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Applied Microbiology and Biotechnology (v.48, #1)
Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222 by A. Singh; O. P. Ward (pp. 1-5).
Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1% v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO3 with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature (15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days.
The lamellar settler – a low-cost alternative for separating the micro-alga Chlorella vulgaris from a cultivation broth? by G. Janelt; P. Bolt; N. Gerbsch; R. Buchholz; M.-G. Cho (pp. 6-10).
Micro-algae, especially Chlorella vulgaris, produce a range of high-value substances and the biomass itself is used for purposes such as feeding in aquaculture. A lamellar settler was designed and built. Its suitability as a low-cost alternative to separate C. vulgaris was investigated. The settler operated semicontinuously in a laboratory photoreactor plant (total volume 9 l). A clearing of 30%–35% and a 50% increase in harvest outflow concentration were observed. The scaled up data for design and construction of a settler for a 200-l production plant were elaborated.
Axenic aerobic biofilms inhibit corrosion of SAE 1018 steel through oxygen depletion by A. Jayaraman; E. T. Cheng; J. C. Earthman; T. K. Wood (pp. 11-17).
Corrosion inhibition of SAE 1018 steel by pure-culture biofilms of Pseudomonas fragi and Escheri-chia coli DH5α has been evaluated in complex Luria-Bertani medium, seawater-mimicking medium, and modified Baar's medium at 30 °C. In batch cultures, both bacteria inhibited corrosion three to six fold compared to sterile controls, and the corrosion was comparable to that observed in anaerobic sterile media. To corroborate this result, a continuous reactor and electrochemical impedance spectroscopy were used to show that both P. fragi K and E. coli DH5α decreased the corrosion rate by 4- to 40-fold as compared to sterile controls; this matched the decrease in corrosion found with sterile medium in the absence of oxygen and with E. coli DH5α grown anaerobically. In addition, the requirement for live respiring cells was demonstrated by the increase in the corrosion rate that was observed upon killing the P. fragi K biofilm in continuous cultures, and it was shown that fermentation products do not cause an increase in corrosion. Hence, pure-culture biofilms inhibit corrosion of SAE 1018 steel by depleting oxygen at the metal surface.
Characterization and optimization of a two-phase partitioning bioreactor for the biodegradation of phenol by L. D. Collins; A. J. Daugulis (pp. 18-22).
A two-phase partitioning bioreactor containing Pseudomonas putida ATCC 11172 was used to degrade high concentrations of phenol in batch and fed-batch mode. The 2-l (nominal volume) partitioning bioreactor employs a 1-l cell-containing aqueous phase, and a 500-ml immiscible and biocompatible second organic phase (2-undecanone), which partitions the toxic substrate into the aqueous phase at a rate based on the metabolic activity of the microorganisms. Using this reactor configuration, operated in batch mode, 10-g phenol was degraded to completion within 84-h. The system was, however, oxygen-limited during the rapid growth phase of the fermentation. A second experiment, using enriched air to prevent oxygen limitation, resulted in the complete degradation of 10-g phenol within 72-h. The use of a sequential feeding strategy, in which a 10-g phenol load was added in sequential 5-g aliquots, resulted in a significant reduction in the lag phase, from 36-h to 12-h, and the consumption of 10-g phenol in 60 h. Finally, fed-batch fermentation was used to attempt to determine the ultimate capacity of the system to degrade phenol. The organic phase was loaded with 10-g phenol, the microorganisms were allowed to consume this aliquot almost to completion, and a second 10-g aliquot was then added. The organic phase was spiked in this manner a total of four times, resulting in the degradation of 46.55-g phenol within 12 days. The system was also monitored for nutrient depletion, and a nutrient-feeding schedule was formulated, in response to the mass of phenol consumed.
Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture by S. Y. Kim; D. K. Oh; K. H. Lee; J. H. Kim (pp. 23-26).
The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h−1. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production.
Purification and characterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051; application to the recovery of bioactive peptides from fusion proteins by sequence-specific digestion by H. Okamoto; T. Fujiwara; E. Nakamura; T. Katoh; H. Iwamoto; H. Tsuzuki (pp. 27-33).
Screening cultures of nonpathogenic microorganisms led us to a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051, which we purified and named BSase. The nucleotide sequence encoding BSase, with a molecular mass of 23 894 Da, completely agreed with that of the mpr gene, which had been reported by Rufo Jr. and Sloma et al. to encode a metalloprotease [J Bacteriol (1990) 172:1019–1023 and 1024–1029 respectively]. However, enzymatic characterization revealed it to have the catalytic triad of a serine protease and not the consensus sequence of a metalloprotease, and it was inhibited by diisopropylfluorophosphate. We therefore consider BSase (mpr) to be a serine protease. In the alignment of the acidic-amino-acid-specific proteases, the proteases from bacilli have a highly conserved histidine residue, which is most important in the histidine triad in the proteases from streptomycetes. Furthermore, Ca2+ was necessary for its activity and stability. BSase cleaved the C-terminal glutamic acid with high specificity and was very stable over a wide pH range. On the basis of these properties, we tried to retrieve a bioactive peptide from a fusion protein by sequence-specific digestion, and succeeded in obtaining the bioactive peptide. BSase was found to be very useful as a tool for selective cleavage.
Production, purification and characterization of glucose oxidase from a newly isolated strain of Penicillium pinophilum by D. Rando; G.-W. Kohring; F. Giffhorn (pp. 34-40).
A number of nutritional factors influencing growth and glucose oxidase (EC 1.1.3.4) production by a newly isolated strain of Penicillium pinophilum were investigated. The most important factors for glucose oxidase production were the use of sucrose as the carbon source, and growth of the fungus at non-optimal pH 6.5. The enzyme was purified to apparent homogeneity with a yield of 74%, including an efficient extraction step of the mycelium mass at pH 3.0, cation-exchange chromatography and gel filtration. The relative molecular mass (M r) of native glucose oxidase was determined to be 154 700 ± 4970, and 77 700 for the denatured subunit. Electron-microscopic examinations revealed a sandwich-shaped dimeric molecule with subunit dimensions of 5.0 × 8.0 nm. Glucose oxidase is a glycoprotein that contains tightly bound FAD with an estimated stoichiometry of 1.76 mol/mol enzyme. The enzyme is specific for d-glucose, for which a K m value of 6.2 mM was determined. The pH optimum was determined in the range pH 4.0–6.0. Glucose oxidase showed high stability on storage in sodium citrate (pH 5.0) and in potassium phosphate (pH 6.0), each 100 mM. The half-life of the activity was considerably more than 305 days at 4 °C and 30 °C, and 213 days at 40 °C. The enzyme was unstable at temperatures above 40 °C in the range pH 2.0–4.0 and at a pH above 7.0.
Reactivation of thermally inactivated enzymes by free and immobilized chaperonin GroEL/ES by T. Teshima; A. Kondo; H. Fukuda (pp. 41-46).
Thermally inactivated bovine deoxyribonuclease I (DNase I) and yeast enolase were reactivated by GroEL/ES from Escherichia coli. In both cases, GroEL/ES was found to have the ability to reactivate inactivated enzymes in an ATP-dependent manner. GroEL/ES can interact with the enzymes that were denatured at high temperature and convert them to the active conformations. To test the applicability of GroEL/ES to the reactivation processes of thermally inactivated enzymes, GroEL/ES was immobilized using formyl-Cellulofine (GroEL/ES-Cellulofine) and its performance was studied. GroEL/ES-Cellulofine retained a sufficiently high ability to reactivate enzymes. Moreover, GroEL/ES-Cellulofine could be used repeatedly, indicating high durability. These results indicate that immobilized chaperonin is effective for reactivation of enzymes that are thermally inactivated in various bioprocesses.
Mannitol dehydrogenase from Rhodobacter sphaeroides Si4: subcloning, overexpression in Escherichia coli and characterization of the recombinant enzyme by A. Schäfer; M. A. Stein; K.-H. Schneider; F. Giffhorn (pp. 47-52).
By polymerase chain reaction mutagenesis techniques, an NdeI restriction site was introduced at the initiation codon of the mannitol dehydrogenase (MDH) gene (mtlK) of Rhodobacter sphaeroides Si4. The mtlK gene was then subcloned from plasmid pAK74 into the NdeI site of the overexpression vector pET24a+ to give plasmid pASFG1. Plasmid pASFG1 was introduced into Escherichia coli BL21(DE3), which was grown in a 1.5-l bioreactor at 37 °C and pH 7.0. Overexpression of MDH in Escherichia coli BL21(DE3) [pASFG1] was determined by enzymatic analysis and sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Under standard growth conditions, E. coli produced considerable amounts of a polypeptide that correlated with MDH in SDS gels, but the activity yield was low. Decreasing the growth temperature to 27 °C and omitting pH regulation resulted in a significant increase in the formation of soluble and enzymatically active MDH up to a specific activity of 12.4 U/mg protein and a yield of 26 000 U/l, which corresponds to 0.38 g/l MDH. This was an 87-fold overexpression of MDH compared to that of the natural host R. sphaeroides Si4, and a 236-fold improvement of the volumetric yield. MDH was purified from E. coli BL21(DE3) [pASFG1] with 67% recovery, using ammo-nium sulfate precipitation, hydrophobic interaction chromatography, and gel filtration. Partial characterization of the recombinant MDH revealed no significant differences to the wild-type enzyme.
The effect of oxidative pretreatment on cellulose degradation by Poria placenta and Trichoderma reesei cellulases by M. Rättö; A.-C. Ritschkoff; L. Viikari (pp. 53-57).
The possible role of hydrogen peroxide in brown-rot decay was investigated by studying the effects of pretreatment of spruce wood and microcrystalline Avicel cellulose with H2O2 and Fe2+ (Fenton's reagent) on the subsequent enzymatic hydrolysis of the substrates. A crude endoglucanase preparation from the brown-rot fungus Poria placenta, a purified endoglucanase from Trichoderma reesei and a commercial Trichoderma cellulase were used as enzymes. Avicel cellulose and spruce dust were depolymerized in the H2O2/Fe2+ treatment. Mainly hemicelluloses were lost in the treatment of spruce dust. The effect of the pretreatment on subsequent enzymatic hydrolysis was found to depend on the nature of the substrate and the enzyme preparation used. Pretreatment with H2O2/Fe2+ clearly increased the amount of enzymatic hydrolysis of spruce dust with both the endoglucanases and the commercial cellulase. In all cases the amount of hydrolysis was increased about threefold. The hydrolysis of Avicel with the endoglucanases was also enhanced, whereas the hydrolysis with the commercial cellulase was decreased.
Efficient synthesis of the blood-coagulation inhibitor hirudin in the filamentous fungus Acremonium chrysogenum by R. Radzio; U. Kück (pp. 58-65).
The isopenicillin-N-synthetase-encoding pcbC gene from the filamentous fungus Acremonium chrysogenum is differentially expressed in strains showing either a high or low cephalosporin C production. For a case study to demonstrate heterologous protein synthesis in A. chrysogenum, we have chosen a synthetic 195-bp gene encoding the thrombin inhibitor hirudin from the leech Hirudo medicinalis. The hirudin gene was fused with the 5′ and 3′ regions of the pcbC gene, resulting in four different expression vectors, which we named pHIR1 to pHIR4. In order to achieve secretion of the heterologous polypeptide, two out of four vectors carry, in addition, secretion signal sequences of an alkaline protease gene originating either from Fusarium sp. or from A. chrysogenum. After DNA-mediated transformation of the two A. chrysogenum strains, transformants were further analysed on the transcriptional and translational level. Irrespective of the vector used for transformation, all transformants show a hirudin-genespecific transcript in Northern hybridizations. In further analysis, hirudin synthesis was determined with a thrombin-inhibition assay, but was detectable only in those strains carrying expression plasmids with the secretion signals. In this case, hirudin was secreted into the culture medium. Transformants from strains with a high cephalosporin C production showed a three- to eightfold higher expression of the hirudin gene compared to low cephalosporin-C-producing strains. The amount of recombinant hirudin was quantified further by ELISA and Western blotting, using a monoclonal antibody directed against recombinant hirudin. Finally, the time course of hirudin gene expression was investigated in a selected transformant that has hirudin activities of 8.0 ATU/ml culture medium. Northern hybridization experiments revealed the highest hirudin transcript level after 2–5 days of cultivation, showing the strongest signal after 3 days. After 4–5 days, we detected the highest hirudin activity, as was confirmed by Western blotting. The level of heterologous hirudin synthesis in A. chrysogenum is discussed in relation to other eukaryotic expression systems.
Optimization of the solubilization and renaturation of fish growth hormone produced by Escherichia coli by M.-H. Hsih; J.-C. Kuo; H.-J. Tsai (pp. 66-72).
Growth hormone (GH) enhances the growth rate of aquacultured fish and shellfish, but it is difficult to extract native GH from fish pituitary glands. However, fish recombinant GH (rGH) can be efficiently synthesized by Escherichia coli cells, although it exists in denatured form in inclusion bodies (IB). We studied the solubilization of IB and the renaturation of rGH to help facilitate the production of a large amount of biologically active rGH. A 100-ml sample of rGH-producing E. coli produced 73.43 ± 5.47 mg IB (dry weight, n = 3) after 20 h induction by 1 mM isopropyl β-o-thiogalactopyranoside. Interestingly, if the bacteria were induced by 0.1 mM β-lactose, 95.3 ± 3.43 mg of IB was obtained. The optimal conditions for denaturation and renaturation of rGH were when IB were solubilized in 6 M guanidine hydrochloride and then dialysed against pH 10 dialysis buffer (50 mM ammonium bicarbonate and 2 mM EDTA) containing 100 mM l-arginine, 2 mM oxidized glutathione and 2 mM reduced glutathione for 24 h at 4 °C in a volume ratio of 3 to 500. At least 20% of the denaturated rGH in IB was renatured. Juvenile black sea bream injected with 0.05 μg/g resultant rGH once every 2 weeks exhibited significant increases (P < 0.05) in weight gain (84%) relative to fish in the control group over a 16-week period. This process is an economical and effective way to obtain an active form of rGH biosynthesized by a prokaryotic system.
Comparison of partial 16S rRNA gene sequences obtained from activated sludge bacteria by E. J. Vainio; A. Moilanen; T. T. Koivula; D. H. Bamford; J. Hantula (pp. 73-79).
The cultivated and uncultivated bacterial communities of an activated sludge plant were studied. Two samples were taken and a total of 516 bacterial isolates were classified into groups using their whole-cell protein patterns. The distribution of bacteria into protein-pattern groups differed significantly between the two samples, suggesting variation in culturable bacterial flora. Partial 16S rRNA gene sequences were determined for representatives of the commonest protein-pattern groups. Most of the sequences obtained were previously unknown, but relatively closely related to known sequences of organisms belonging to the α, β or γ subclasses of the proteobacteria, the first two subclasses being predominant. This classification of bacteria isolated on a diluted nutrient-rich medium differed from recent culture-dependent studies using nutrient-rich media. The uncultivated bacterial community was studied by analyzing ten partial 16S rRNA gene sequences cloned directly from activated sludge. None of the cloned sequences was identical to those determined for culturable organisms; or to those in the GenBank database. They were, however, related to the α or β subclasses of the proteobacteria, or to the gram-positive bacteria with a high G+C DNA content.
Isolation of new variants of surfactin by a recombinant Bacillus subtilis by S. Nakayama; S. Takahashi; M. Hirai; M. Shoda (pp. 80-82).
A recombinant Bacillus subtilis MI113(pC115), carrying a gene responsible for the production of surfactin and iturin A cloned from B. subtilis RB14C, produced new surfactin variants, in addition to the already reported surfactin, when MI113(pC115) was cultured in solid-state fermentation of soybean curd residue (okara) as a substrate. All variants isolated by HPLC were characterized.
Biotransformation of linoleic acid with the Candida tropicalis M25 mutant by D. Fabritius; H.-J. Schäfer; A. Steinbüchel (pp. 83-87).
Linoleic acid was transformed by mutant Candida tropicalis M25 and transformations were studied in batch and fed-batch cultures. Cofermentations with palmitic acid as inducer of the fatty acid degradation pathway were performed. Besides the (Z),(Z)-octadeca-6,9-dienedioic acid, (Z),(Z)-3-hydroxyoctadeca-9,12-dienedioic acid and (Z),(Z)-3-hydroxytetradeca-5,8-dienedioic acid were obtained as the main fermentation products. The maximum concentrations of (Z),(Z)-octadeca-6,9-dienedioic acid and (Z),(Z)-3-hydroxyoctadeca-9,12-dienedioic acid reached values of 6.4 g/l and 6.9 g/l respectively. The structures of the products were characterized by chemical and spectroscopic methods. The configuration of the double bonds was not changed during bioconversion. As only one regioisomer of the hydroxylated fatty acid was detected, the hydroxylation is site-specific.
Production of poly(β-hydroxybutyrate-β-hydroxyvalerate) copolymer from sugars by Azotobacter salinestris by W. J. Page; N. Bhanthumnavin; J. Manchak; M. Ruman (pp. 88-93).
Azotobacter salinestris, a sodium-dependent, microaerophilic N2-fixing soil bacterium, formed polyhydroxyalkanoate copolymers comprised of β-hydroxybutyric acid and 9–12 mol% β-hydroxyvaleric acid (HV) during growth on sugars. Increased HV content was achieved by feeding valeric acid to the culture growing on glucose, but propionic acid could be directed to HV formation only when it served as the sole C source. Polymer production in nitrogen-fixing cells was increased at higher aeration, provided that a complex organic nitrogen source was also present, but there was no HV in the polymer. HV production was increased to 28 mol% in nitrogen-fixing cells when aeration was lower and acetate was provided with glucose in the medium. Enzymes leading to the production of polyhydroxyalkanoate copolymers were found to be similar in A. salinestris and Azotobacter vinelandii, but A. vinelandii is unable to form HV from propionate or from sugars without valeric acid addition. A biochemical scheme is proposed for the production of HV in A. salinestris, whereby the glyoxylate bypass assimilates acetate to generate succinate, which may be converted into propionyl-CoA for HV synthesis. The results suggest that it may be possible to control the molar yield of HV formed from sugars by A. salinestris.
Choline and acetylcholine: novel cationic osmolytes in Lactobacillus plantarum by E. P. W. Kets; M. Nierop Groot; E. A. Galinski; J. A. M. De Bont (pp. 94-98).
The aim of this work was to study the physiological response of Lactobacillus plantarum subjected to osmotic stress in the presence of three structurally related compatible solutes. Either betaine, choline or acetylcholine was accumulated by osmotically stressed cells when provided in the chemically defined medium. Choline and acetylcholine were accumulated to maximum concentrations of 139 and 222 μmol g (dry weight) of cells−1 respectively and were not converted to betaine. Addition of 0.5 mM choline or 0.5 mM acetylcholine to the medium increased the growth rates of cells in media with various amounts of added sodium chloride. Both choline and acetylcholine are positively charged compounds; therefore, it was presumed that charged intracellular solutes could counterbalance the excess of positive charge. Intracellular inorganic ion levels (K+, SO2− 4, PO3− 4 and Cl−) of cells cultured under conditions of osmotic stress remained similar in the presence of either betaine, choline or acetylcholine. However, cells cultured in the presence of choline or acetylcholine accumulated an additional quantity of approximately 125 or 200 μmol.glutamate (dry weight) cells−1 respectively, as compared to cells grown in the presence of betaine. Hence glutamate appears to be the counterion for choline and acetylcholine. This is the first study demonstrating accumulation of choline and acetylcholine in lactic acid bacteria subjected to osmotic stress.
Effect of the addition of Peptostreptococcus productus ATCC35244 on the gastro-intestinal microbiota and its activity, as simulated in an in vitro simulator of the human gastro-intestinal tract by L. Nollet; I. Vande Velde; W. Verstraete (pp. 99-104).
Peptostreptococcus productus ATCC35244, a reductive acetogenic strain, was added daily over 9 successive days to the fourth vessel (ascending colon) of the SHIME, a six-stage reactor system simulating the in vivo continuous culture conditions of the human gastro-intestinal tract. Final numbers of organisms (cfu)/ml reactor contents (c) were attained such that log10 c = 6.9 ± 0.1. The addition caused the CH4 production to decrease below the detection limit while total gas and CO2 production in the fifth (transverse colon) and sixth reactor (descending colon) were lowered and the acetic acid concentration was augmented. Ending the supplementation caused CH4 production to re-establish within 4 days, while CO2 production increased much more slowly. The concentration of acetic acid only started to decrease after 7 days. The results indicate that P. productus, upon regular administration, is able to compete with methanogens for H2 in the gastro-intestinal microbial ecosystem because of its reductive acetogenic character.
Creating auxotrophic mutants in Methylophilus methylotrophus AS1 by combining electroporation and chemical mutagenesis by C. S. Kim; T. K. Wood (pp. 105-108).
Stable auxotrophic mutants of the methylotroph Methylophilus methylotrophus AS1 were obtained by a novel mutagenesis technique in which electroporation is used to transport the chemical mutagen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) across the cell membrane. By combining chemical mutagenesis with electroporation and screening single colonies for auxotrophy in 36 different amino acids and growth factors, 3 auxotrophs per 156 colonies screened were obtained, whereas no auxotrophs were found with chemical mutagenesis alone. MNNG mutagen toxicity was also increased in the methylotroph with this novel mutagenesis technique (death rate 96% compared to 79%). This technique did not increase the mutation rate for strain Escherichia coli BK6 which responds well to simple exposure to the mutagen.
Superior survival and degradation of dibenzo-p-dioxin and dibenzofuran in soil by soil-adapted Sphingomonas sp. strain RW1 by M. Megharaj; R.-M. Wittich; R. Blasco; D. H. Pieper; K. N. Timmis (pp. 109-114).
The dibenzo-p-dioxin(DD)- and dibenzofuran(DF)-degrading bacterium, Sphingomonas sp. strain RW1, was tagged by insertion of a mini-Tn5 lacZ transposon in order to follow its fate in complex laboratory soil systems. The tagged strain was tested for its ability to survive in soil and degrade DF and DD applied at a concentration of 1 mg/g. Bacteria pre-adapted to soil conditions were found to survive better in DF- and DD-amended soil and degrade the substrate more efficiently than bacteria that had not been subjected to pre-adaptation. The concentration of soil-applied DF and DD, individually and in combination, decreased to less than 2% of the original concentrations within 3 weeks of addition of the RW1 derivative, accompanied by a short, but significant exponential increase in RW1 viable cells. During the same period the native bacterial population in soil was stable while viable fungi declined.
Initiation of [36Cl]hexachlorobenzene dechlorination in three different soils under artificially induced anaerobic conditions by P. Rosenbrock; R. Martens; F. Buscot; J. C. Munch (pp. 115-120).
The potential for reductive dechlorination of hexachlorobenzene was investigated in samples of three different, naturally oxic soils held under conditions of high oxygen deficiency. The soils were water-saturated and the influence on dechlorination of adding different electron donors, a surfactant and an anaerobic microbial consortium was tested. The influence of supplied electron donors seems to depend on the organic matter content of the soils. Dechlorination in the organic-matter-rich soil from Maulach was not affected by amendment with organic electron donors. A release of about 40% chloride within 140 days was observed for this soil in all biotic-treated assays. By contrast, the organic-matter-poor soil of Eppingen showed no dechlorination in unamended assays. However, when it was supplemented with organic electron donors dechlorination of 2%–37% occurred within 140 days, depending on the type of electron donor. Complex substrate (wheat strawdust), from which carbon is slowly liberated, gave the best results. These two soils had an indigenous dechlorinating anaerobic microflora, whereas the third soil (Rastatt) required inoculation with an anaerobic consortium for dechlorination. The addition of electron donors alone did not cause dechlorination in this sandy soil. The addition of a surfactant (Tween 80) to increase the bioavailability of hexachlorobenzene did not enhance dechlorination. This process was not inhibited by inherent alternative electron acceptors in soil (NO3−, SO4 2−, Fe3+). The dechlorination did not require methanogenic conditions.
Thermophilic biodegradation of BTEX by two consortia of anaerobic bacteria by C.-I. Chen; R. T. Taylor (pp. 121-128).
Two thermophilic anaerobic bacterial consortia (ALK-1 and LLNL-1), capable of degrading the aromatic fuel hydrocarbons, benzene, toluene, ethylbenzene, and the xylenes (BTEX compounds), were developed at 60 °C from the produced water of ARCO'S Kuparuk oil field at Alaska and the subsurface water at the Lawrence Livermore National Laboratory gasoline-spill site, respectively. Both consortia were found to grow at 45–75 °C on BTEX compounds as their sole carbon and energy sources with 50 °C being the optimal temperature. With 3.5 mg total BTEX added to sealed 50-ml serum bottles, which contained 30 ml mineral salts medium and the consortium, benzene, toluene, ethylbenze, m-xylene, and an unresolved mixture of o- and p-xylenes were biodegraded by 22%, 38%, 42%, 40%, and 38%, respectively, by ALK-1 after 14 days of incubation at 50 °C. Somewhat lower, but significant, percentages of the BTEX compounds also were biodegraded at 60 °C and 70 °C. The extent of biodegradation of these BTEX compounds by LLNL-1 at each of these three temperatures was slightly less than that achieved by ALK-1. Use of [ring-14C]toluene in the BTEX mixture incubated at 50 °C verified that 41% and 31% of the biodegraded toluene was metabolized within 14 days to water-soluble products by ALK-1 and LLNL-1, respectively. A small fraction of it was mineralized to 14CO2. The use of [U-14C]benzene revealed that 2.6%–4.3% of the biodegraded benzene was metabolized at 50 °C to water-soluble products by the two consortia; however, no mineralization of the degraded [U-14C]benzene to 14CO2 was observed. The biodegradation of BTEX at all three temperatures by both consortia was tightly coupled to sulfate reduction as well as H2S generation. None was observed when sulfate was omitted from the serum bottles. This suggests that sulfate-reducing bacteria are most likely responsible for the observed thermophilic biodegradation of BTEX in both consortial cultures.
Biodegradation dynamics of aromatic compounds from waste air in a trickle-bed reactor by D. Hekmat; A. Linn; M. Stephan; D. Vortmeyer (pp. 129-134).
Biodegradation of polyalkylated benzenes from waste air was investigated experimentally in a laboratory-scale trickle-bed reactor. The study focussed on the description of process dynamics with regard to its behavior during quasi-steady-state, the start-up of the reactor, the influence of the nitrogen source and the response to cyclical starvation periods. A stable long-term operation of the bioreactor could be achieved by maintaining a sufficient nitrogen source at any time and by removing excess biomass, i.e. mainly extracellular polymeric substances, from the system from time to time. The start-up period of the bioreactor using an adapted mixed population was as short as 48 h and reproducible. A clear dependence of the biodegradation on the available nitrogen concentration in the liquid phase was observed with experiments where ammonium was added as pulses. A dynamic operation mode with cyclical starvation periods was applied simulating a two-shift operation of a waste-air purification plant over 7 days a week. This cyclical operation led to an increase of the integral biodegradation performance. The characteristic bioreactor response time to the dynamic operation with starvation periods was about 6–7 h and conformed with the observations of other studies.