiota including the archaea missed by inoculation of reference media. Implementation of the SAB medium in veterinary and medical microbiology laboratories will ease the routine culture-based detection of methanogenic archaea in clinical and environmental specimens.