An improved enzyme-linked immunosorbent assay for whole-cell determination of methanogens in samples from anaerobic reactors.

An enzyme-linked immunosorbent assay was developed for the detection of whole cells of methanogens in samples from anaerobic continuously stirred tank digesters treating slurries of solid waste. The assay was found to allow for quantitative analysis of the most important groups of methanogens in samples from anaerobic digesters in a reproducible manner. Polyclonal antisera against eight strains of methanogens were employed in the test. The specificities of the antisera were increased by adsorption with cross-reacting cells. The reproducibility of the assay depended on the use of high-quality microtiter plates and the addition of dilute hydrochloric acid to the samples. In an experiment on different digester samples, the test demonstrated a unique pattern of different methanogenic strains present in each sample. The limited preparatory work required for the assay and the simple assay design make the test well suited for routine analysis of large numbers of samples and thus for process surveillance during operation of biogas digesters.     

Extremely thermophilic cellulolytic anaerobes from icelandic hot springs

Anaerobic enrichment cultures with Avicel as substrate and inoculated with biomat samples from Icelandic hot springs were cultured at 70 ° or 78 °C and examined for the presence of microorganisms that produce extracellular cellulolytic and xylanolytic enzymes. From four enrichments grown at 78 °C eighteen strains were isolated. Five of the strains were screened for their substrate utilization, and on the basis of differences in morphology and substrates used, the two most unique strains were selected for further characterization. All cellulolytic cultures were rod-shaped and non-sporeforming. Motility was not observed. Cells stained gram-negative at various stages of the growth phase. During growth on Avicel, most cultures produced acetate as the major fermentation product, with smaller amounts of lactic acid and ethanol. Carbon dioxide and hydrogen were also produced. The phenotypic characteristics of the enrichment cultures and of isolates are described and assessed in relation to temperature and pH in the hot spring environment. A comparison is made between Icelandic strains isolated in our laboratory and strains isolated from hot springs from other parts of the world. The biotechnological potential of this group of bacteria is briefly discussed.     

Threshold Acetate Concentrations for Acetate Catabolism by Aceticlastic Methanogenic Bacteria

Marked differences were found for minimum threshold concentrations of acetate catabolism by Methanosarcina barkeri 227 (1.180 mM), Methanosarcina mazei S-6 (0.396 mM), and a Methanothrix sp. (0.069 mM). This indicates that the aceticlastic methanogens responsible for the conversion of acetate to methane in various ecosystems might be different, depending on the prevailing in situ acetate concentrations.     

Temperature Compensation in Methanosarcina barkeri by Modulation of Hydrogen and Acetate Affinity

The affinity of Methanosarcina barkeri 227 for acetate and hydrogen at different incubation temperatures was investigated. Increasing the temperature from 20 to 37°C resulted in a 4.5-fold increase in K_m for acetate and a 4.8-fold increase for hydrogen. The corresponding increase in V_max for acetate was 8.3-fold (5.4-fold for hydrogen). This response implied a decrease in the temperature coefficient (Q₁₀) and hence a decrease in the temperature dependency as a function of decreasing substrate concentration.     

Production of lactic acid from whey using hydrolysed whey protein as nitrogen source

Summary Hydrolysed whey (lactose 4% w/vol) was fermented in laboratory reactors for production of lactic acid. By using a non0sterile unsupplemented continuous process with a hydraulic retention time of 10 hours, lactic acid was produced in a concentration of approx. 2.5 w/vol % with less than 0.1% volatile fatty acids and less than 0.2% lactose/galactose present in the effluent.     

Ecological factors affect the level and scaling of avian BMR

The basal rate of metabolism (BMR) in 533 species of birds, when examined with ANCOVA, principally correlates with body mass, most of the residual variation correlating with food habits, climate, habitat, a volant or flightless condition, use or not of torpor, and a highland or lowland distribution. Avian BMR also correlates with migratory habits, if climate and a montane distribution is excluded from the analysis, and with an occurrence on small islands if a flightless condition and migration are excluded. Residual variation correlates with membership in avian orders and families principally because these groups are behaviorally and ecologically distinctive. However, the distinction between passerines and other birds remains a significant correlate of avian BMR, even after six ecological factors are included, with other birds having BMRs that averaged 74% of the passerine mean. This combination of factors accounts for 97.7% of the variation in avian BMR. Yet, migratory species that belong to Anseriformes, Charadriiformes, Pelecaniformes, and Procellariiformes and breed in temperate or polar environments have mass-independent basal rates equal to those found in passerines. In contrast, penguins belong to an order of polar, aquatic birds that have basal rates lower than passerines because their flightless condition depresses basal rate. Passerines dominate temperate, terrestrial environments and the four orders of aquatic birds dominate temperate and polar aquatic environments because their high BMRs facilitate reproduction and migration. The low BMRs of tropical passerines may reflect a sedentary lifestyle as much as a life in a tropical climate. Birds have BMRs that are 30-40% greater than mammals because of the commitment of birds to an expensive and expansive form of flight.     

Responses of the biogas process to pulses of oleate in reactors treating mixtures of cattle and pig manure

The effect of oleate on the anaerobic digestion process was investigated. Two thermophilic continuously stirred tank reactors (CSTR) were fed with mixtures of cattle and pig manure with different total solid (TS) and volatile solid (VS) content. The reactors were subjected to increasing pulses of oleate. Following pulses of 0.5 and 1.0 g oleate/L, the most distinct increase in volatile fatty acid (VFA) concentrations were observed in the reactor with the lowest TS/VS content. This suggests a higher adsorption of oleate on the surfaces of biofibers in the reactor with the highest TS/VS and a less pronounced inhibition of the anaerobic digestion process. On the other hand, addition of 2.0 g oleate/L severely inhibited the process in both reactors, and a significant increase in all VFA concentrations combined with an immediate drop in methane production was noticed. However, 20 days after the reactors had been exposed to oleate both reactors showed a lower VFA concentration along with a higher methane production than before the pulses. This indicates that oleate had a stimulating effect on the overall process. The improved acetogenic and methanogenic activity in the reactors was confirmed in batch activity tests. In addition to this, toxicity tests revealed that the oleate pulses induced an increase in the tolerance level of acetotrophic methanogens towards oleate. When evaluating the usability of different process parameters (i.e., VFA and methane production) as indicators of process recovery, following the inhibition by oleate, propionate was found to be most suitable.     

Thermodesulfobacterium hveragerdense sp. nov., and Thermodesulfovibrio islandicus sp. nov., two thermophilic sulfate reducing bacteria isolated from a Icelandic hot spring

Two thermophilic non-sporeforming sulfate-reducing bacteria (SRB) were isolated from microbial mats collected from an Icelandic hot spring. Strain JSP was a gram negative rod, with an average cell size of 2.8 x 0.5 microm. No flagella were found. Growth occurred between 55 and 74 degrees C with an optimum between 70 and 74 degrees C at pH 7.0. The G+C content was 40 mol%. Strain R1Ha3 was a gram negative vibrio-shaped rod with an average cell size of 1.7 x 0.4 microm. Motility was observed mediated by one polar flagellum. The growth optimum at pH 7.0 was 65 degrees C, and growth occurred between 45 and 70 degrees C. The G+C content was 38 mol%. In the presence of sulfate, both strains used lactate, pyruvate and H2 as electron donors. In addition, strain R1Ha3 used formate. Pyruvate was the only substrate supporting fermentative growth of both strains. Growth occurred with sulfate as well as thiosulfate as electron acceptors. Furthermore, strain R1Ha3 reduced nitrate and strain JSP reduced sulfite. Neither of the strains were able to oxidize lactate completely to CO2 and neither of the strains contained desulfoviridin. 16S rDNA sequencing placed strain JSP in the genus Thermodesulfobacterium and strain R1Ha3 in the genus Thermodesulfovibrio. Based on the DNA-DNA hybridization studies and differences in morphology and physiology to their closest relatives the two new isolates were considered as new species. Strain JSP is named Thermodesulfobacterium hveragerdense and strain R1Ha3 Thermodesulfovibrio islandicus.     

The genes coding for the hsp70 (dnaK) molecular chaperone machine occur in the moderate thermophilic archaeon Methanosarcina thermophila TM-1

The hsp70(dnaK) locus of the moderate thermophilic archaeon Methanosarcina thermophila TM-1 was cloned, sequenced, and tested in vitro to measure gene induction by heat and ammonia, i.e., stressors pertinent to the biotechnological ecosystem of this methanogen that plays a key role in anaerobic bioconversions. The locus' genes and organization, 5'-grpE-hsp70(dnaK)-hsp40 (dnaJ)-trkA-3', are the same as those of the closely related mesophile Methanosarcina mazei S-6, but different from those of the only other archaeon for which comparable sequence data exist, the thermophile Methanobacterium thermoautotrophicum deltaH, from another genus, in which trkA is not part of the locus. The proteins encoded in the TM-1 genes are very similar to the S-6 homologs, but considerably less similar to the deltaH proteins. The TM-1 Hsp70(DnaK) protein has the 23-amino acid deletion--by comparison with homologs from gram-negative bacteria first described in the S-6 molecule and later found to be present in all homologs from archaea and gram positives. The genes responded to a temperature elevation in a manner that demonstrated that they are heat-shock genes, functionally active in vivo. Ammonia also induced a heat-shock type of response by hsp70(dnaK), and a similar response by trkA. The data suggest that the moderate thermophile TM-1 has an active Hsp70(DnaK)-chaperone machine in contrast to hyperthermophilic archaea, and that trkA is a stress gene, inasmuch as it responds like classic heat-shock genes to stressors that induce a typical heat-shock response.