Anaerobic waste stabilization

The present knowledge of the microbiology, physiology and regulation of anaerobic digestion in conventional or advanced processes is reviewed. In all systems the carbon flow from biopolymers to biogas is determined by syntrophic interactions of fermentative or acetogenic bacteria with methanogens at the level of interspecies hydrogen transfer. Inhibitors or heavy metal ions may interfere at different levels.The stabilization of waste at mesophilic and thermophilic temperatures is compared and the process stability as well as the inactivation of pathogens is discussed. Characteristics of conventional digestion systems and of recently developed advanced processes with solids and liquids uncoupling are compared and selection criteria with respect to the type of sludge are outlined.Areas of future research for a better understanding of the biochemistry, the physiology and the regulation of the degradation of pollutants are suggested.     

Cloning and overexpression of a thermostable signal peptide peptidase (SppA) from Thermoplasma volcanium GSS1 in E. coli

In this study, a gene coding for thermophilic serine protease of the ClpP class from the thermoacidophilic archaeon Thermoplasma volcanium (Tpv) was cloned and expressed in Escherichia coli. The primary sequence and domain analysis of this enzyme showed similarities (50–60% similarity) to signal peptide peptidases (SppA) of bacteria and other archaea. An increase of about tenfold in the activity was achieved by overexpression of Tpv SppA in E. coli, as detected by enzyme assays conducted using Ala-Ala-Phe-pNa and N-Suc-Ala-Ala-Pro-Phe-pNA as substrates. The recombinant enzyme, purified using an anion exchange column chromatography, displayed an apparent molecular mass of 26 kDa on SDS-PAGE analysis. Purified Tpv SppA was active in a broad range of pH and temperature with maximal activity at 60°C and between pH 7.5 and pH 8.0. The activity of the enzyme was strongly inhibited by inhibitors typical for serine proteases, i.e., chymostatin and PMSF. The activity of the Tpv SppA and the stability at high temperature were significantly enhanced in the presence of 5 mM Ca²⁺ ions. Our multiple sequence alignment data revealed a conserved Ser/Lys catalytic dyad in Tpv SppA that comprised Ser76 (nucleophile) and Lys128 (general base) residues. A search for a transmembrane domain using automated programs did not predict any signal peptide associated with the Tpv SppA and, therefore, suggested a cytoplasmic location for this enzyme.     

Description and validation of a rapid (1 h) flow cytometry test for enumerating thermophilic bacteria in milk powders

AIMS: The aim of this study was to develop a rapid assay for enumerating thermophilic bacteria in milk powder. METHODS AND RESULTS: The BactiFlow flow cytometer was used to count bacteria based on esterase activity in viable bacterial cells. A protocol for total viable bacteria was modified by heat-treating the sample to selectively label thermophilic bacteria. Samples of milk powder dissolved in 0.1% peptone were treated with 0.8% ethylenediaminetetraacetic acid to reduce background interference because of denatured milk proteins. Either thermophilic bacteria were added to the dissolved milk powder or milk powder solutions were incubated at 55 degrees C for 2-3 h to enrich the natural thermophile population for testing. Results from the BactiFlow were compared with traditional plate count results. CONCLUSIONS: Thermophilic bacteria in milk powder can be enumerated within 1 h using the BactiFlow flow cytometer. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbiological test results obtained within 1 h can potentially be used to monitor manufacturing processes, effectively trace problems and provide confidence in the manufacture of product.     

<Emphasis Type="Italic">Anoxybacillus rupiensis</Emphasis> sp. Nov., a novel thermophilic bacterium isolated from Rupi basin (Bulgaria)

Three strains of a novel thermophilic, strictly aerobic, Gram-positive, spore-forming hemo-organotrophic bacterium were isolated from three hot springs in the region of Rupi basin, Bulgaria as producers of amylolytic enzymes. Their 16S rRNA gene sequences (first 500 nucleotides) were very similar (99.8%). Strains were able to ferment a wide spectrum of carbohydrates such as sugars, polyols, and polysaccharides like xylan, glycogen and starch. Optimal growth was observed at 55–58°C, and pH at 6.0–6.5. Phylogenetic analysis of the whole 16S rRNA gene sequence clustered the strain R270^T with the representatives of the genus Anoxybacillus and with Geobacillus tepidamans. The G + C content of the genomic DNA was 41.7%. DNA–DNA hybridization analysis revealed low homology with the closest relatives (32.0 mol% homology to Geobacillus tepidamans). Fatty acid profile (major fatty acids iso-C15:0 and iso-C17:0) confirmed the affiliation of the strain to the genus Anoxybacillus. On the basis of the data presented here, we propose that strain R270^T, represents a new species of the genus Anoxybacillus for which, we recommend the name Anoxybacillus rupiensis sp. nov. (=DSM 17127^T = NBIMCC 8387^T). The 16S rRNA gene sequence data of a strain R270^T have been deposited in the EMBL databases under the accession number AJ879076.     

Biosynthesis of secreted ribonucleases of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> and <Emphasis Type="Italic">Bacillus circulans</Emphasis> under nitrogen starvation

The level of biosynthesis of secreted guanyl-specific ribonucleases (RNases) of Bacillus intermedius (binases) and Bacillus circulans (RNases Bci) by recombinant B. subtilis strains increases under nitrogen starvation. The promoter of the binase gene carries the sequences homologous to the recognition sites of the regulatory protein TnrA, which regulates gene expression under growth limitation by nitrogen. Using the B. subtilis strain defective in protein TnrA, it has been shown that the regulatory protein TnrA is involved in the regulation of expression of the binase gene and the gene of RNase Bci. The TnrA regulation of expression of the RNase Bci gene is indirect, probably by means of the regulatory protein PucR. Thus, it has been established that at least two regulatory mechanisms activate the expression of the genes encoding the secreted RNases of spore-forming bacteria: a system of proteins homologous to the B. subtilis PhoP-PhoR, and regulation by a protein similar to the B. subtilis TnrA regulatory protein.     

Isolations of α-glucosidase-producing thermophilic bacilli from hot springs of Turkey

From 42 different hot springs in six provinces belonging to distinct geographical regions of Turkey, 451 thermophilic bacilli were isolated and 67 isolates with a high amylase activity were selected to determine the α-glucosidase production capacities by using pNPG as a substrate. α-Glucosidase production capacities of the isolates varied within the range from 77.18 to 0.001 U/g. Eleven of our thermophilic bacilli produced α-glucosidase at significant levels comparable with that of the reference strains tested; thus, five strains, F84b (77.18 U/g), A333 (48.64 U/g), F84a (36.64 U/g), E134 (32.09 U/g), and A343 (10.79 U/g), were selected for further experiments. 16S rDNA sequence analysis revealed that these selected isolates all belonged to thermophilic bacilli 16S rDNA genetic group 5, four of them representing the genus Geobacillus, while strain A343 had an uncultured bacterium as the closest relative. Changes in α-glucosidase levels in the intracellular and extracellular fractions were determined during 48-h cultivation of A333, A343, F84a, F84b, E134, and the reference strain G. stearothermophilus ATCC 12980. According to α-glucosidase production type and enzyme levels in intracellular and extracellular fractions, Geobacillus spp. A333, F84a, and F84b were defined as extracellular enzyme producers, whereas the thermophilic bacterium A343 was found to be an intracellular α-glucosidase producer, similar to ATCC 12980 strain. Geobacillus sp. E134 differed in α-glucosidase production type from all tested isolates and the reference strain; it was described as a membrane-associated cell-bound enzyme producer. In this study, apart from screening a great number of new thermophilic bacilli from the hot springs of Turkey, which have not yet been thoroughly studied, five new thermostable α-1,4-glucosidase-producing bacilli that have biotechnological potential with α-glucosidases located at different cell positions were obtained. The text was submitted by the authors in English.     

Iron binding effects on the kinetic stability and unfolding energetics of a thermophilic phenylalanine hydroxylase from Chloroflexus aurantiacus

The effects of non-heme iron binding on the function, structure, and stability of a monomeric phenylalanine hydroxylase from the thermophile Chloroflexus aurantiacus (caPAH) were investigated. Comparative studies on holo (iron-bound) and apo (iron-depleted) caPAH indicated that iron(II) binding does not significantly affect the overall structure of the enzyme. Thermal denaturation studies performed using differential scanning calorimetry showed that the unfolding reaction was kinetically controlled and that holo-caPAH displayed a large increase in thermal stability (approximately 15 °C upshift in the T _m value) compared with the apoenzyme. Analysis using a simple irreversible two-state model also showed a higher kinetic stability for holo-caPAH at optimal growth temperature (denaturing approximately 8 times more slowly than the apo form at 55 °C). Experiments performed in the presence of urea in combination with structure–energetics calculations suggest that iron binding reduces the change in accessible surface area exposed in the unfolding transition state (from approximately 36% to approximately 5% of the total change in accessible surface area) and also the surface involved in water-unsatisfied broken internal contacts (solvation barriers). Additional comparative analyses using phenylalanine hydroxylase from mesophilic and psychrophilic organisms suggest that, in addition to its catalytic role, the non-heme iron serves to enhance the kinetic stability of phenylalanine hydroxylase at the optimal growth temperature of the organism. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Production of lactic acid from paper sludge using acid-tolerant, thermophilic Bacillus coagulan strains

Production of lactic acid from paper sludge was studied using thermophilic Bacillus coagulan strains 36D1 and P4-102B. More than 80% of lactic acid yield and more than 87% of cellulose conversion were achieved using both strains without any pH control due to the buffering effect of CaCO₃ in paper sludge. The addition of CaCO₃ as the buffering reagent in rich medium increased lactic acid yield but had little effect on cellulose conversion; when lean medium was utilized, the addition of CaCO₃ had little effect on either cellulose conversion or lactic acid yield. Lowering the fermentation temperature lowered lactic acid yield but increased cellulose conversion. Semi-continuous simultaneous saccharification and co-fermentation (SSCF) using medium containing 100 g/L cellulose equivalent paper sludge without pH control was carried out in serum bottles for up to 1000 h. When rich medium was utilized, the average lactic acid concentrations in steady state for strains 36D1 and P4-102B were 92 g/L and 91.7 g/L, respectively, and lactic acid yields were 77% and 78%. The average lactic acid concentrations produced using semi-continuous SSCF with lean medium were 77.5 g/L and 77.0 g/L for strains 36D1 and P4-102B, respectively, and lactic acid yields were 72% and 75%. The productivities at steady state were 0.96 g/L/h and 0.82 g/L/h for both strains in rich medium and lean medium, respectively. Our data support that B. coagulan strains 36D1 and P4-102B are promising for converting paper sludge to lactic acid via SSCF.     

Thermophilic biohydrogen production by an anaerobic heat treated-hot spring culture

Batch experiments were conducted to investigate the thermophilic biohydrogen production using an enrichment culture from a Turkish hot spring. Following the enrichment, the culture was heat treated at 100 °C for 10 min to select for spore-forming bacteria. H₂ production was accompanied by production of acetate, butyrate, lactate and ethanol. H₂ production was associated by acetate–butyrate type fermentation while accumulation of lactate and ethanol negatively affected the H₂ yield. H₂ production was highest in the temperature range from 49.6 to 54.8 °C and optimum values for initial pH and concentrations of iron, yeast extract and glucose were 6.5, 40 mg/l, 4–13.5 g/l, respectively. PCR–DGGE profiling showed that the heat treated culture consisted of species closely affiliated to genus Thermoanaerobacterium.