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Applied Microbiology and Biotechnology (v.47, #6)
Development of a low-cost fermentation medium for ethanol production from biomass by K. L. Kadam; M. M. Newman (pp. 625-629).
Nutrient cost is an important aspect in the fermentation of biomass to ethanol. With a goal of developing a cost-effective fermentation medium, several industrially available nutrient sources were evaluated for their effectiveness in the simultaneous saccharification and fermentation of pretreated poplar with Saccharomyces cerevisiae D5A. These studies showed that a low-cost medium containing 0.3% corn steep liquor and 2.5 mM MgSO4 · 7H2O was similar in performance to a nutrient-rich medium. Besides its low cost, this alternative medium consists of components that are available on a commercial scale, thereby making it industrially relevant.
Activity of Pseudomonas cepacia lipase in organic media is greatly enhanced after immobilization on a polypropylene support by Gaëlle Pencreac'h; Jacques C. Baratti (pp. 630-635).
The purified lipase from Pseudomonas cepacia was used as free and immobilized enzyme preparation for hydrolysis of p-nitrophenyl palmitate (pNPP) and p-nitrophenyl acetate (pNPA) in organic media. The free enzyme was mixed with bovine serum albumin and lyophilized. Immobilization was on porous polypropylene. Conditions where diffusional limitations of the substrate were not limiting the reaction rate were defined. The specific activity of the lipase was greatly enhanced upon immobilization: 16.5- and 7.8-fold for pNPP and pNPA respectively. Both the free and immobilized lipases followed Michaelis–Menten kinetics in organic solvent despite the heterogeneity (solid/liquid) of the reaction mixture. For pNPP, the activation factor upon immobilization came mainly from a reduction in K m, app while k cat was increased for pNPA.
Anaerobic leaching of covellite by Thiobacillus ferrooxidans by E. Donati; C. Pogliani; J. L. Boiardi (pp. 636-639).
Thiobacillus ferrooxidans was able to grow under anaerobic conditions on copper sulphide with ferric ion as the electron acceptor. The dissolution of covellite under these conditions (68% after 35 days) was higher than values observed aerobically in cultures with similar media composition and almost as high as under aerobic conditions without iron. From these results we propose a mechanism for anaerobic bioleaching of covellite in the presence of ferric iron and speculate that it may occur in leach dumps where the oxygen concentration is, as reported elsewhere, very low.
Effects of support material on the pattern of volatile fatty acid accumulation at overload in anaerobic digestion of semi-solid waste by L. Björnsson; B. Mattiasson; T. Henrysson (pp. 640-644).
Anaerobic degradation of a semi-solid waste with a total solids content of 4% particulate matter, much of it insoluble, was investigated in four laboratory-scale reactors. Two of the reactors were equipped with different textile materials for immobilisation of microorganisms, while the other two were used as continuously-stirred-tank reactor references. A constant organic loading rate and hydraulic retention time were used in the start-up period; the hydraulic retention time was then decreased and the effects of this change were monitored. Volatile fatty acid (VFA) concentration and pH were chosen as indicators of the microbial status in the reactors. The reactors with support material showed a greater resistance to overload than did the continuously-stirred-tank reactors. This is in agreement with many studies undertaken on the anaerobic treatment of wastewater. However, no problems with clogging occurred, showing that a support material is also applicable in systems treating waste containing large amounts of insoluble, particulate matter. The pH was comparable to VFA for indicating an approaching process failure. However, the pattern of VFA accumulation was qualitatively different between the reactors with and without support material. Obviously the metabolic pattern of mixed cultures changes when the microorganisms are immobilised.
The influence of β-cyclodextrin on the kinetics of 1-en-dehydrogenation of 6α-methylhydrocortisone by Arthrobacter globiformis cells by V. V. Fokina; A. V. Karpov; I. A. Sidorov; V. A. Andrjushina; A. Y. Arinbasarova (pp. 645-649).
β-Cyclodextrin changes the kinetic peculiarities of microbial 1-en-dehydrogenation of 6α-methylhydrocortisone: the reaction rate, degree of conversion and respiratory-chain activity are increased. Respiratory-chain activity in the presence of β-cyclodextrin is increased both in the presence and in the absence of 6α-methylhydrocortisone. A mathematical model is proposed to describe the kinetics of the process. This model suggests the formation of different multi-component complexes consisting of inclusion complexes and the functional unit involving the enzyme and the respiratory chain. All the parameters of the model were estimated by data fitting. In the model framework the formation of multi-component complexes leads to an increase of the maximal reaction rate and to a decrease of substrate inhibition. An approach is suggested for optimisation of 6α-methylhydrocortisone 1-en-dehydrogenation in the presence of β-cyclodextrin. The optimal concentrations of 6α-methylhydrocortisone and β-cyclodextrin have been calculated.
Isolation and characterization of highly (R)-specific N-acetyl-1-phenylethylamine amidohydrolase, a new enzyme from Arthrobacter aurescens AcR5b by M. Graf; A. Brunella; M. Kittelmann; K. Laumen; O. Ghisalba (pp. 650-657).
A new amidohydrolase deacetylating several N-acetyl-1-phenylethylamine derivatives (R)-specifically was found in Arthrobacter aurescens AcR5b. The strain was isolated from a wet haystack by enrichment culture with (R)-N-acetyl-1-phenylethylamine as the sole carbon source. (R) and (S )-N-acetyl-1-phenylethylamine do not serve as inducers for acylase formation. By improving the growth conditions the enzyme production was increased 47-fold. The amidohydrolase was purified to homogeneity leading to a 5.2-fold increase of the specific activity with a recovery of 67%. A molecular mass of 220 kDa was estimated by gel filtration. Sodium dodecyl sulfate/polyacrylamide gel electrophorosis shows two subunits with molecular masses of 16 kDa and 89 kDa. The optimum pH and temperature were pH 8 and 50 °C, respectively. The enzyme was stable in the range of pH 7–9 and at temperatures up to 30 °C. The enzyme activity was inhibited by Cu2+, Co2+, Ni2+, and Zn2+, and this inhibition was reversed by EDTA.M
Mode of depolymerisation of hemicellulose by various mannanases and xylanases in relation to their ability to bleach softwood pulp by G. M. Gübitz; D. Haltrich; B. Latal; W. Steiner (pp. 658-662).
Endo-mannanases and endo-xylanases cleave different heteromannans and xylans yielding mainly dimers and trimers of the corresponding sugars as end-products. However, in the early stages of hydrolysis, four purified mannanases and four xylanases from fungal and bacterial origin, examined in this study, showed a different pattern of released oligomers (determined up to the pentamers). Furthermore, some of these enzymes showed a preference for cleaving the polysaccharides in the middle of the chain while others acted more at the end. When the increase in the specific fluidity of mannan and xylan solutions per reducing sugar released (K v) was measured against the bleaching effect of the enzymes on softwood kraft pulp, a correlation was found. A xylanase from Penicillium simplicissimum (K v = 0.15 l mPa−1s−1g−1) and a mannanase from Sclerotium rolfsii (K v = 0.12 l mPa−1s−1g−1) applied in a O(QX)P bleaching sequence (O = oxygen delignification, X = treatment with hemicellulolytic enzymes, Q = chelation of metals, P = treatment with hydrogen peroxide in alkaline solution) gave a high brightness increase of 3.0% and 1.9% ISO respectively. A less significant brightness increase was obtained with enzymes showing lower K v values, such as a xylanase from Schizophyllum commune (Kv = 0.051 l mPa−1s−1g−1, 0.2% ISO) and a bacterial mannanase (K v = 0.061 l mPa−1s−1g−1,0.5% ISO).
Cloning and expression of the Δ9 fatty acid desaturase gene from Cryptococcus curvatus ATCC 20509 containing histidine boxes and a cytochrome b 5 domain by P. A. E. P. Meesters; J. Springer; G. Eggink (pp. 663-667).
To allow genetic modification of the fatty acid biosynthesis routes in the lipid-accumulating yeast Cryptococcus curvatus the Δ9 fatty acid desaturase gene was cloned and characterized. The 1668-bp gene encodes a protein of 556 amino acids with a calculated molecular mass of 62 kDa. The gene shows strong homology to previously cloned Δ9 fatty acid desaturase genes from yeast and rat. Homology includes three histidine boxes characteristic for membrane-bound desaturases and a cytochrome b 5 domain responsible for electron transport. The Δ9 desaturase gene has a high G+C content of 61% and displays a codon usage different from that of Saccharomyces cerevisiae, but similar to that of the basidiomycete Schizophyllum commune. Expression of the Δ9 desaturase gene of C. curvatus ATCC 20509 was studied in the presence of different fatty acids in the growth medium. Repression of desaturase mRNA signals was found if fatty acids with a double bond at the Δ9 position were present. Fatty acids with a double bond at another position (Δ10 or Δ6) or saturated fatty acids had no effect on the transcription of the cloned gene.
Enrichment strategies for nitrile-hydrolysing bacteria by N. Layh; B. Hirrlinger; A. Stolz; H.-J. Knackmuss (pp. 668-674).
A series of enrichments with different nitriles as sole source of nitrogen was performed in order to obtain a relationship between the selective nitrogen source and (i) the enzyme systems that are synthesized by the isolates and (ii) the enzyme specificities for the utilization of the nitriles. Bacteria were enriched with 2-phenylpropionitrile, 2-(2-methoxyphenyl)propionitrile, 2-phenylbutyronitrile, ibuprofen nitrile, naproxen nitrile, ketoprofen nitrile, ketoprofen amide, benzonitrile, or naphthalenecarbonitrile as sole nitrogen source and succinate as sole source of carbon and energy. 2-Phenylpropionitrile as nitrogen source resulted predominantly in the enrichment of gram-negative bacteria, which harboured nitrilase and in some cases also amidase activity. In contrast, with the other nitriles used, a substantial majority of gram-positive strains, mainly of the genus Rhodococcus, were isolated. These strains contained predominantly a nitrile hydratase/amidase system. The nitrilases and nitrile hydratases showed R or S selectivity with generally poor optical yields. In contrast, the amidases were almost exclusively S-selective, often forming the optically pure acids with an enantiomeric excess above 99%. The conversion of different nitriles by the isolates was compared. The nitrile-hydrolysing systems of the new isolates usually showed high activity against those nitriles that were used for the enrichment of the bacteria.
Pentachlorophenol biodegradation kinetics of an oligotrophic fluidized-bed enrichment culture by E. S. Melin; J. F. Ferguson; J. A. Puhakka (pp. 675-682).
A fluidized-bed reactor (FBR) was used to enrich an aerobic chlorophenol-degrading microbial culture. Long-term continuous-flow operation with low effluent concentrations selected oligotrophic microorganisms producing good-quality effluent for pentachlorophenol(PCP)-contaminated water. PCP biodegradation kinetics was studied using this FBR enrichment culture. The results from FBR batch experiments were modeled using a modified Haldane equation, which resulted in the following kinetic constants: q max = 0.41 mg PCP mg protein−1 day−1, K S = 16 μg l−1, K i = 5.3 mg l−1, and n = 3.5. These results show that the culture has a high affinity for PCP but is also inhibited by relatively low PCP concentrations (above 1.1 mg PCP l−1). This enrichment culture was maintained over 1 year of continuous-flow operation with PCP as the sole source of carbon and energy. During continuous-flow operation, effluent concentrations below 2 μg l−1 were achieved at 268 min hydraulic retention time (t HR) and 2.5 mg PCP l−1 feed concentration. An increase in loading rate by decreasing t HR did not significantly deteriorate the effluent quality until a t HR decrease from 30 min to 21 min resulted in process failure. Recovery from process failure was slow. Decreasing the feed PCP concentration and increasing t HR resulted in an improved process recovery.
Rosmarinic acid production by Lavandula vera MM cell-suspension culture by M. Ilieva; A. Pavlov (pp. 683-688).
The time courses of growth and rosmarinic acid production by Lavandula vera MM cell suspension were investigated. The uptake of the main nutrients (sucrose, nitrogen, phosphorus, K, Ca, Mg) was followed during cultivation and the data on the physiology of the L. vera MM cell culture are presented. It was established that the cell culture synthesizes rosmarinic acid during the linear phase of growth for a relatively short period (between the 4th and 8th days of cultivation). The influence of sucrose concentration in the nutrient medium on cell growth and accumulation of rosmarinic acid by L. vera MM cell culture was investigated. The results showed that 7% sucrose in the nutrient medium ensured a steady growth of the cell suspension and increased the yield of rosmarinic acid (29.2 g/l dry biomass and 507.5 mg/l rosmarinic acid compared to 13.0 g/l dry biomass and 68.6 mg/l rosmarinic acid for the control cultivation with 3% sucrose).
Effects of yeast extract and glucose on xanthan production and cell growth in batch culture of Xanthomonas campestris by Yang-Ming Lo; Shang-Tian Yang; David B. Min (pp. 689-694).
Although available kinetic data provide a useful insight into the effects of medium composition on xanthan production by Xanthomonas campestris, they cannot account for the synergetic effects of carbon (glucose) and nitrogen (yeast extract) substrates on cell growth and xanthan production. In this work, we studied the effects of the glucose/yeast-extract ratio (G/YE) in the medium on cell growth and xanthan production in various operating modes, including batch, two-stage batch, and fed-batch fermentations. In general, both the xanthan yield and specific production rate increased with increasing G/YE in the medium, but the cell yield and specific growth rate decreased as G/YE increased. A two-stage batch fermentation with a G/YE shift from an initial low level (2.5% glucose/0.3% yeast extract) to a high level (5.0% glucose/0.3% yeast extract) at the end of the exponential growth phase was found to be preferable for xanthan production. This two-stage fermentation design both provided fast cell growth and gave a high xanthan yield and xanthan production rate. In contrast, fed-batch fermentation with intermittent additions of glucose to the fermentor during the stationary phase was not favorable for xanthan production because of the relatively low G/YE resulting in low xanthan production rate and yield. It is also important to use a moderately high yeast extract concentration in the medium in order to reach a high cell density before the culture enters the stationary phase. A high cell density is also important to the overall xanthan production rate.
The de novo production of drosophilin A (tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (tetrachloro-1,4-dimethoxybenzene) by ligninolytic basidiomycetes by P. J. M. Teunissen; H. J. Swarts; J. A. Field (pp. 695-700).
Ligninolytic basidiomycetes were screened for their ability to produce the tetrachlorinated hydroquinone metabolites drosophilin A (DA, tetrachloro-4-methoxyphenol) and drosophilin A methyl ether (DAME, tetrachloro-1,4-dimethoxybenzene). Five fungal strains produced these metabolites in detectable amounts, including strains from Bjerkandera and Peniophora, which are genera not previously known for DA or DAME production. Phellinus fastuosus ATCC26.125 had the highest and most reliable production of DA and DAME in peptone medium, respectively 15–60 μM and 4–40 μM. This fungus was used to study culture conditions that could increase DAME production. A fourfold increase in DAME production was found after the addition of hydroquinone to growing cultures of P. fastuosus. Therefore, hydroquinone is postulated to be a possible biosynthetic precursor of DAME in the fungus. Antagonising P. fastuosus by adding filter-sterilised culture fluid of a competing fungus, Phlebia radiata, increased DAME production significantly by tenfold. This result suggests that DAME production is elicited by compounds present in the culture fluid of P. radiata, indicating that DAME has an antibiotic function in P. fastuosus.
The biotechnology of hydrogen production by Nostoc flagelliforme grown under chemostat conditions by Rixa R. Lichtl; Michael J. Bazin; David O. Hall (pp. 701-707).
The potential of using N2-fixing cyanobacteria to produce hydrogen photobiologically has stimulated research on the physiology and biotechnology of species exhibiting high H2 production rates over long periods of time. In this work Nostoc flagelliforme, a terrestrial N2-fixing cyanobacterium, has been examined to establish its physiology and potential for H2 production under controlled conditions. Cell filaments of N. flagelliforme were purified and grown in liquid culture to optimize its H2 metabolism. In batch-grown cultures the activity of nitrogenase, the key enzyme for H2 production in N2-fixing organisms, was found to be high only during a short phase of exponential growth. A chemostat system was thus constructed for long-term experiments using continuous cultures, with the aim of exploiting the exponential growth phase. The dilution rate (D) and environmental factors, such as N2 concentration in the gas phase and temperature, significantly influenced H2 production. Cells grown continuously under the optimized conditions of D = 0.022 h−1, 34 °C and 5.1 kPa N2 in the gas phase exhibited H2 production rates that were more than four times higher than the maximal rates under standard batch growth conditions.
Influence of cell immobilization on the production of benzaldehyde and benzyl alcohol by the white-rot fungi Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens by Carmen Lapadatescu; Gilles Feron; Catherine Vergoignan; Aleth Djian; Alain Durand; Pascal Bonnarme (pp. 708-714).
Three white-rot basidiomycetes, Bjerkandera adusta, Ischnoderma benzoinum and Dichomitus squalens, were cultivated on a liquid medium supplemented with l-phenylalanine, a precursor for benzaldehyde (bitter almond aroma) and benzyl alcohol. Remarkable amounts of benzaldehyde (587 mg l−1) were found in cultures of B. adusta. Immobilization of this fungus on polyurethane foam cubes allowed an 8.3-fold increase of the production of benzaldehyde and a 15-fold increase of the productivity as compared with non-immobilized cells. Aryl-alcohol oxidase activity was only detected in B. adusta. This activity was also significantly enhanced in immobilized cells, suggesting that it plays an important role in benzaldehyde biosynthesis. Conversely, consistent amounts of benzyl alcohol (340 mg l−1 for B. adusta and I. benzoinum and 100 mg l−1 for D. squalens) were produced by the three fungi when immobilized. Laccase activity was found only in the strains I. benzoinum and D. squalens. This activity was markedly enhanced in free cells cultures. Immobilization of the fungi did not promote benzyl alcohol production by comparison with free cell cultures (500 mg l−1).
Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides by P. Schmitt; C. Vasseur; V. Phalip; D. Q. Huang; C. Diviès; H. Prévost (pp. 715-718).
The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation.
Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction by Marie-Josée Lévesque; Sylvie La Boissière; Jean-Christophe Thomas; Réjean Beaudet; Richard Villemur; Sylvie La Boissière (pp. 719-725).
A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 μg soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time.
Batch and fed-batch bioreactor cultivations of a Thermus species with thermophilic BTEX-degrading activity by C.-I. Chen; R. T. Taylor (pp. 726-733).
The thermophilic bacterium, Thermus species ATCC 27978, which is capable of aerobically degrading benzene, toluene, ethylbenzene, and the xylenes (BTEX), was cultured in 5-1 fermentors on a Castenholz salts-tryptone medium. This bacterium can be cultivated more conveniently at 45 °C, a temperature substantially lower than its optimal growth temperature (approx. 60 °C). Yet, the washed harvested cells from such cultures display the same initial BTEX-degrading activity as those when Thermus sp. is grown at its higher optimal temperature. Two bioreactor cultivation modes, batch and fed batch, were investigated. More biomass and more BTEX-degrading activity (assayed at 60 °C) were generated in fed-batch cultures than in the growth-limited batch cultures. The former yielded a biomass concentration of 2.5 g dry cell weight (DCW) l−1 and whole-cell degrading specific activities of 7.6 ± 1.3, 10.1 ± 1.9, 9.8 ± 2.1, 2.3 ± 0.5, and 4.6 ± 0.9 nmol degraded (mg DCW)−1 min−1 for benzene, toluene, ethylbenzene, m-xylene, and the o- plus p-xylenes (unresolved mixture), respectively. Although the formation of cellular BTEX-degrading activity is growth-associated, a slow to moderate specific growth rate of 0.02–0.07 h−1 favors the production of BTEX-degrading activity, while a high growth rate, of the order of 0.16 h−1, is detrimental to its production. The washed harvested Thermus sp. cells were capable of degrading BTEX over a broad range of thermophilic incubation temperatures, 45–77 °C.
Nitrification, denitrification, and dechlorination in bleached kraft pulp mill wastewater by E. Kostyál; E.-L. Nurmiaho-Lassila; J. A. Puhakka; M. Salkinoja-Salonen (pp. 734-741).
This study deals with combining the biologi cal removal of organic halogens with the removal of nitrogen from bleached kraft pulp mill wastewater in fluidized-bed reactors under nitrifying and denitrifying conditions. Untreated and biotreated bleached kraft pulp mill wastewaters had no detrimental effect on nitrification or denitrification. The nitrifying biofilm reactor, pregrown on synthetic inorganic feed with ammonia, removed without a lag phase adsorbable organic halogens [7.2 mg Cl (g biomass volatile solids)−1day−1] from bleached kraft pulp mill wastewater and selected chlorophenols from synthetic wastewater. Electron microscopical examination of the biofilm showed that bacteria, morphologically similar to the nitrifying species Nitrosomonas or Nitrobacter, and Nitrosospira were dominant. The denitrifying fluidized-bed reactor, pregrown on nitrate and methanol, denitrified without a lag phase bleached kraft pulp mill wastewater. Under denitrifying conditions, 35% of the total organic carbon content of untreated bleached kraft pulp mill waste water was removed. The reducing power delivered by untreated bleached kraft pulp mill wastewater for denitrification was 2 mmol electrons/mmol carbon mineralized. Dechlorination under denitrifying conditions was negligible.
Reductive dechlorination of chlorophenols in slurries of low-organic-carbon marine sediments and subsurface soils by Dorothy D. Hale Boothe; John E. Rogers; Juergen Wiegel (pp. 742-748).
The reductive dechlorination of 2,4- and 3,4-dichlorophenol (DCP) was studied in slurries of marine sediments and subsurface soils with dissolved organic carbon concentrations less than 1 ppm. Dechlorination was markedly greater in marine sediment slurries than in subsoil slurries, although similar products were observed in each case. From 25% to 98% of the 2,4- and 3,4-DCP (6.5 μm/l) added to most marine slurries was converted to 4- and 3-chlorophenol (CP) respectively, within 30 weeks. In contrast 2,4-DCP was dechlorinated to 4-CP (>90%) in only 1 of 24 replicate subsoil slurries after 32 weeks of incubation. Dechlorination was observed within 2 weeks when yeast extract was added to subsoil slurries; yeast extract additions also stimulated dechlorination in marine sediments but to a lesser extent. The intermediate monochlorophenol products did not persist in marine slurries but did persist in the subsoil slurries. It was concluded that the total organic carbon at a site is not always a good predictor of the site's ability to support dechlorination activity.