Optimum Conditions for Transformation of Maleic Acid by Immobilized Cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260

Immobilized cells of Alcaligenes xylosoxidanssubsp. xylosoxidans260 transformed 98% of the maleic acid (initial concentration of 5.0 g/l medium) under periodic conditions for 48 h. Free cells transformed only 26% of the substrate in 96 h. Immobilized cells of a selected S-variant ofA. xylosoxidanstransformed the maleate (30.0 g/l) entirely in 96 h during batch cultivation and only 15.0 g/l of the maleate in continuous cultivation at a flow rate of 0.03 h^–1.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid from a mixture of xylose and glucose by co-cultivation of lactic acid bacteria

The production of optically pure lactic acid in a high yield from xylose or a mixture of xylose and glucose, which is a model hydrolysate of lignocellulose, is described. In a single cultivation, Enterococcus casseliflavus produced 38 g/l of lactic acid with an optical purity of 96% enantiomeric excess (ee) and 6.4 g/l of acetic acid from 50 g/l of xylose when MRS medium was used. When a mixture of 50 g/l of xylose and 100 g/l of glucose was used as the carbon source in a cultivation of E. casseliflavus alone, glucose was converted to lactic acid in the early phase of the cultivation but xylose was hardly consumed. In a co-cultivation where E. casseliflavus and Lactobacillus casei specific for glucose were simultaneously inoculated, little or no lactic acid was produced after the glucose was almost consumed. A co-cultivation with two-stage inoculation (in which E. casseliflavus was added at a cultivation time of 40 h after L. casei cells were inoculated) resulted in complete consumption of 50 g/l of xylose and 100 g/l of glucose. In the co-cultivation, 95 g/l of lactic acid with a high optical purity of 96% ee was obtained at 192 h. Such a co-cultivation using two microorganisms specific for each sugar is considered to be one promising cultivation technique for the efficient production of lactic acid from a sugar mixture derived from lignocellulose.     

Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans

A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H2O----HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, greater than 97% as determined by amino acid sequencing) was estimated to be greater than 300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.     

Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans.

A cyanide-metabolizing bacterium, strain DF3, isolated from soil was identified as Alcaligenes xylosoxidans subsp. denitrificans. Whole cells and cell extracts of strain DF3 catalyzed hydrolysis of cyanide to formate and ammonia (HCN + 2H2O----HCOOH + NH3) without forming formamide as a free intermediate. The cyanide-hydrolyzing activity was inducibly produced in cells during growth in cyanide-containing media. Cyanate (OCN-) and a wide range of aliphatic and aromatic nitriles were not hydrolyzed by intact cells of A. xylosoxidans subsp. denitrificans DF3. Strain DF3 hydrolyzed cyanide with great efficacy. Thus, by using resting induced cells at a concentration of 11.3 mg (dry weight) per ml, the cyanide concentration could be reduced from 0.97 M (approximately 25,220 ppm) to less than 77 nM (approximately 0.002 ppm) in 55 h. Enzyme purification established that cyanide hydrolysis by A. xylosoxidans subsp. denitrificans DF3 was due to a single intracellular enzyme. The soluble enzyme was purified approximately 160-fold, and the first 25 NH2-terminal amino acids were determined by automated Edman degradation. The molecular mass of the active enzyme (purity, greater than 97% as determined by amino acid sequencing) was estimated to be greater than 300,000 Da. The cyanide-hydrolyzing enzyme of A. xylosoxidans subsp. denitrificans DF3 was tentatively named cyanidase to distinguish it from known nitrilases (EC 3.5.5.1) which act on organic nitriles.     

Production of L-DOPA from pyrocatechol and DL-serine by bioconversion using immobilized Erwinia herbicola cells

Summary Immobilized cells of Erwinia herbicola were used for L-DOPA production from pyrocatechol and DL-serine. Optimal conditions have been defined and utilized in batch and continuous reactors. A maximal volumetric productivity of 0.46 g/l.h in L-DOPA was obtained with a conversion yield of 18% (L-DOPA concentration 2.3 g/l).     

Vertical Rotating Immobilized Cell Reactor of the bacteriumZymomonas mobilis for stable long-term continuous ethanol production

Summary Vertical Rotating Immobilized Cell Reactor was designed and built for glucose conversion into ethanol. Immobilized biomass units withZ. mobilis cells attached into polyurethane foam discs were fixed along a rotating shaft inside the bioreactor. The effect of rotation speed on the concentration of immobilized biomass was studied. Stability of the bioreactor over long-term operation was dependent on the concentration of the immobilized biomass. With fermentation carried out at 6 rpm a constant active immobilized cell concentration of only 34.5 g/l was maintained and used to convert up to 140 g glucose/l into more than 70 g ethanol/l with a volumetric ethanol productivity of 63 g/l/h.     

Alcoholic fermentation by agar-immobilized yeast cells

Immobilized yeast cells in agar gel beads were used in a packed bed reactor for the production of ethanol from cane molasses at 30°C, pH 4.5. The maximum productivity, 79.5g ethanol/l.h was obtained with 195g/l reducing sugar as feed. Substrate (64.2%) was utilized at a dilution of 1.33h^-1. The immobilized cell reactor was operated continuously at a constant dilution rate of 0.67h^-1 for 100 days. The maximum specific ethanol productivity and specific sugar uptake rate were 0.610g ethanol/g cell.h and 1.275g sugar/g cell.h, respectively.     

Comparison of free and immobilized Pichia pastoris cells for conversion of ethanol to acetaldehyde

Free and immobilized cells of Pichia pastoris were used to convert ethanol to acetaldehyde in small-scale batch reactors. Immobilized cells were less active than free cells (V(max) free = 7.81 g/L h, V(max) immobilized = 3.17 g/L h) due to a number of factors including end product inhibition and diffusional limitations. Immobilized cells were more resistant to heat denaturation both in the presence and absence of ethanol. Immobilized cells retained more of their activity during repeated batch cycles than did free cells.     

Regeneration of NAD(P)H by immobilized whole cells of Clostridium butyricum under hydrogen high pressure

Immobilized whole cells of Clostridium butyricum reduced both NAD(+) and NADP(+) in the presence of hydrogen at a pressure of 100 atm. The NAD(+) and NADP(+) reduction activities were 4.45 and 4.30 U/g dry cells, respectively [U = NAD(P)H regenerated, mu mol/min]. The amount of NADH regenerated by immobilized cells increased with increasing hydrogen pressure above 10 atm. Immobilized cells (6 mg dry cells) of Cl. butyricum completely converted NAD(+) (6.4 mumole) to NADH for 5 h, whereas only 60% of NAD(+) were reduced by free cells. Immobilized cells retained 89% activity after the 5-h reactions were repeated 4 times. L-Alanine was continuously produced at the rate of 12.8 mumol/min g dry cells from hydrogen, ammonium, and pyruvate with immobilized Cl. butyricum-alanine dehydrogenase.     

Enhanced chrysene degradation by halotolerant Achromobacter xylosoxidans using Response Surface Methodology

Degradation of chrysene, a four ring High Molecular Weight (HMW) Polycyclic Aromatic Hydrocarbon (PAH) is of intense environmental interest, being carcinogenic, teratogenic and mutagenic. Multiple PAH degrading halotolerant Achromobacter xylosoxidans was isolated from crude oil polluted saline site. Response Surface Methodology (RSM) using Central Composite Design (CCD) of Bushnell-Haas medium components was successfully employed for optimization resulting 40.79% chrysene degradation on 4th day. The interactions between variables as chrysene and glucose concentrations, pH and inoculum size on degradation were examined by RSM. Under optimum conditions, A. xylosoxidans exhibited 85.96% chrysene degradation on 5th day. The optimum values predicted by RSM were confirmed through confirmatory experiments. It was also noted that pH and glucose as co-substrate play a dynamic role in enhancement of chrysene degradation. Hence, A. xylosoxidans can be further used for subsequent microcosm and in situ experiments for its potential to remediate PAH contaminated saline and non-saline soils.     

Use of maleic acid by mixed cultures of microorganisms

In a mixed batch culture, Alcaligenes xylosoxidans subsp. xylosoxidans 260 transformed maleic acid into malic acid. Bacillus subtilis 271 used malic acid as a substrate, thus stimulating further transformation of maleic acid. Both bacterial cultures dissociated with the formation of R, S, and M forms. At a concentration of 5.0 g/l, maleic acid was utilized maximally by RS and SS forms of the association A. xylosoxidans and Bacillus subtilis. At concentrations 15.0 and 25.0 g/l, maleic acid was utilized maximally by SS and MS forms of the mixed culture, respectively. Association of bacteria A. xylosoxidans and B. subtilis was not stable under flow conditions water.     

Glycerol as substrate for biomass and β-carotene production byRhodotorula lactosa

Glycerol was used as the sole carbon and energy source for growingRhodotorula lactosa. The maximum biomass yield (0.53 g/g substrate) was obtained after 20 h with 21.5 g glycerol/l; growth was inhibited with 28.0 g glycerol/l and cell morphology was changed. At this time, the cells were not pigmented. After 48 h of cultivation, -carotene was at 1.8 mg/g dry cells, yielding 22.0 mg/l. When cells were grown for 20 h, washed, suspended in distilled water and aerated for 24 hours, more -carotene (2.66 mg/g dry cells or 28.0 mg/l of the original culture) was produced. Cell protein content after 48 h was 36 to 38% (w/w) before extraction and 45 to 47% (w/w) for acetone-extracted cells.     

Alkanesulfonate degradation by novel strains of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and Rhodococcus sp., and evidence for an ethanesulfonate monooxygenase in A. xylosoxidans strain AE4

Novel isolates of Achromobacter xylosoxidans, Tsukamurella wratislaviensis and a Rhodococcus sp. are described. These grew with short-chain alkanesulfonates as their sole source of carbon and energy. T. wratislaviensis strain SB2 grew well with C(3)-C(6) linear alkanesulfonates, isethionate and taurine, Rhodococcus sp. strain CB1 used C(3)-C(10) linear alkanesulfonates, taurine and cysteate, but neither strain grew with ethanesulfonate. In contrast, A. xylosoxidans strain AE4 grew well with ethanesulfonate, making it the first bacterium to be described which can grow with this compound. It also grew with unsubstituted C(3)-C(5) alkanesulfonates and isethionate. Hydrolysis was excluded as a mechanism for alkanesulfonate metabolism in these strains; and evidence is given for a diversity of uptake and desulfonatase systems. We provide evidence for an initial monooxygenase-dependent desulfonation in the metabolism of ethanesulfonate and propanesulfonate by A. xylosoxidans strain AE4.     

Preparation of lactose free milk by fermentation using immobilizedSaccharomyces fragilis

Summary Saccharomyces fragilis cells (40% w/v) were immobilized in 2% Ca-alginate and were used in a batch process for the removal of lactose from milk by fermentation. Immobilized cells (10 g) could completely desugarate 100 mL of milk in 3.5 h. The immobilized preparation was used repeatedly in 15 batches without decrease in the activity.     

Ethanol production from fructose in continuous culture by free and flocculent cells of Zymomonas mobilis

Summary Zymomonas mobilis strain ZM4 was used for ethanol production from fructose (100 g/l) in continuous culture with a mineral (containing Ca pantothenate) or a rich (containing yeast extract) mediium. With both media high conversion yields were observed but the ethanol productivity was limited by the low biomass content of the fermentor. A new flocculent strain of Z.mobilis (ZM4F) was cultivated in a CSTR with an internal settler and showed a maximal productivity of 93 g/l.h (fructose conversion of 80%). When the fructose conversion was 96% an ethanol productivity of 85.6 g/l.h with an ethanol yield of 0.49 g/g (96% of theoretical) was observed.     

Effect of microbial competition on the survival and activity of 2,4-D-degrading Alcaligenes xylosoxidans subsp. denitrificans added to soil

In laboratory experiments samples of natural or chloroform-fumigated soils were inoculated with an Alcaligenes xylosoxidans subsp. denitrificans which is able to use 2,4-dichlorophenoxyacetic acid (2,4-D) as a sole carbon source. Biotic factors affecting survival and activity of the inoculant were determined. In natural soil the numbers and activity of Alc. xylosoxidans declines in few days. The strain proliferated only when it was inoculated immediately after soil fumigation. Its activity 15 d after inoculation was then twice its initial activity. When inoculation of fumigated samples was delayed, the numbers of Alc. xylosoxidans declined, but its activity was higher than in the natural soil. Addition of soil bacteria or fungi resulted in a reduction in the numbers and activity of Alc. xylosoxidans. These results suggest that microbial competition for nutrients and biological spaces causes the decline in the population and activity of inoculant added to soil.     

NreB from Achromobacter xylosoxidans 31A Is a nickel-induced transporter conferring nickel resistance

There are two distinct nickel resistance loci on plasmid pTOM9 from Achromobacter xylosoxidans 31A, ncc and nre. Expression of the nreB gene was specifically induced by nickel and conferred nickel resistance on both A. xylosoxidans 31A and Escherichia coli. E. coli cells expressing nreB showed reduced accumulation of Ni(2+), suggesting that NreB mediated nickel efflux. The histidine-rich C-terminal region of NreB was not essential but contributed to maximal Ni(2+) resistance.     

Decolorizing activity of malachite green and its mechanisms involved in dye biodegradation by Achromobacter xylosoxidans MG1

An Achromobacter xylosoxidans MG1 strainisolated from the effluent treatment plant of a textile and dyeing factory from Yunnan Province in China was found capable of decolorizing the malachite green dye at a high efficacy. Strain MG1 reduced 86% malachite green at the concentration of 2,000 mg/l within 1 h, representing a greater ability for decolorizing and a higher tolerance of this compound than all previously reported bacteria. Color removal was optimal at pH 6 and 38°C. Further experimental evidences demonstrated that both cytoplasmic and extracellular biodegradation contributed to the decolorization of malachite green. Nested PCR was employed to identify the candidate genes responsible for malachite green decolorization, and we identified a cytoplasmic triphenylmethane reductase gene with 100% amino acid similarity to the corresponding gene in Citrobacter sp. strain. In contrast to our expectation, the addition of metyrapone had little effect on the cytoplasmic biodegradation, suggesting that cytochrome P450 was not involved in the high-performance reduction. The extracellular biodegradation was likely attributable to the secretion of extracellular proteases and some heat-resistant compounds.     

Continuous neomycin production by immobilized cells of Streptomyces marinensis NUV-5 in an airlift bioreactor

Neomycin production by free and calcium alginate immobilized cells was investigated in an airlift reactor. The average volumetric productivity with continuous fermentation (72.97 mg/l/h) was greater than with free cells (45.05 mg/l/h). The total neomycin produced with continuous fermentation was 62% greater than with that of free cells. Immobilized Streptomyces particles showed a half-life of 42 days during continuous fermentation under airlift conditions.     

The treatment of gaseous benzene by two-phase partitioning bioreactors: a high performance alternative to the use of biofilters

A 2-l (1-l working volume) two-phase partitioning bioreactor (TPPB) was used as an integrated scrubber/bioreactor in which the removal and destruction of benzene from a gas stream was achieved by the reactor's organic/aqueous liquid contents. The organic solvent used to trap benzene was n-hexadecane, and degradation of benzene was achieved in the aqueous phase using the bacterium Alcaligenes xylosoxidans Y234. A gas stream with a benzene concentration of 340 mg l(-1) at a flow rate of 0.414 l h(-1) was delivered to the system at a loading capacity of 140 g m(-3) h(-1), and an elimination capacity of 133 g m(-3 )h(-1) was achieved (the volume in this term is the total liquid volume of the TPPB). This elimination capacity is between 3 and 13 times greater than any benzene elimination achieved by biofiltration, a competing biological air treatment strategy. It was also determined that the evaluation of TPPB performance in terms of elimination capacity should include the cell mass present in the system, as this is a readily controllable quantity. A specific benzene utilization rate of 0.57 g benzene (g cells)(-1) h(-1) was experimentally determined in a bioreactor with a cell concentration that varied dynamically between 0.2 and 1 g l(-1). If it assumed that this specific benzene utilization rate (0.57 g g(-1) h(-1)) is independent of cell concentration, then a TPPB operated at high cell concentrations could potentially achieve elimination capacities several hundred times greater than those obtained with biofilters.