Zuzana Mladenovska

Effect of temperature increase from 55 to 65°C on performance and microbial population dynamics of an anaerobic reactor treating cattle manure

The effect of a temperature increase from 55 to 65 degrees C on process performance and microbial population dynamics were investigated in thermophilic, lab-scale, continuously stirred tank reactors. The reactors had a working volume of 31 and were fed with cattle manure at an organic loading rate of 3 g VS/l reactor volume/d. The hydraulic retention time in the reactors was 15 days. A stable reactor performance was obtained for periods of three retention times both at 55 degrees C and 65 degrees C. At 65 degrees C methane yield stabilized at approximately 165ml/g VS/d compared to 200 ml/g VS/d at 55 degrees C. Simultaneously, the level of total volatile fatty acids, VFA, increased from being below 0.3 g/l to 1.8-2.4 g acetate/l. The specific methanogenic activities (SMA) of biomass from the reactors were measured with acetate, propionate, butyrate, hydrogen, formate and glucose. At 65 degrees C. a decreased activity was found for glucose-, acetate-, butyrate- and formate-utilizers and no significant activity was measured with propionate. Only the hydrogen-consuming methanogens showed an enhanced activity at 65 degrees C. Numbers of cultivable methanogens, estimated by the most probable number (MPN) method, were significantly lower on glucose, acetate and butyrate at the increased operational temperature, while the numbers of hydrogenotrophic methanogens remained unchanged. No viable propionate-degrading bacteria were enriched at 65 C. Use of ribosomal oligonucleotide probes showed that an increase in temperature resulted in a decreased contribution of the rRNA of the domain bacteria from 74-79 to 57-62% of the universal probe, while the rRNA of the domain archaea, increased from 18-23 to 34-36%.

Isolation and characterization of Caldicellulosiruptor lactoaceticus sp. nov., an extremely thermophilic, cellulolytic, anaerobic bacterium

An anaerobic, extremely thermophilic, cellulolytic, non-spore-forming bacterium, strain 6A, was isolated from an alkaline hot spring in Hveragerði, Iceland. The bacterium was non-motile, rod-shaped (1.5–3.5x0.7 μm) and occurred singly, in pairs or in chains and stained gram-negative. The growth temperature was between 50 and 78°C with a temperature optimum near 68°C. Growth occurred between pH 5.8 and 8.2 with an optimum near 7.0. The bacterium fermented microcrystalline cellulose (Avicel) and produced lactate, acetate and H2 as the major fermentation products, and CO2 and ethanol occurred as minor fermentation products. Only a restricted number of carbon sources (cellulose, xylan, starch, pectin, cellobiose, xylose, maltose and lactose) were used as substrates. During growth on Avicel, the bacterium produced free cellulases with carboxymethylcellulase and avicelase activity. The G+C content of the cellular DNA of strain 6A was 35.2±0.8 mol%. Complete 16S rDNA sequence analysis showed that strain 6A was phylogenetically related to Caldicellulosiruptor saccharolyticus. It is proposed that the isolated bacterium be named Caldicellulosiruptor lactoaceticus sp. nov.

Kinetics of propionate conversion in anaerobic continuously stirred tank reactors.

Abstract The kinetic parameters of anaerobic propionate degradation by biomass from 7 continuously stirred tank reactors differing in temperature, hydraulic retention time and substrate composition were investigated. In substrate‐depletion experiments (batch) the maximum propionate degradation rate, A max, and the half saturation constant, K m , were initially estimated by applying the integrated Michaelis‐Menten equation. A max was in the range from 22.8 to 29.1 μmol gVS−1 h−1 while K m was in the range from 0.46–0.95 mM. In general, A max gave a good reflection of the reactor performances. Secondly, the accuracy of the applied method was evaluated by use of radiotracer methodology. A max was found to be 14–15% lower in the substrate‐depletion experiment than in the radioisotope experiment due to endogenous propionate production. By including the endogenous propionate production, a 42–49% lower K m was estimated. The results demonstrate that the rate of endogenous substrate (propionate) production should be taken into account when estimating kinetic parameters in biomass from manure‐based anaerobic reactors.

ALIS-FLP: amplified ligation selected fragment-length polymorphism method for microbial genotyping.

A DNA fingerprinting method known as ALIS‐FLP (amplified ligation selected fragment‐length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA‐PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.