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Applied Microbiology and Biotechnology (v.49, #2)

A covalent two-step immobilization technique using itaconic anhydride by L. Fischer; F. Peißker (pp. 129-135).

β-Glucosidase from almonds (EC 3.2.1.21) was covalently immobilized by a two-step technique. In the first step, double bonds were introduced into the β-glucosidase by derivatization with itaconic anhydride. In separate studies with α-N-protected l-amino acids, it was established that itaconic anhydride acylated mainly primary amino groups of lysines and, to a much lesser extent hydroxyl groups of tyrosines and sulfhydryl groups of cysteines. The acylated β-glucosidase showed no loss of activity and the K _m decreased from 3.6 mM to 2.6 mM when p-nitrophenyl β-d-glucopyranoside was used as the substrate. In the second step, the derivatized β-glucosidase was co-polymerized radically with N,N′-methylenebisacrylamide in buffer solution. The resulting acrylamide immobilizate possessed a much better storage stability at 30–56 °C when compared to β-glucosidase immobilized on Eupergit C. However, the specific activity was higher with the Eupergit immobilizate. Free and acrylamide-immobilized β-glucosidase were used for glucosylation of chloramphenicol by transglucosylation in 20% (v/v) acetonitrile at 37 °C. The acrylamide immobilizate demonstrated a great enhancement of stability and approximately 50% more chloramphenicol β-glucoside was obtained after 5 h.

A chromatographic and mass-spectrometric approach for the analysis of lipase-produced thioester derivatives by D. Cavaillé-Lefebvre; D. Combes; B. Rehbock; R. G. Berger (pp. 136-140).

The synthesis of thioethyl 2-methylpropanoate, butanoate, 3-methylbutanoate, hexanoate, and of thiobutyl propanoate, butanoate, and pentanoate was achieved via esterification of ethanethiol or butanethiol with short-chain acids using an immobilised lipase from Rhizomucor miehei. High acid conversions were obtained (47% for thiobutyl pentanoate). The analysis of educts and products was carried out by reverse-phase liquid chromatography (HPLC), gas chromatography (GC), GC/mass spectrometry, and GC/olfactometry. All of the thioesters imparted a similar, onion-like smell, which remained unchanged on dilution. The thresholds of thioesters derived from the same thiol were loosely correlated with the size of the acid moiety: the larger the molecular mass the higher the threshold. Surprisingly, higher odour thresholds were obtained for the branched-chain thioesters than for their linear analogues.

Cloning and disruption of the b-isopropylmalate dehydrogenase gene (LEU2 ) of Pichia stipitis with URA3 and recovery of the double auxotroph by P. Lu; B. P. Davis; J. Hendrick; T. W. Jeffries (pp. 141-146).

Transformation of Pichia stipitis is required to advance genetic studies and development of xylose metabolism in this yeast. To this end, we used P. stipitis URA3 (PsURA3) to disrupt P. stipitis LEU2 in a P. stipitis ura3 mutant. A highly fermentative P. stipitis mutant (FPL-DX26) was selected for resistance to 5′-fluoroorotic acid to obtain P. stipitis FPL-UC7 (ura3-3). A URA3:lacZ“pop-out” cassette was constructed containing PsURA3 flanked by direct repeats from segments of the lacZ reading frame. The P. stipitis LEU2 gene (PsLEU2) was cloned from a P. stipitis CBS 6054 genomic library through homology to Saccharomyces cerevisiae LEU2, and a disruption cassette was constructed by replacing the PsLEU2 reading sequence with the PsURA3:lacZ cassette. FPL-UC7 (ura3-3) was transformed with the disruption cassette, and a site-specific integrant was identified by selecting for the Leu⁻ Ura⁺ phenotype. The ura3 marker was recovered from this strain by plating cells onto 5′-fluoroorotate and screening for spontaneous URA3 deletion mutants. Excision of the flanked PsURA3 gene resulted in the Leu⁻Ura⁻ phenotype. The double auxotrophs are stable and can be transformed at a high frequency by PsLEU2 or PsURA3 carried on autonomous-replication-sequence-based plasmids.

An autoselection system in recombinant Kluyveromyces lactis enhances cloned gene stability and provides freedom in medium selection by H.-P. Hsieh; N. A. Da Silva (pp. 147-152).

An autoselection system for increasing plasmid stability in Kluyveromyces lactis, based on the blockage of the pyrimidine de novo and salvage pathways, was investigated. In a manner analogous to that used in Saccharomyces cerevisiae, a putative “fur1” mutation was selected in a uraA K. lactis strain using 5-fluorouracil and 5-fluorocytosine plates. Survival of the mutant required expression of a plasmid-borne URA3 gene regardless of the culture medium employed, verifying the efficacy of this autoselection system in K. lactis. The expression of heterologous invertase, encoded by the S. cerevisiae SUC2 gene, was studied during long-term sequential batch cultures (70 generations) in complex yeast/peptone/glucose medium. The fur1 mutant successfully retained the plasmid; invertase specific activity remained above 90% of the initial level. Furthermore, no mutation reversion was observed. In contrast, for the control non-fur1 strain, only 4% of the cells retained the plasmid after 70 generations, and invertase specific activity dropped to less than 10% of the initial level. Experiments comparing growth and activity in different media indicated the potential for improving productivity through medium enrichment using this autoselection system.

Chromosomal polymorphism and adaptation to specific industrial environments of Saccharomyces strains by A. C. Codón; T. Benítez; M. Korhola (pp. 154-163).

Several industrial Saccharomyces strains, including bakers', wine, brewers' and distillers' yeasts, have been characterized with regards to their DNA content, chromosomal polymorphism and homologies with the DNA of laboratory strains. Measurement of the DNA contents of cells suggested that most of the industrial yeasts were aneuploids. Polymorphisms in the electrophoretic chromosomal pattern were so large that each strain could be individually identified. However, no specific changes relating to a particular group were observed. Hybridization using different probes from laboratory strains was very strong in all cases, indicating that all industrial strains possess a high degree of DNA homology with laboratory yeasts. Probes URA3, CUP1, LEU2, TRP1, GAL4 or ADC1 demonstrated the presence of one or two bands, two especially in bakers' strains. Also, results indicate that all hybridized genes are located on the same chromosomes both in laboratory and industrial strains. Translocation from chromosome VIII to XVI seems to have occurred in a distillers' strain, judging by the location of the CUP1 probe. Finally, when the SUC2 probe is used, results indicate a very widespread presence of the SUC genes in only bakers' and molasses alcohol distillers' strains. This clearly suggests that amplification of SUC genes is an adaptive mechanism conferring better fitness upon the strains in their specific industrial conditions. The widespread presence of Ty1 and Ty2 elements as well as Y′ subtelomeric sequences could account for the inter- and intrachromosomal changes detected.

Increased toxicity of modified mosquitocidal binary toxins of Bacillus sphaericus expressed in Escherichia coli by S. Ahmad; A. Selvapandiyan; R. K. Bhatnagar (pp. 164-167).

The binary mosquitocidal genes of 51-kDa and 42-kDa proteins isolated from Bacillus sphaericus 1593 have been expressed at moderate levels in Escherichia coli employing the pQE expression system. The expressed proteins are readily visible in Coomassie-blue-stained protein gels. The recombinant E. coli cells expressing toxic proteins were toxic towards Culex larvae. During the assembly of crystals in B. sphaericus, the 42-kDa toxin is first cleaved at the N-terminal end by a specific B. sphaericus protease. To express the toxins in E. coli the B.sphaericus specific protease-recognition site was deleted at the N-terminal end of the 42-kDa toxin, thereby mimicking the structure of the toxin as present in the crystal. This modification resulted in a twofold increase in the toxicity of the E. coli cells expressing the modified 42-kDa toxin as a constituent of the binary toxin. Our results demonstrate the utility of this modification for heterologous expression of the binary toxin genes from B. sphaericus.

The production of exopolysaccharides by Aureobasidium pullulans in fermenters with low-shear configurations by P. A. Gibbs; R. J. Seviour (pp. 168-174).

No increases in exopolysaccharide (EPS) yields in Aureobasidium pullulans were observed when grown with reduced-shear impellers instead of standard Rushton turbines in the same vessel. However, yields were dramatically reduced when the organism was grown in an airlift reactor. This fall in production could be counteracted by improving fluid circulation through the placement of impellers within the draught tube, a strategy that resulted in the highest EPS concentration (approx. 13 g l⁻¹) of all the fermenter configurations tested.

Oat β-glucan and xylan hydrolysates as selective substrates for Bifidobacterium and Lactobacillus strains by J. Jaskari; P. Kontula; A. Siitonen; H. Jousimies-Somer; T. Mattila-Sandholm; K. Poutanen (pp. 175-181).

Novel oligomers that resist digestion in the upper gut were prepared from oat mixed-linked β-glucan and xylan by enzymatic hydrolysis with lichenase of Bacillus subtilis and xylanase of Trichoderma reesei respectively. The low-molecular-mass hydrolysis products of β-glucan and xylan were compared with fructooligomers and raffinose in their ability to provide growth substrates for probiotic (Lactobacillus and Bifidobacterium) and intestinal (Bacteroides, Clostridium and Escherichia coli) strains in vitro. A degradation profile of each carbohydrate and total sugar consumption were analysed with HPLC, and bacterial growth rate with an automatic turbidometer, the Bioscreen C system. β-Glucooligomers and xylooligomers both enhanced the growth of health-promoting probiotic strains as compared with intestinal bacterial growth, but not to a significant level. Raffinose stimulated the probiotic strains significantly, whereas fructooligomers induced high average growth for intestinal bacteria also.

Keywords: Key words Oat; β-Glucooligomers; Xylooligomers; Fructooligomers; Raffinose; Prebiotic

β-Glycosidase (amygdalase and linamarase) from Endomyces fibuliger (LU677): formation and crude enzyme properties by L. Brimer; M. J. R. Nout; G. Tuncel (pp. 182-188).

In our previous studies, the yeast Endomyces fibuliger LU677 was found to degrade amygdalin in bitter apricot seeds. The present investigation shows that E. fibuliger LU677 produces extracellular β-glycosidase activity when grown in malt extract broth (MEB). Growth was very good at 25 °C and 30 °C and slightly less at 35 °C. When grown in MEB of pH 5 and pH 6 with addition of 0, 10 or 100 ppm amygdalin, E. fibuliger produced only slightly more biomass at pH 5, and was only slightly inhibited in the presence of amygdalin. Approximately, 60% of the added amygdalin was degraded (fastest at 35 °C) during an incubation period of 5 days. Supernatants of cultures grown at 25 °C and pH 6 for 5 days were tested for the effects of pH and temperature on activity (using amygdalin, linamarin and prunasin as substrates). Prunase activity had two pH optima (pH 4 and pH 6), amygdalase and linamarase only one each at pH 6 and pH 4–5 respectively. The linamarase activity evolved earlier than amygdalase (2 days and 4 days respectively). The data thus indicate the presence of at least two different glycosidases having different pH optima and kinetics of excretion. In the presence of amygdalin, lower glycosidase activities were generally produced. However, the amygdalin was degraded from the start of the growth, strongly indicating an uptake of amygdalin by the cells. The temperature optimum for all activities was at 40 °C. Activities of amygdalase (assayed at pH 4) and linamarase (at pH 6) evolving during the growth of E. fibuliger were generally higher in cultures grown at 25 °C and 30 °C. TLC analysis of amygdalin degradation products show a two-stage sequential mechanism as follows: (1) amygdalin to prunasin and (2) prunasin to cyanohydrin.

Modelling the kinetics of growth of Acetobacter aceti in discontinuous culture: influence of the temperature of operation by I. de Ory; L. Enrique Romero; D. Cantero (pp. 189-193).

Acetic acid fermentation is the biochemical process by which, under strict conditions of aerobiosis, Acetobacter aceti oxidises the ethanol contained in alcoholic substrates into acetic acid. This paper studies the effect of temperature on the specific growth rate of the microorganisms (μ _C), in particular, the mathematical modelling of this process, with the aim of developing previous studies of the mathematical relationships between μ _C of A. aceti and the concentrations of substrate (ethanol), product (acetic acid) and dissolved oxygen. Until now this relationship has not been widely studied, and only a few studies have looked at the influence of temperature on growth kinetics of this bacteria. We have developed an extensive experimental system, to determine precisely the influence of temperature on the maximum specific growth rate.

Metabolism of EDTA and its metal chelates by whole cells and cell-free extracts of strain BNC1 by T. Klüner; D. C. Hempel; B. Nörtemann (pp. 194-201).

The influence of metal ions on the metabolism of ethylenediaminetetraacetate (EDTA) by whole cells and cell-free extracts of strain BNC1 was investigated. Metal-EDTA chelates with thermodynamic stability constants below 10¹² were readily mineralized by whole cells with maximum specific turnover rates of 15 (MnEDTA) to 20 (Ca-, Mg-, and BaEDTA) μmol g protein⁻¹ min⁻¹. With the exception of ZnEDTA, chelates with stability constants greater than 10¹² were not oxidized at a significant rate. However, it was shown for Fe(III)EDTA that even strong complexes can be degraded after pretreatment by addition of calcium and magnesium salts in the pH range 9–11. The range of EDTA chelates converted by cell-free extracts of strain BNC1 did not depend on their thermodynamic stabilities. The EDTA chelates of Ba²⁺, Co²⁺, Mg²⁺, Mn²⁺, and Zn²⁺ were oxidized whereas Ca-, Cd-, Cu-, Fe-, Pb-, and SnEDTA were not. The first catabolic enzyme appears to be an EDTA monooxygenase since it requires O₂, NADH, and FMN for its activity and yields glyoxylate and ethylenediaminetriacetate as products. The latter is further degraded via N,N′-ethylenediaminediacetate. The maximum specific turnover rate with MgEDTA, the favoured EDTA species, was 50–130 μmol g protein⁻¹ min⁻¹, and the K _m value was 120 μmol/l (K _s for whole cells = 8 μmol/l). Whole cells as well as cell-free extracts of strain BNC1 also converted several structural analogues of EDTA.

The effect of growth conditions on the biodegradation of tributyl phosphate and potential for the remediation of acid mine drainage waters by a naturally-occurring mixed microbial culture by R. A. P. Thomas; L. E. Macaskie (pp. 202-209).

The biodegradation of tributyl phosphate (Bu₃-P, TBP), releasing phosphate at a high enough concentration locally to precipitate uranium from solution, was demonstrated by a mixed culture consisting primarily of pseudomonads. The effect of various parameters on Bu₃-P biodegradation by growing cells is described. Growth at the expense of Bu₃-P as the carbon and phosphorus source occurred over a pH range from 6.5 to 8, and optimally at pH 7. Bu₃-P biodegradation was optimal at 30 °C, reduced at 20 °C and negligible at 4 °C and 37 °C. Incorporation of Cu or Cd inhibited, and Ni, Co and Mn reduced its degradation. Inorganic phosphate (above 10 mM) and kerosene (up to 1 g/l) reduced Bu₃-P biodegradation significantly, but nitrate had no effect. Sulphate (10–100 mM) was inhibitory. When pregrown biomass was used the fastest rates of tributyl and dibutyl phosphate biodegradation were 25 μmol h⁻¹ mg protein⁻¹ and 37 μmol h⁻¹ mg protein⁻¹ respectively. Microcarrier-immobilised biomass decontaminated uranium-bearing acid mine waste water by uranium phosphate precipitation at the expense of Bu₃-P hydrolysis in the presence of 35 mM SO₄ ²⁻. At pH 4.5, 79% of the UO₂ ²⁺ was removed at a flow rate of 1.4 ml/h on a 7-ml test column.

Potential for biodegradation of hydrocarbons in soil from the Ross Dependency, Antarctica by J. Aislabie; M. McLeod; R. Fraser (pp. 210-214).

Oil spills occur in the Antarctic when fuel oils such as JP8 jet fuel are moved or stored. Hydrocarbons, both n-alkanes and aromatic compounds, have been detected in oil-contaminated soils of the Ross Dependency. In such areas hydrocarbon-degrading microbes, if naturally occurring, could be used for clean-up. Soil samples from oil-impacted and control sites were analysed for hydrocarbon-degrading microbes and for a range of parameters known to limit biodegradative activity. Soils were analysed for water content, pH, concentrations of nutrients (N and P) and electrical conductivity. Numbers of culturable heterotrophic bacteria and hydrocarbon degraders were greater in some of the oil-contaminated samples. Mineralisation studies with ¹⁴C-radiolabelled hexadecane and naphthalene demonstrated that nitrogen amendments significantly enhanced hydrocarbon mineralisation rates in an oil-impacted soil.

Treatment of cyanide-containing wastewater from the food industry in a laboratory-scale fixed-bed methanogenic reactor by H. Siller; J. Winter (pp. 215-220).

During the process of producing cassava starch from Manihot esculenta roots, large amounts of cyanoglycosides were released, which rapidly decayed to CN⁻ following enzymatic hydrolysis. Depending on the varying cyanoglycoside content of the cassava varieties, the cyanide concentration in the wastewater was as high as 200 mg/l. To simulate anaerobic stabilization, a wastewater with a chemical oxygen demand (COD) of about 20 g/l was prepared from cassava roots and was fermented in a fixed-bed methanogenic reactor. The start-up phase for a 99% degradation of low concentrations of cyanide (10 mg/l) required about 6 months. After establishment of the biofilm, a cyanide concentration of up to 150 mg CN⁻/l in the fresh wastewater was degraded during anaerobic treatment at a hydraulic retention time of 3 days. All nitrogen from the degraded cyanide was converted to organic nitrogen by the biomass of the effluent. The cyanide-degrading biocoenosis of the anaerobic reactor could tolerate shock concentrations of cyanide up to 240 mg CN⁻/l for a short time. Up to 5 mmol/l NH₄Cl (i.e. 70 mg N/l = 265 mg NH₄Cl/l) in the fresh wastewater did not affect cyanide degradation. The bleaching agent sulphite, however, had a negative effect on COD and cyanide removal. For anaerobic treatment, the maximum COD space loading was 12 g l⁻¹ day⁻¹, equivalent to a hydraulic retention time of 1.8 days. The COD removal efficiency was around 90%. The maximum permanent cyanide space loading was 50 mg CN⁻ l⁻¹ day⁻¹, with tolerable shock loadings up to 75 mg CN⁻ l⁻¹day⁻¹. Under steady-state conditions, the cyanide concentration of the effluent was lower than 0.5 mg/l.

Use of random amplified polymorphic DNA markers for the detection of Azospirillum strains in soil microcosms by S. Fancelli; M. Castaldini; M. T. Ceccherini; C. Di Serio; R. Fani; E. Gallori; M. Marangolo; N. Miclaus; M. Bazzicalupo (pp. 221-225).

Probes for the detection of Azospirillum strains were obtained from DNA fragments generated by random amplification of polymorphic DNA (RAPD) and tested to assess their specificity towards DNA extracted from pure cultures. The most specific probe, referred to as α4, produced a hybridization signal only with amplified DNA of A. lipoferum ATCC29731. This strain was inoculated, together with two other Azospirillum strains, in soil microcosms of different complexity and its presence tested with the probe α4. This probe confirmed its high specificity with amplified DNA extracted from the soil microcosm and in the presence of other A. lipoferum strains, indicating that the strategy for bacterial detection, based on RAPD markers, is useful for monitoring the presence of a particular strain under environment-like conditions. Other RAPD-derived probes, when tested on soil samples, did not show the same level of specificity as that shown on DNA from pure cultures. This result suggests that some precautions are necessary in the choice of a really specific RAPD marker. In a further development of this strategy, the α4 probe was sequenced and two pairs of “nested” primers were designed, which enabled a diagnostic polymerase chain reaction from soil samples that was specific for the A. lipoferum species.

Metabolic transformations and characterisation of the sludge community in an enhanced biological phosphorus removal system by M. Christensson; L. L. Blackall; T. Welander (pp. 226-234).

Enhanced biological phosphorus removal was performed in a continuous laboratory-scale two-reactor system with sludge recirculation over a 75-day period. Influent wastewater was a synthetic medium based on acetate, and the sludge age was kept at 12 days. The adapted sludge stored poly-β-hydroxyalkanoic acids (PHA) in the anaerobic reactor with a conversion ratio of 1.45 PHA/acetic acid (based on chemical O₂ demand: COD/COD) and gave ratio of a phosphate-P release to acetic acid uptake of 0.51 P/CH₃COOH (w/w). Fractionation of anaerobic and aerobic sludges showed that the main part of phosphorus taken up, was eluted in the trichloroacetic acid fraction indicating that it was polyphosphate. A total of 60% of the phosphorus in the aerobic sludge was solubilized in the trichloroacetic acid fraction, whereas this fraction accounted for only 32% of the phosphorus in the anaerobic sludge. Only 4% of the total phosphorus in the aerobic sludge and 2% in the anaerobic sludge was found in the EDTA fraction, indicating low amounts of metal-bound phosphates. Isolation on acetate-based agar medium showed that Acinetobacter strains were present in the sludge. However, a more complete analysis of the bacterial community of the sludge was obtained by creating a clone library based on the 16S rRNA gene. A total of 51 partial clone sequences were phylogenetically evaluated. The predominating group was found in the high-(G+C) (mol%) gram-positive bacterial subphylum (31% of the sequenced clones), while the gamma proteobacteria only constituted 9.8% of the clones.

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Applied Microbiology and Biotechnology (v.49, #2)