The isolation and microbial community analysis of hydrogen producing bacteria from activated sludge

AIMS: To profile the fractions of bacteria in heat-treated activated sludge capable of producing hydrogen and subsequently to isolate those organisms and confirm their ability to produce hydrogen. METHODS AND RESULTS: Profiling the community composition of the microflora in activated sludge using 16S rRNA gene-directed polymerase chain reaction-denaturing gradient gel electrophoresis suggested that a majority of bacteria were various Clostridium species. This was confirmed by clone library analysis, where 80% of the cloned inserts were Clostridium sp. A total of five isolates were established on solid media. Three of them, designated as W1, W4 and W5, harboured the hydrogenase gene as determined by PCR and DNA sequence analysis (99% similarity). These isolates were similar to Clostridium butyricum and Clostridium diolis as determined by 16S rRNA gene sequence. A maximum hydrogen production yield of 220 ml H(2) g(-1) glucose was achieved by W5, which was grown on improved mineral medium by batch fermentation without pH adjustment and nitrogen sparging during fermentation. Accumulation of malic acid and fumaric acid during hydrogen fermentation might lead to higher hydrogen yields for W4 and W5. W1 is the first reported Clostridium species that can tolerate microaerobic conditions for producing hydrogen. CONCLUSION: Clostridium species in heat-treated activated sludge were the most commonly identified bacteria responsible for hydrogen production. Specific genetic markers for strains W1, W4 and W5 would be of great utility in investigating hydrogen production at the molecular level. Two previously described primer sets targeting hydrogenase genes were shown not to be specific, amplifying other genes from nonhydrogen producers. SIGNIFICANCE AND IMPACT OF THE STUDY: Clostridium species isolated from heat-treated activated sludge were confirmed as hydrogen producers during dark hydrogen fermentation. The isolates will be useful for studying hydrogen production from wastewater, including the process of gene regulation and hydrogenase activity.     

Characterization of microbial biofilms in a thermophilic biogas system by high-throughput metagenome sequencing

DNAs of two biofilms of a thermophilic two-phase leach-bed biogas reactor fed with rye silage and winter barley straw were sequenced by 454-pyrosequencing technology to assess the biofilm-based microbial community and their genetic potential for anaerobic digestion. The studied biofilms matured on the surface of the substrates in the hydrolysis reactor (HR) and on the packing in the anaerobic filter reactor (AF). The classification of metagenome reads showed Clostridium as most prevalent bacteria in the HR, indicating a predominant role for plant material digestion. Notably, insights into the genetic potential of plant-degrading bacteria were determined as well as further bacterial groups, which may assist Clostridium in carbohydrate degradation. Methanosarcina and Methanothermobacter were determined as most prevalent methanogenic archaea. In consequence, the biofilm-based methanogenesis in this system might be driven by the hydrogenotrophic pathway but also by the aceticlastic methanogenesis depending on metabolite concentrations such as the acetic acid concentration. Moreover, bacteria, which are capable of acetate oxidation in syntrophic interaction with methanogens, were also predicted. Finally, the metagenome analysis unveiled a large number of reads with unidentified microbial origin, indicating that the anaerobic degradation process may also be conducted by up to now unknown species.     

Taxonomic composition and gene content of a methane-producing microbial community isolated from a biogas reactor

A total community DNA sample from an agricultural biogas reactor continuously fed with maize silage, green rye, and small proportions of chicken manure has recently been sequenced using massively parallel pyrosequencing. In this study, the sample was computationally characterized without a prior assembly step, providing quantitative insights into the taxonomic composition and gene content of the underlying microbial community. Clostridiales from the phylum Firmicutes is the most prevalent phylogenetic order, Methanomicrobiales are dominant among methanogenic archaea. An analysis of Operational Taxonomic Units (OTUs) revealed that the entire microbial community is only partially covered by the sequenced sample, despite that estimates suggest only a moderate overall diversity of the community. Furthermore, the results strongly indicate that archaea related to the genus Methanoculleus, using CO(2) as electron acceptor and H(2) as electron donor, are the main producers of methane in the analyzed biogas reactor sample. A phylogenetic analysis of glycosyl hydrolase protein families suggests that Clostridia play an important role in the digestion of polysaccharides and oligosaccharides. Finally, the results unveiled that most of the organisms constituting the sample are still unexplored.     

Performance and microbial community analysis of two-stage process with extreme thermophilic hydrogen and thermophilic methane production from hydrolysate in UASB reactors

The two-stage process for extreme thermophilic hydrogen and thermophilic methane production from wheat straw hydrolysate was investigated in up-flow anaerobic sludge bed (UASB) reactors. Specific hydrogen and methane yields of 89 ml-H₂/g-VS (190 ml-H₂/g-sugars) and 307 ml-CH₄/g-VS, respectively were achieved simultaneously with the overall VS removal efficiency of 81% by operating with total hydraulic retention time (HRT) of 4 days . The energy conversion efficiency was dramatically increased from only 7.5% in the hydrogen stage to 87.5% of the potential energy from hydrolysate, corresponding to total energy of 13.4 kJ/g-VS. Dominant hydrogen-producing bacteria in the H₂-UASB reactor were Thermoanaerobacter wiegelii, Caldanaerobacter subteraneus, and Caloramator fervidus. Meanwhile, the CH₄-UASB reactor was dominated with methanogens of Methanosarcina mazei and Methanothermobacter defluvii. The results from this study suggest the two stage anaerobic process can be effectively used for energy recovery and for stabilization of hydrolysate at anaerobic conditions.     

Monitoring of horizontal gene transfer from agricultural microorganisms to soil bacteria and analysis of microbial community in soils

To investigate the possibility of horizontal gene transfer between agricultural microorganisms and soil microorganisms in the environment, Bacillus subtilis KB producing iturin and the PGPR recombinant strain Pseudomonas fluorescens MX1 were used as model microorganisms. The soil samples of cucumber or tomato plants cultivated in pots and the greenhouse for a six month period were investigated by PCR, real-time PCR, Southern hybridization, and terminal restriction fragment length polymorphism (T-RFLP) fingerprinting. Our data from Southern blotting and TRFLP patterns suggest that the model bacteria do not give significant impacts on the other bacteria in the pots and greenhouse during cultivation.     

Comparison of six methods for the recovery of PCR-compatible microbial DNA from an agricultural biogas plant

Applied Microbiology and Biotechnology - Six different commercial methods were compared to evaluate their efficiency in recovering high quantity/quality PCR compatible microbial DNA from an...     

Physiological state of a microbial community in a biomass recycle reactor

The transition in physiological state was investigated between a carbon-limited chemostat population and microbes growing very slowly in a biomass recycle reactor. The mixed microbial population was metabolizing a mixture of biopolymers and linear alkylbenzene sulfonate, formulated to represent the organic load in graywater. Biomass increased 30-fold during the first 14 days after a shift from chemostat to biomass recycle mode. The ratios of ATP and RNA to cell protein decreased over the first days but then remained constant. The specific rate of CO₂ production by microbes in the reactor decreased 6-fold within 24 h after the shift, and respiratory potentials declined 2–3 fold during the first 7 days. Whereas chemostat cultures used equal proportions of organic carbon substrate for catabolism and anabolism, the proportion of organic substrate oxidized to CO₂ rose from 62 to 82% over the first 8 days in a biomass recycle reactor, and eventually reached 100% as this reactor population exhibited no net growth. Biomass recycle populations removed from the system and subjected to a nutritional shift-up did not immediately initiate exponential growth. The physiological state of cells in the biomass recycle reactor may be distinct from those grown in batch or continuous culture, or from starved cells. Received 02 June 1997/ Accepted in revised form 20 February 1998     

Biofilm microbial community of a thermophilic trickling biofilter used for continuous biohydrogen production

Molecular methods were employed to investigate the microbial community of a biofilm obtained from a thermophilic trickling biofilter reactor (TBR) that was operated long-term to produce H(2). Biomass concentration in the TBR gradually decreased as reactor bed height increased. Despite this difference in biomass concentration, samples from the bottom and middle of the TBR bed revealed similar microbial populations as determined by PCR-DGGE analysis of 16S rRNA genes. Nucleotide sequences of most DGGE bands were affiliated with the classes Clostridia and Bacilli in the phylum Firmicutes, and the most dominant bands showed a high sequence similarity to Thermoanaerobacterium thermosaccharolyticum.     

Comparison of signature lipid methods to determine microbial community structure in compost

The microbial community structure changes substantially during the composting process and simple methods to follow these changes can potentially be used to estimate compost maturity. In this study, two such methods, the microbial identification (MIDI) method and the ester-linked (EL) procedure to determine the composition of long-chain fatty acids, were applied to compost samples of different age. The ability of the two methods to describe the microbial succession was evaluated by comparison with phospholipid fatty acid (PLFA) analysis on the same samples.Samples were taken from a 200-l laboratory compost reactor, treating source-separated organic household waste. During the initial stages of the process, the total concentration of fatty acids in compost samples treated with the EL and MIDI methods was many times higher than with the PLFA method. This was probably due to the presence of fatty acids from the organic material in the original waste. However, this substantial difference between PLFA and the other two methods was not found later in composting. Although the PLFA method gave the most detailed information about the growth and overall succession of the microbial community, the much simpler MIDI and EL methods also successfully described the shift from the initially dominating straight chain fatty acids to iso- and anteiso branched, 10 Me branched and cyclopropane fatty acids in the later stages of the process. Thus, the MIDI and EL extraction methods appear to be suitable for analysis of microbial FAME profiles in compost, particularly in the later stages of the process.     

Emergy analysis of biogas production and electricity generation from small-scale agricultural digesters

An emergy analysis was performed to assess the relative sustainability and environmental impact of small-scale energy production using Taiwanese model plug-flow anaerobic digesters to treat livestock manure in Costa Rica. Emergy analysis quantifies all inputs to a system by converting them to solar energy equivalents, thus allowing for direct comparison of the diverse inputs of renewable energies, human labor and economic goods needed to construct and maintain anaerobic digestion systems. The digesters were located on the campus of EARTH University, Costa Rica, and the biogas was utilized to power a 40 kW generator that supplies electricity for farm operations. Separate emergy analyses were performed for the biogas production and the combination of biogas production and generation of electricity. Manure was the largest input in both analyses, accounting for 85.3% of the annual emergy input for biogas production and 66.9% for electricity generation from the biogas. The fraction of emergy inputs from renewable sources (ΦR) was 66% for biogas production and 52% for electricity generation from the biogas. The transformities of biogas and electricity generation from the biogas were 5.23E+04 sej/J and 1.01E+06 sej/J respectively. The emergy yield ratios (EYR) were 2.93 for biogas production and 2.07 for electricity generation indicating that these digesters efficiently match purchased resources and renewable energies to produce energy from livestock manure. The generation of electricity from the biogas resulted in a decrease in the emergy sustainability index (ESI) from 5.67 to 2.22 and an increase in the environmental loading ratio (ELR) from 0.52 to 0.93. Using a generator to convert the biogas to electricity does decrease the sustainability of the system, largely due to the high emergy value associated with the electrical generation equipment and machinery, but these results demonstrate that the production of biogas and the generation of electricity from Taiwanese model digesters in Costa Rica are environmentally sustainable processes that result in the production of energy that is largely dependent on renewable and recycled energies.     

Anaerobic digestion of spent mushroom substrate under thermophilic conditions: performance and microbial community analysis

Applied Microbiology and Biotechnology - Spent mushroom substrate (SMS) is the residue of edible mushroom production occurring in huge amounts. The SMS residue can be digested for biogas production...     

Yield and protein quality of thermophilic Bacillus spp. biomass related to thermophilic aerobic digestion of agricultural wastes for animal feed supplementation

Bacillus spp. responsible for thermophilic aerobic digestion (TAD) of agricultural wastes were studied for their growth rate, yield and protein quality (amino acid profile) under conditions that approximate full-scale waste digestion as pointers to the capacity of TAD to achieve protein enrichment of wastes for reuse in animal feeding. Specific growth rates of the thermophiles varied with temperature and aeration rates. For Bacillus coagulans, the highest specific growth rate was 1.98 muh(-1); for Bacillus licheniformis 2.56 muh(-1) and for Bacillus stearothermophilus 2.63 muh(-1). Molar yield of B. stearothermophilus on glucose increased with temperature to a peak of 0.404 g g(-1) at 50 degrees C before declining. Peak concentration of overflow metabolite (acetate) increased from 10 mmol at 45 degrees C to 34 mmol at 65 degrees C before declining. Accumulation of biomass in all three isolates decreased with increase in temperature while protein content of biomass increased. Highest biomass protein (79%) was obtained in B. stearothermophilus at 70 degrees C. Content of most essential amino acids of the biomass improved with temperature. Amino acid profile of the biomass was comparable to or superior to the FAO standard for SCP intended for use in animal feeding. Culture condition (waste digestion condition) may be manipulated to optimize protein yield and quality of waste digested by TAD for recycling in animal feed.     

Isolation from yeast of a metabolically active water-soluble form of ergosterol

A water-soluble complex containing ergosterol together with a component of yeast has been isolated. The complex can be isolated from commercial yeast extract to which ergosterol has been added or directly from whole yeast cells. The complexing component has the properties of a large polysaccharide and the binding between the sterol and the polysaccharide appears to be noncovalent. The complex is easily prepared and is stable in aqueous solution; ergosterol in this solution is metabolically available to yeast cells to which it is added.     

Adaption of microbial community during the start-up stage of a thermophilic anaerobic digester treating food waste

A successful start-up enables acceleration of anaerobic digestion (AD) into steady state. The microbial community influences the AD performance during the start-up. To investigate how microbial communities changed during the start-up, microbial dynamics was analyzed via high-throughput sequencing in this study. The results confirmed that the AD was started up within 25 d. Thermophilic methanogens and bacterial members functioning in hydrolysis, acidogenesis, and syntrophic oxidation became predominant during the start-up stage, reflecting a quick adaption of microorganisms to operating conditions. Such predominance also indicated the great contribution of these members to the fast start-up of AD. Redundancy analysis confirmed that the bacterial abundance significantly correlated with AD conditions. The stable ratio of hydrogenotrophic methanogens to aceticlastic methanogens is also important to maintain the stability of the AD process. This work will be helpful to understand the contribution of microbial community to the start-up of AD.     

Diversity of the resident microbiota in a thermophilic municipal biogas plant

Biogas plants continuously convert biological wastes mainly into a mixture of methane, CO₂ and H₂O—a conversion that is carried out by a consortium of bacteria and archaea. Especially in the municipal plants dedicated towards waste treatment, the reactor feed may vary considerably, exposing the resident microbiota to a changing variety of substrates. To evaluate how and if such changes influence the microbiology, an established biogas plant (6,600 m³, up to 600 m³ biogas per h) was followed over the course of more than 2 years via polymerase chain reaction–denaturing gradient gel electrophoresis of 16S rRNA genes and subsequent sequencing. Both the bacterial and the archaeal community remained stable over the investigation. Of the bacterial consortium, about half of the sequences were in decreasing order of occurrence: Thermoacetogenium sp., Anaerobaculum mobile, Clostridium ultunense, Petrotoga sp., Lactobacillus hammesii, Butyrivibrio sp., Syntrophococcus sucromutans, Olsenella sp., Tepidanaerobacter sp., Sporanaerobacter acetigenes, Pseudoramibacter alactolyticus, Lactobacillus fuchuensis or Lactobacillus sakei, Lactobacillus parabrevis or Lactobacillus spicheri and Enterococcus faecalis. The other half matched closely to ones from similar habitats (thermophilic anaerobic methanogenic digestion). The archaea consisted of Methanobrevibacter sp., Methanoculleus bourgensis, Methanosphaera stadtmanae, Methanimicrococcus blatticola and uncultured Methanomicrobiales. The role of these species in methane production is discussed.