Biofilm development during the start-up period of anaerobic biofilm reactors: the biofilm Archaea community is highly dependent on the support material

To evaluate the impact of the nature of the support material on its colonization by a methanogenic consortium, four substrata made of different materials: polyvinyl chloride, 2 polyethylene and polypropylene were tested during the start-up of lab-scale fixed-film reactors. The reactor performances were evaluated and compared together with the analysis of the biofilms. Biofilm growth was quantified and the structure of bacterial and archaeal communities were characterized by molecular fingerprinting profiles (capillary electrophoresis-single strand conformation polymorphism). The composition of the inoculum was shown to have a major impact on the bacterial composition of the biofilm, whatever the nature of the support material or the organic loading rate applied to the reactors during the start-up period. In contrast, the biofilm archaeal populations were independent of the inoculum used but highly dependent on the support material. Supports favouring Archaea colonization, the limiting factor in the overall process, should be preferred.     

Competition and coexistence of sulfate-reducing bacteria,acetogens and methanogens in a lab-scale anaerobic bioreactor as affected by changing substrate to sulfate ratio

The microbial population structure and function of natural anaerobic communities maintained in lab-scale continuously stirred tank reactors at different lactate to sulfate ratios and in the absence of sulfate were analyzed using an integrated approach of molecular techniques and chemical analysis. The population structure, determined by denaturing gradient gel electrophoresis and by the use of oligonucleotide probes, was linked to the functional changes in the reactors. At the influent lactate to sulfate molar ratio of 0.35 mol mol⁻¹, i.e., electron donor limitation, lactate oxidation was mainly carried out by incompletely oxidizing sulfate-reducing bacteria, which formed 80–85% of the total bacterial population. Desulfomicrobium- and Desulfovibrio-like species were the most abundant sulfate-reducing bacteria. Acetogens and methanogenic Archaea were mostly outcompeted, although less than 2% of an acetogenic population could still be observed at this limiting concentration of lactate. In the near absence of sulfate (i.e., at very high lactate/sulfate ratio), acetogens and methanogenic Archaea were the dominant microbial communities. Acetogenic bacteria represented by Dendrosporobacter quercicolus-like species formed more than 70% of the population, while methanogenic bacteria related to uncultured Archaea comprising about 10–15% of the microbial community. At an influent lactate to sulfate molar ratio of 2 mol mol⁻¹, i.e., under sulfate-limiting conditions, a different metabolic route was followed by the mixed anaerobic community. Apparently, lactate was fermented to acetate and propionate, while the majority of sulfidogenesis and methanogenesis were dependent on these fermentation products. This was consistent with the presence of significant levels (40–45% of total bacteria) of D. quercicolus-like heteroacetogens and a corresponding increase of propionate-oxidizing Desulfobulbus-like sulfate-reducing bacteria (20% of the total bacteria). Methanogenic Archaea accounted for 10% of the total microbial community.     

The vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu

This study was conducted to characterize the vertical distribution of bacterial and archaeal communities in the water and sediment of Lake Taihu, which underwent a change in trophic status from oligotrophic to hypertrophic in last half of the 20th century. The results revealed that the bacterial communities in different layers of sediment sample were very similar, and were related to Alpha -, Beta -, Gamma - and Deltaproteobacteria, Nitrospira, Bacteroidetes, Firmicutes, Gemmatimonadetes, Verrucomicrobia, Chlorobi, Actinobacteria and Acidobacteria . In contrast, the archaeal communities varied greatly with depth. The archaeal communities were primarily related to Euryarchaeota and Crenarchaeota , with methanogenic Archaea accounting for approximately 2–35% of the total Archaea. Additionally, sequences related to putative ammonia-oxidizing Archaea and ammonia-oxidizing Bacteria were detected in different layers of sediment samples. The abundance of Archaea, Bacteria, methanogenic Archaea and Nitrospira was further characterized by real-time PCR.     

Influence of microwave radiation on bacterial community structure in biofilm

Organic matter exposed to microwave radiation triggers standard thermal effects as well as a range of so called non-thermal effects. The present work examined the non-thermal effects of microwave radiation on the biofilm in bioreactors with immobilised biomass. Microwave-exposed and conventionally heated reactors were used and both groups of reactors operated on analogous technological parameters. The analysis of the treated sewage demonstrated a significant increase in nitrification and denitrification efficiency in the bioreactors treated with microwave radiation. An analysis of bacteria diversity based on DGGE method showed significantly different bacterial communities developed in the reactors exposed to the microwave radiation in comparison to the control reactors. Moreover, bacterial richness measured by Shannon index was significantly higher in the microwave treated samples (Mann–Whitney's test, p < 0.05). These findings indicate that microwave radiation can affect the structure and function of bacterial communities independent of thermal effects.     

Influence of environmental conditions on methanogenic compositions in anaerobic biogas reactors

The influence of environmental parameters on the diversity of methanogenic communities in 15 full-scale biogas plants operating under different conditions with either manure or sludge as feedstock was studied. Fluorescence in situ hybridization was used to identify dominant methanogenic members of the Archaea in the reactor samples; enriched and pure cultures were used to support the in situ identification. Dominance could be identified by a positive response by more than 90% of the total members of the Archaea to a specific group- or order-level probe. There was a clear dichotomy between the manure digesters and the sludge digesters. The manure digesters contained high levels of ammonia and of volatile fatty acids (VFA) and were dominated by members of the Methanosarcinaceae, while the sludge digesters contained low levels of ammonia and of VFA and were dominated by members of the Methanosaetaceae. The methanogenic diversity was greater in reactors operating under mesophilic temperatures. The impact of the original inoculum used for the reactor start-up was also investigated by assessment of the present population in the reactor. The inoculum population appeared to have no influence on the eventual population.     

Microbial communities involved in biogas production from wheat straw as the sole substrate within a two-phase solid-state anaerobic digestion

Microbial communities involved in biogas production from wheat straw as the sole substrate were investigated. Anaerobic digestion was carried out within an up-flow anaerobic solid-state (UASS) reactor connected to an anaerobic filter (AF) by liquor recirculation. Two lab-scale reactor systems were operated simultaneously at 37 °C and 55 °C. The UASS reactors were fed at a fixed organic loading rate of 2.5 g L⁻¹ d⁻¹, based on volatile solids. Molecular genetic analyses of the bacterial and archaeal communities within the UASS reactors (digestate and effluent liquor) and the AFs (biofilm carrier and effluent liquor) were conducted under steady-state conditions. The thermophilic UASS reactor had a considerably higher biogas and methane yield in comparison to the mesophilic UASS, while the mesophilic AF was slightly more productive than the thermophilic AF. When the thermophilic and mesophilic community structures were compared, the thermophilic system was characterized by a higher Firmicutes to Bacteroidetes ratio, as revealed by 16S rRNA gene (rrs) sequence analysis. The composition of the archaeal communities was phase-separated under thermophilic conditions, but rather stage-specific under mesophilic conditions. Family- and order-specific real-time PCR of methanogenic Archaea supported the taxonomic distribution obtained by rrs sequence analysis. The higher anaerobic digestion efficiency of the thermophilic compared to the mesophilic UASS reactor was accompanied by a high abundance of Firmicutes and Methanosarcina sp. in the thermophilic UASS biofilm.     

Microbiology and performance of a methanogenic biofilm reactor during the start-up period

Aims:  To understand the interactions between anaerobic biofilm development and process performances during the start-up period of methanogenic biofilm reactor.
Methods and Results:  Two methanogenic inverse turbulent bed reactors have been started and monitored for 81 days. Biofilm development (adhesion, growth, population dynamic) and characteristics (biodiversity, structure) were investigated using molecular tools (PCR–SSCP, FISH-CSLM). Identification of the dominant populations, in relation to process performances and to the present knowledge of their metabolic activities, was used to propose a global scheme of the degradation routes involved. The inoculum, which determines the microbial species present in the biofilm influences bioreactor performances during the start-up period. FISH observations revealed a homogeneous distribution of the Archaea and bacterial populations inside the biofilm.
Conclusion:  This study points out the link between biodiversity, functional stability and methanogenic process performances during start-up of anaerobic biofilm reactor. It shows that inoculum and substrate composition greatly influence biodiversity, physiology and structure of the biofilm.
Significance and Impact of the Study:  The combination of molecular techniques associated to a biochemical engineering approach is useful to get relevant information on the microbiology of a methanogenic growing biofilm, in relation with the start-up of the process.     

Microbial community analysis of rectal methanogens and sulfate reducing bacteria in two non-human primate species

Background  Methanogenesis by methanogenic Archaea and sulfate reduction by sulfate reducing bacteria (SRB) are the major hydrogenotrophic pathways in the human colon. Methanogenic status of mammals is suggested to be under evolutionary rather than dietary control. However, information is lacking regarding the dynamics of hydrogenotrophic microbial communities among different primate species.
Methods  Rectal swabs were collected from 10 sooty mangabeys ( Cercocebus atys ) and 10 baboons ( Papio hamadryas ). The diversity and abundance of methanogens and SRB were examined using PCR-denaturing gradient gel electrophoresis (DGGE) and real-time quantitative PCR (qPCR).
Results  The DGGE results revealed that intestinal Archaea and SRB communities differ between mangabeys and baboons. Phylogenetic analyses of Archaea DGGE bands revealed two distinct clusters with one representing a putative novel order of methanogenic Archaea. The qPCR detected a similar abundance of methanogens and SRB.
Conclusions  Intestinal Archaea and SRB coexist in these primates, and the community patterns are host species-specific.     

Molecular diversity analysis of rumen methanogenic Archaea from goat in eastern China by DGGE methods using different primer pairs

Aims:  To screen a pair of primers suitable for denaturing gradient gel electrophoretic (DGGE) analysis of ruminal methanogenic Archaea and to detect the archaeal communities in the rumen of goat.
Methods and Results:  Nine primer pairs for 16S rDNA of methanogenic Archaea , including six for directed polymerase chain reaction (PCR) and three for nested PCR were first evaluated by PCR amplification of the total DNA from rumen fluids and bacteria. The DGGE analysis of rumen fluids was then conducted with three primer sets (344fGC/915r, 1106fGC/1378r and 519f/915rGC) of the nine pairs tested. Good separation and quality of patterns were obtained in DGGE analysis with primer pairs 1106fGC/1378r and 519f/915rGC. A total of 40 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic Archaea while primer pair 519f/915rGC had better amplification ranges than the other two primer pairs.
Conclusions:  The procedure of DGGE analysis with primer pair 519f/915rGC was more suitable for investigating methanogenic archaeal community in the rumen. The dominant methanogenic Archaea in the rumen of goat was Methanobrevibacter sp. and an unidentified methanogenic Archaea .
Significance and Impact of the Study:  One pair of primers suitable for DGGE analysis of ruminal methanogenic Archaea was obtained and the molecular diversity of ruminal methanogenic Archaea in goat was investigated by PCR-DGGE.     

Methane Production and Methanogenic Archaea in the Digestive Tracts of Millipedes (Diplopoda)

Methane production by intestinal methanogenic Archaea and their community structure were compared among phylogenetic lineages of millipedes. Tropical and temperate millipedes of 35 species and 17 families were investigated. Species that emitted methane were mostly in the juliform orders Julida, Spirobolida, and Spirostreptida. The irregular phylogenetic distribution of methane production correlated with the presence of the methanogen-specific mcrA gene. The study brings the first detailed survey of methanogens’ diversity in the digestive tract of millipedes. Sequences related to Methanosarcinales, Methanobacteriales, Methanomicrobiales and some unclassified Archaea were detected using molecular profiling (DGGE). The differences in substrate preferences of the main lineages of methanogenic Archaea found in different millipede orders indicate that the composition of methanogen communities may reflect the differences in available substrates for methanogenesis or the presence of symbiotic protozoa in the digestive tract. We conclude that differences in methane production in the millipede gut reflect differences in the activity and proliferation of intestinal methanogens rather than an absolute inability of some millipede taxa to host methanogens. This inference was supported by the general presence of methanogenic activity in millipede faecal pellets and the presence of the 16S rRNA gene of methanogens in all tested taxa in the two main groups of millipedes, the Helminthophora and the Pentazonia.     

Comparison of microwaves to fluidized sand baths for heating tubular reactors for hydrothermal and dilute acid batch pretreatment of corn stover

Heating of batch tubular reactors with fluidized sand baths and with microwaves resulted in distinctive sugar yield profiles from pretreatment and subsequent enzymatic hydrolysis of corn stover at the same time, temperature, and dilute sulfuric acid concentration combinations and hydrothermal pretreatment conditions. Microwave heated pretreatment led to faster xylan, lignin, and acetyl removal as well as earlier xylan degradation than sand baths, but maximum sugar recoveries were similar. Solid state CP/MAS NMR revealed that microwave heating was more effective in altering cellulose structural features especially in breakdown of amorphous regions of corn stover than sand bath heating. Enzymatic hydrolysis of pretreated corn stover was improved by microwave heating compared to sand bath heating. Mechanisms were proposed to explain the differences in results for the two systems and provide new insights into pretreatment that can help advance this technology.     

Effect of seed sludge microbial community and activity on the performance of anaerobic reactors during the start-up period

In this study, two laboratory-scale anaerobic batch reactors started up with different inoculum sludges and fed with the same synthetic wastewater were monitored in terms of performance and microbial community shift by denaturant gradient gel electrophoresis fingerprinting and subsequent cloning, sequencing analysis in order to reveal importance of initial quality of inoculum sludge for operation of anaerobic reactors. For this purpose, two different seed sludge were evaluated. In Reactor1 seeded with a sludge having less diverse microbial community (19 operational taxonomic unit (OTU’s) for Bacterial and 8 OTU’s for Archaeal community, respectively) and a methanogenic activity of 150 ml CH₄ g TVS⁻¹ day⁻¹, a chemical oxygen demand (COD) removal efficiency of 78.8 ± 4.17% was obtained at a substrate to microorganism (S/X) ratio of 0.38. On the other hand, Reactor2, seeded with a sludge having a much more diverse microbial community (24 OTU’s for Bacterial and 9 OTU’s for Archaeal communities, respectively) and a methanogenic activity, 450 ml CH₄ g TVS⁻¹ day⁻¹, operated in the same conditions showed a better start-up performance; a COD removal efficiency of over 98% at a S/X ratio of 0.53. Sequence analysis of Seed2 revealed the presence of diverse fermentative and syntrophic bacteria, whereas excised bands of Seed1 related to fermentative and sulfate/metal-reducing bacteria. This study revealed that a higher degree of bacterial diversity, especially the presence of syntrophic bacteria besides the abundance of key species such as methanogenic Archaea may play an important role in the performance of anaerobic reactors during the start-up period.     

Intra-habitat Differences in the Composition of the Methanogenic Archaeal Community between the Microcystis-Dominated and the Macrophyte-Dominated Bays in Taihu Lake

A regime shift between a macrophyte-dominated clear state and a phytoplankton-dominated turbid state can have considerable impact on ecosystem structure and function of shallow lakes. However, very little is known about the response of the methanogenic archaeal community in the sediment during this regime shift. We investigated the methanogenic archaeal community at two sites in the large, shallow, eutrophic Taihu Lake over the course of one year. One site is located in Meiliang Bay and is dominated by Microcystis blooms, and the other site is located in East Taihu Bay and is dominated by aquatic macrophytes. Terminal restriction fragment length polymorphism (T-RFLP) and phylogenetic analyses of archaeal 16S rRNA genes were used to analyze the methanogenic community. Higher ratio of methanogens in Archaea was observed in East Taihu Bay than in Meiliang Bay. The methanogenic archaeal community was dominated by the Methanobacteriales and the LDS cluster in macrophytes-dominated East Taihu Bay, while it was dominated by the Methanosarcinaceae, Methanobacteriales, and the LDS cluster in Microcystis-dominated Meiliang Bay. Clustering analysis of all of the samples revealed differences in the composition of the methanogenic archaeal communities between the two sites that were independent of seasonal variations. Further statistical analysis indicated that the chlorophyll a (Chla) concentration had a profound impact on the composition of the methanogenic archaeal community in Meiliang Bay, whereas it was primarily influenced by total organic carbon (TOC) levels in East Taihu Bay. Overall, this investigation demonstrates that intra-habitat differences in the composition of methanogenic archaeal communities are likely driven by changes in the available organic materials.     

Bacterial community structure in experimental methanogenic bioreactors and search for pathogenic clostridia as community members

Microbial conversion of organic waste or harvested plant material into biogas has become an attractive technology for energy production. Biogas is produced in reactors under anaerobic conditions by a consortium of microorganisms which commonly include bacteria of the genus Clostridium. Since the genus Clostridium also harbors some highly pathogenic members in its phylogenetic cluster I, there has been some concern that an unintended growth of such pathogens might occur during the fermentation process. Therefore this study aimed to follow how process parameters affect the diversity of Bacteria in general, and the diversity of Clostridium cluster I members in particular. The development of both communities was followed in model biogas reactors from start-up during stable methanogenic conditions. The biogas reactors were run with either cattle or pig manures as substrates, and both were operated at mesophilic and thermophilic conditions. The structural diversity was analyzed independent of cultivation using a PCR-based detection of 16S rRNA genes and genetic profiling by single-strand conformation polymorphism (SSCP). Genetic profiles indicated that both bacterial and clostridial communities evolved in parallel, and the community structures were highly influenced by both substrate and temperature. Sequence analysis of 16S rRNA genes recovered from prominent bands from SSCP profiles representing Clostridia detected no pathogenic species. Thus, this study gave no indication that pathogenic clostridia would be enriched as dominant community members in biogas reactors fed with manure.     

Phylogenetic analysis of housekeeping Archaeal proteins and early stages of Archaea evolution

The most probable horizontal gene transfer events in the evolution of Archaea are reconstructed based on the comparison of phylogenetic trees of housekeeping orthologous protein families with consensus phylogenies of Archaea. The existence of these phenomena suggests that the common ancestor of Archaea was of methanogenic and hyperthermophilic nature and dwelt in communities with a high level of ecological integration.     

Thermal action of 2.45 GHz microwaves on the cytoplasm of Chinese hamster cells

In order to demonstrate possible specific effects of microwaves at the cellular level V-79 Chinese hamster cells were exposed to 2.45-GHz radiation at power levels of 20–200 mW/cm² and at specific absorption rates of 10–100 mW/g. Intracellular cytoplasmic changes were observed by fluorescence polarization using a method based on the intracellular enzymatic hydrolysis of nonfluorescent fluorescein diacetate (FDA). At levels of absorbed energy below 90 J/g, modifications of microviscosity and mitochondrial state were absent, but a slight stimulation of enzymatic hydrolysis of FDA was observed which may be explained by microwave-induced alterations of cellular membranes possibly due to differences in heating pattern of microwaves compared to water-bath heating. At levels of absorbed energy above 90 J/g, the decrease of enzymatic hydrolysis of FDA, increase in degree of polarization, and increase of permeation of the fluorescent marker correlated well with the decrease in cell viability as measured by the exclusion of trypan blue. At equal absorbed energy, microwaves were found to exert effects comparable to classical heating except that permeation was slightly more affected by microwave than by classical heating. This suggests that membrane alteration produced by microwaves might differ from those induced by classical heating or that microwaves may have heated the membrane to higher temperatures than did classical heating.     

Roles of microorganisms other than Clostridium and Enterobacter in anaerobic fermentative biohydrogen production systems--a review

Anaerobic fermentative biohydrogen production, the conversion of organic substances especially from organic wastes to hydrogen gas, has become a viable and promising means of producing sustainable energy. Successful biological hydrogen production depends on the overall performance (results of interactions) of bacterial communities, i.e., mixed cultures in reactors. Mixed cultures might provide useful combinations of metabolic pathways for the processing of complex waste material ingredients, thereby supporting the more efficient decomposition and hydrogenation of biomass than pure bacteria species would. Therefore, understanding the relationships between variations in microbial composition and hydrogen production efficiency is the first step in constructing more efficient hydrogen-producing consortia, especially when complex and non-sterilized organic wastes are used as feeding substrates. In this review, we describe recent discoveries on bacterial community composition obtained from dark fermentation biohydrogen production systems, with emphasis on the possible roles of microorganisms that co-exist with common hydrogen producers.     

Efficient treatment of garbage slurry in methanogenic bioreactor packed by fibrous sponge with high porosity

Adding a supporting material to a methanogenic bioreactor treating garbage slurry can improve efficiency of methane production. However, little is known on how characteristics (e.g., porosity and hydrophobicity) of the supporting material affect the bioreactor degrading garbage slurry. We describe the reactor performances and microbial communities in bioreactors containing hydrophilic or hydrophobic sheets, or fibrous hydrophilic or hydrophobic sponges. The porosity affected the efficiency of methane production and solid waste removal more than the hydrophilic or hydrophobic nature of the supporting material. When the terminal restriction fragment length polymorphism technique was used at a lower organic loading rate (OLR), microbial diversities in the suspended fraction were retained on the hydrophobic, but not the hydrophilic, sheets. Moreover, real-time quantitative polymerase chain reaction (PCR) performed at a higher OLR revealed that the excellent performance of reactors containing fibrous sponges with high porosity (98%) was supported by a clear increase in the numbers of methanogens on these sponges, resulting in larger total numbers of methanogens in the reactors. In addition, the bacterial communities in fractions retained on both the hydrophobic and hydrophilic fibrous sponges differed from those in the suspended fraction, thus increasing bacterial diversity in the reactor. Thus, higher porosity of the supporting material improves the bioreactor performance by increasing the amount of methanogens and bacterial diversity; surface hydrophobicity contributes to maintaining the suspended microbial community.     

Destruction and injury of Escherichia coli during microwave heating under vacuum

AIMS: To study the effect of 2450 MHz microwave radiation under vacuum (vacuum microwave or VM) on survival and injury of Escherichia coli and to search for possible nonthermal effects associated with VM. METHODS AND RESULTS: Destruction kinetics of E. coli in peptone water were determined in a continuous-flow vacuum system, heated by convection heating in a water bath or with microwaves (VMs). Vacuum was used to control the boiling point of water and to maintain temperature in the bacterial suspensions at specified levels (49-64 degrees C). CONCLUSIONS: z-Value in the water bath treatment was 9.1 degrees C while for VM at 510 and 711 W it was 6.2 and 5.9 degrees C, suggesting that E. coli is more sensitive to temperature changes under microwave heating. Arrhenius calculations of the activation energies of the destruction reactions suggest that the mechanism of destruction in VM may be different from that of conventional heat. The number of injured micro-organisms showed no significant differences among treatments. SIGNIFICANCE AND IMPACT OF THE STUDY: The impact of temperature on E. coli destruction was different when microwaves were the medium of heat transfer, suggesting the existence of factors other than heat contributing to the lethal effect of VM.