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Applied Microbiology and Biotechnology (v.71, #4)

The role of PHB metabolism in the symbiosis of rhizobia with legumes by Maria A. Trainer; Trevor C. Charles (pp. 377-386).

The carbon storage polymer poly-β-hydroxybutyrate (PHB) is a potential biodegradable alternative to plastics, which plays a key role in the cellular metabolism of many bacterial species. Most species of rhizobia synthesize PHB but not all species accumulate it during symbiosis with legumes; the reason for this remains unclear, although it was recently shown that a metabolic mutant of a nonaccumulating species retains the capacity to store PHB in symbiosis. Although the precise roles of PHB metabolism in these bacteria during infection, nodulation, and nitrogen fixation are not determined, the elucidation of these roles will influence our understanding of the metabolic nature of the symbiotic relationship. This review explores the progress that was made in determining the biochemistry and genetics of PHB metabolism. This includes the elucidation of the PHB cycle, variations in PHB metabolism among rhizobial species, and the implications of these variations, while proposing a model for the role of PHB metabolism and storage in symbiosis.

Biohybrid nanosystems with polymer nanofibers and nanotubes by A. Greiner; J. H. Wendorff; A. L. Yarin; E. Zussman (pp. 387-393).

Advanced techniques for the preparation of nanofibers, core shell fibers, hollow fibers, and rods and tubes from natural and synthetic polymers with diameters down to a few nanometers have recently been established. These techniques, among them electro- and co-electrospinning and specific template methods, allow the incorporation not only of semiconductor or catalytic nanoparticles or chromophores but also enzymes, proteins, microorganism, etc., directly during the preparation process into these nanostructures in a very gentle way. One particular advantage is that biological objects such as, for instance, proteins can be immobilized in a fluid environment within these polymer-based nano-objects in such a way that they keep their native conformation and the corresponding functions. The range of applications of such biohybrid nanosystems is extremely broad, for instance, in the areas of biosensorics, catalysis, drug delivery, or optoelectronics.

Proteolytic systems of lactic acid bacteria by Kirsi Savijoki; Hanne Ingmer; Pekka Varmanen (pp. 394-406).

Lactic acid bacteria (LAB) have a very long history of use in the manufacturing processes of fermented foods and a great deal of effort was made to investigate and manipulate the role of LAB in these processes. Today, the diverse group of LAB includes species that are among the best-studied microorganisms and proteolysis is one of the particular physiological traits of LAB of which detailed knowledge was obtained. The proteolytic system involved in casein utilization provides cells with essential amino acids during growth in milk and is also of industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products. For the most extensively studied LAB, Lactococcus lactis, a model for casein proteolysis, transport, peptidolysis, and regulation thereof is now established. In addition to nutrient processing, cellular proteolysis plays a critical role in polypeptide quality control and in many regulatory circuits by keeping basal levels of regulatory proteins low and removing them when they are no longer needed. As part of the industrial processes, LAB are challenged by various stress conditions that are likely to affect metabolic activities, including proteolysis. While environmental stress responses of LAB have received increasing interest in recent years, our current knowledge on stress-related proteolysis in LAB is almost exclusively based on studies on L. lactis. This review provides the current status in the research of proteolytic systems of LAB with industrial relevance.

Effect of additives on gas-phase catalysis with immobilised Thermoanaerobacter species alcohol dehydrogenase (ADH T) by A. H. Trivedi; A. C. Spiess; T. Daussmann; J. Büchs (pp. 407-414).

This paper presents a strategy for preparing an efficient immobilised alcohol dehydrogenase preparation for a gas-phase reaction. The effects of additives such as buffers and sucrose on the immobilisation efficiency (residual activity and protein loading) and on the gas-phase reaction efficiency (initial reaction rate and half-life) of Thermoanaerobacter sp. alcohol dehydrogenase were studied. The reduction of acetophenone to 1-phenylethanol under in situ cofactor regeneration using isopropanol as co-substrate was used as a model reaction at fixed reaction conditions (temperature and thermodynamic activities). A strongly enhanced thermostability of the enzyme in the gas-phase reaction was achieved when the enzyme was immobilised with 50 mM phosphate buffer (pH 7) containing sucrose five times the protein amount (on weight/weight basis). This resulted in a remarkable productivity of 200 g L⁻¹ day⁻¹ even at non-optimised reaction conditions. The interaction of additives with the enzyme and water affects the immobilisation and gas-phase efficiencies of the enzyme. However, it was not possible to predict the effect of additives on the gas-phase reaction efficiency even after knowing their effect on the immobilisation efficiency.

Increase in hyaluronic acid production by Streptococcus equi subsp. zooepidemicus strain deficient in β-glucuronidase in laboratory conditions by Ján Krahulec; Jana Krahulcová (pp. 415-422).

Streptococcus equi subsp. zooepidemicus is known to produce a hyaluronic acid capsule to resist the host immune defense. As the structure of the polysaccharide is identical to the one produced by humans, the bacteria S. equisubsp. zooepidemicusis used in biotechnological production of hyaluronic acid. In our laboratory we prepared mutated strains that are β-glucuronidase deficient. Comparing the wild-type strain, which is positive in β-glucuronidase activity, with the mutated strains named clone1 and clone2 in laboratory conditions, we observed that β-glucuronidase influences the production of hyaluronic acid considerably and the molecular weight of hyaluronan slightly. The production of hyaluronic acid by the mutated strains is higher by approximately 20% and the molecular weight is larger by about 2%. The significant increase in the production of hyaluronic acid and the slight increase in the molecular weight are probably caused by an absence of free β-glucuronic acid, due to its removal from the non-reducing termini of the polysaccharide by β-glucuronidase. The presence of free β-glucuronic acid would likely induce the expression of the β-glucuronic-acid-utilizing operon, which in turn would reflect into a misuse of energy in the glucose-rich media.

Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440 by Zhiyong Sun; Juliana A. Ramsay; Martin Guay; Bruce A. Ramsay (pp. 423-431).

Four automatic substrate feeding strategies were developed and investigated in this study to obtain rapid, repeatable, and reliable high cell densities of Pseudomonas putida KT2440 from glucose. Growth yield data of the key nutrients, Y _X/Glucose, Y _X/NH4, Y _X/PO4, Y _X/Mg, and Y _CO2/Glucose, were determined to be 0.41, 5.44, 13.70, 236, and 0.65 g g⁻¹, respectively. Although standard exponential feeding strategy worked well when the predetermined μ was set at 0.25 h⁻¹, an exponential glucose feeding strategy with online μ _max estimation resulted in a higher average biomass productivity (3.4 vs 2.8 g l⁻¹ h⁻¹). A CO₂ production rate based pulse glucose feeding strategy also resulted in good overall productivity (3.0 g l⁻¹ h⁻¹) and can be used as an alternative to pH-stat or DO-stat feeding. A cumulative CO₂ production based continuous feed with real-time cumulative glucose consumption estimation resulted in much higher biomass productivity (4.3 g l⁻¹ h⁻¹) and appears to be an excellent and reliable approach to fully automating high-cell-density fed-batch cultivation of P. putida.

Bioconversion of agrowastes by Lentinula edodes: the high potential of viticulture residues by Rigoberto Gaitán-Hernández; Martín Esqueda; Aldo Gutiérrez; Alfonso Sánchez; Miguel Beltrán-García; Gerardo Mata (pp. 432-439).

The production of four strains of edible mushroom Lentinula edodes was evaluated through solid-state fermentation (SSF) of vineyard pruning (VP), barley straw (BS), and wheat straw (WS). Biological efficiency, proximal composition, and energy value of the fruiting bodies, as well as substrate chemical changes after harvest, were determined. The shortest primordium formation time (28 days), highest biological efficiency (93.25%), highest yield (37.46%), and shortest production cycle (6 days) were observed in VP. The fruiting bodies obtained from VP had high energy value (379.09 to 392.95 kcal) and contents of protein (12.37 to 17.19%), but low contents of fat (1.82 to 2.15%). After SSF, phenol concentration decreased on VP (1.2 mmol/L) and BS (0.31 mmol/L), but on WS remained practically the same. Hemicellulose decreased in all substrates; cellulose increased on WS and decreased in the rest of the treatments. Lignin decreased on WS and BS, but its concentration increased on VP. The variability observed in the degradation capacity of lignocellulosic components was influenced by the substrate's nature, environmental factors, and genetic factors among strains. VP has great potential for shiitake production due to its low cost, short production cycles, and high biological efficiency.

An aqueous–organic two-phase bioprocess for efficient production of the natural aroma chemicals 2-phenylethanol and 2-phenylethylacetate with yeast by M. M. W. Etschmann; J. Schrader (pp. 440-443).

The natural aroma chemicals 2-phenylethanol (2-PE) and 2-phenylethylacetate (2-PEAc) are of high industrial relevance and can be produced from l-phenylalanine in a yeast-based process with growth-associated product formation. Due to product inhibition, in situ product removal is mandatory to obtain economically interesting concentrations. A fed-batch approach using polypropylene glycol 1200 as in situ extractant and the precursor in a saturated concentration led to the highest 2-PE productivity reported for a bioprocess so far. With Kluyveromyces marxianus CBS 600, 26.5 g/l 2-PE and 6.1 g/l 2-PEAc in the organic phase were obtained, corresponding to space–time yields of 0.33 and 0.08 g/l h, respectively.

A homologue of cathepsin L identified in conditioned medium from Sf9 insect cells by Eva Lindskog; Ingrid Svensson; Lena Häggström (pp. 444-449).

Gelatin zymography revealed the presence of proteolytic activity in conditioned medium (CM) from a serum-free, non-infected Spodoptera frugiperda, Sf9 insect cell culture. Two peptidase bands at about 49 and 39 kDa were detected and found to be proform and active form of the same enzyme. The 49-kDa form was visible on zymogram gels in samples of CM taken on days 4 and 5 of an Sf9 culture, while the 39-kDa form was seen on days 6 and 7. On basis of the inhibitor profile and substrate range, the enzyme was identified as an Sf9 homologue of cathepsin L, a papain-like cysteine peptidase. After lowering the pH of Sf9 CM to 3.5, an additional peptidase band at 22 kDa appeared. This peptidase showed the same inhibitor profile, substrate range and optimum pH (5.0) as the 39-kDa form, indicating that Sf9 cathepsin L has two active forms, at 39 and 22 kDa. Addition of the cysteine peptidase inhibitor E-64c to an Sf9 culture inhibited all proteolytic activities of Sf9 cathepsin L but did not influence the proliferation of Sf9 cells.

Stable transformation of Chromobacterium violaceum with a broad-host-range plasmid by L. Broetto; R. Cecagno; F. H. Sant'Anna; S. Weber; I. S. Schrank (pp. 450-454).

Stable transformants of Chromobacterium violaceum were obtained by high-voltage electroporation with a 7-kilobase binary plasmid. The technique was reliable, reproducible, and simple, with efficiencies of 10⁵ transformants/μg of plasmid DNA. The electrical conditions that resulted in the highest efficiencies were short pulse length (4.4–4.5 ms) and high voltage (12.5 kV/cm). The numbers of transformants were almost the same during the growth exponential phase (variation at optical density) and resulted in the highest efficiencies at DNA concentration of 250 pg/ml. Saturation appeared to begin at 4 μg/ml of DNA. This method of C. violaceum transformation should enhance the genetic and biotechnological research by providing a valuable, widely used procedure of introducing DNA into this bacterium.

Functional expression system for cytochrome P450 genes using the reductase domain of self-sufficient P450RhF from Rhodococcus sp. NCIMB 9784 by Miho Nodate; Mitsutoshi Kubota; Norihiko Misawa (pp. 455-462).

Cytochrome P450RhF from Rhodococcus sp. NCIMB 9784 is a self-sufficient P450 monooxygenase. We report here a simple system for the functional expression of various P450 genes using the reductase domain of this P450RhF, which comprises flavin mononucleotide- and nicotinamide adenine dinucleotide phosphate binding motifs and a [2Fe2S] ferredoxin-like center. Vector pRED was constructed, which carried the T7 promoter, cloning sites for a P450, a linker sequence, and the P450RhF reductase domain, in this order. The known P450 genes, encoding P450cam from Pseudomonas putida (CYP101A) and P450bzo from an environmental metagenome library (CYP203A), were expressed on vector pRED as soluble fusion enzymes with their natural spectral features in Escherichia coli. These E. coli cells expressing the P450cam and P450bzo genes could convert (+)-camphor and 4-hydroxybenzoate into 5-exo-hydroxycamphor and protocatechuate (3,4-dihydroxybenzoate), respectively (the expected products). Using this system, we also succeeded in directly identifying the function of P450 CYP153A as alkane 1-monooxygenase for the first time, i.e., E. coli cells expressing a P450 CYP153A gene named P450balk, which was isolated form Alcanivorax borkumensis SK2, converted octane into 1-octanol with high efficiency (800 mg/l). The system presented here may be applicable to the functional identification of a wide variety of bacterial cytochromes P450.

Production of a lipolytic enzyme originating from Bacillus halodurans LBB2 in the methylotrophic yeast Pichia pastoris by Santosh O. Ramchuran; Virginia A. Vargas; Rajni Hatti-Kaul; Eva Nordberg Karlsson (pp. 463-472).

A gene encoding a lipolytic enzyme amplified from the alkaliphilic bacterium Bacillus halodurans LBB2 was cloned into the pPICZαB vector and integrated into the genome of the protease deficient yeast strain Pichia pastoris SMD1168H. This previously undescribed enzyme was produced in active form, and cloning in frame with the Saccharomyces cerevisiae secretion signal (α-factor) enabled extracellular accumulation of correctly processed enzyme, with an apparent molecular mass of 30 kDa. In shake-flask cultivations, very low production levels were obtained, but these were significantly improved by use of a “batch-induced” cultivation technique which allowed a maximum enzyme activity of 14,000 U/l using p-nitrophenyl butyrate (C-4) as a substrate and a final extracellular lipolytic enzyme concentration of approximately 0.2 g/l. Partial characterization of the produced enzyme (at pH 9) revealed a preference for the short-chain ester (C-4) and significant but lower activity towards medium (C5-C6) and long (C16 and C18) fatty acid chain-length esters. In addition, the enzyme exhibited true lipase activity (7,300 U/l) using olive oil as substrate and significant levels of phospholipase activity (6,400 U/l) by use of a phosphatidylcholine substrate, but no lysophospholipase activity was detected using a lysophosphatidylcholine substrate.

Isolation and characterization of a carotenoid oxygenase gene from Chlorella zofingiensis (Chlorophyta) by Jun-Chao Huang; Yan Wang; Gerhard Sandmann; Feng Chen (pp. 473-479).

The green alga Chlorella zofingiensis produces large amounts of the valuable ketocarotenoid astaxanthin under dark, heterotrophic growth conditions, making it potentially employable for commercial production of astaxanthin as feed additives, colorants, and health products. Here, we report the identification and characterization of a β-carotene oxygenase (CRTO) gene that is directly involved in the biosynthesis of ketocarotenoids in C. zofingiensis. The open reading frame of the crtO gene, which is interrupted by three introns of 243, 318, and 351 bp, respectively, encodes a polypeptide of 312 amino acid residues. Only one crtO gene was detected in the genome of C. zofingiensis. Furthermore, the expression of the crtO gene was transiently up-regulated upon glucose treatment. Functional complementation in Escherichia coli showed that the coding protein of the crtO gene not only exhibits normal CRTO activity by converting β-carotene to canthaxanthin via echinenone, but also displays a high enzymatic activity of converting zeaxanthin to astaxanthin via adonixanthin. Based on the bifunctional CRTO, a predicted pathway for astaxanthin biosynthesis in C. zofingiensis is described, and the CRTO is termed as carotenoid 4,4′-β-ionone ring oxygenase.

Sequencing and expression analysis of the sakacin P bacteriocin produced by a Lactobacillus sakei strain isolated from naturally fermented sausages by Rosalinda Urso; Kalliopi Rantsiou; Carlo Cantoni; Giuseppe Comi; Luca Cocolin (pp. 480-485).

A Lactobacillus sakei strain, designated as I151 and isolated from naturally fermented sausages, was found to produce the sakacin P bacteriocin which is active against Listeria monocytogenes. In this study, we performed the sequencing of the gene cluster involved in the production of the sakacin P, and we followed the expression of the sppA gene, encoding for the bacteriocin, in vitro, using Rogosa–Sharpe medium, and in situ, inoculating the strain in fermented sausages as starter culture. The results obtained underlined the high similarity (>99%) of the entire sakacin P gene cluster from the L. sakei studied here with others present in strains of L. sakei already described. Moreover, from the expression experiments, it was shown that the gene is expressed during the exponential phase and that production procedures typical of fermented sausages are not turning off the expression of the gene encoding the bacteriocin. The capability of the strain studied to produce sakacin P during production is considered an advantage for its use as starter culture to improve the safety aspect of traditional fermented sausages produced in Italy.

Expression of the urease gene of Agaricus bisporus: a tool for studying fruit body formation and post-harvest development by Matthijs J. M. Wagemaker; Daniel C. Eastwood; Chris van der Drift; Mike S. M. Jetten; Kerry Burton; Leo J. L. D. Van Griensven; Huub J. M. Op den Camp (pp. 486-492).

Fruit body initials of Agaricus bisporus contain high levels of urea, which decrease in the following developmental stages until stage 4 (harvest) when urea levels increase again. At storage, the high urea content may affect the quality of the mushroom, i.e. by the formation of ammonia from urea through the action of urease (EC 3.5.1.5). Despite the abundance of urea in the edible mushroom A. bisporus, little is known about its physiological role. The urease gene of A. bisporus and its promoter region were identified and cloned. The coding part of the genomic DNA was interrupted by nine introns as confirmed by cDNA analysis. The first full homobasidiomycete urease protein sequence obtained comprised 838 amino acids (molecular mass 90,694 Da, pI 5.8). An alignment with fungal, plant and bacterial ureases revealed a high conservation. The expression of the urease gene, measured by Northern analyses, was studied both during normal development of fruit bodies and during post-harvest senescence. Expression in normal development was significantly up-regulated in developmental stages 5 and 6. During post-harvest senescence, the expression of urease was mainly observed in the stipe tissue; expression decreased on the first day and remained at a basal level through the remaining sampling period.

Cloning of novel laccase isozyme genes from Trametes sp. AH28-2 and analyses of their differential expression by Y. Z. Xiao; Y. Z. Hong; J. F. Li; J. Hang; P. G. Tong; W. Fang; C. Z. Zhou (pp. 493-501).

Three novel laccase isozyme genes, lacA, lacB, and lacC, have been identified from basidiomycete Trametes sp. AH28-2. These genes display a high similarity with other basidiomycete laccases at the amino acid level. An inferred TATA box and several putative CAAT, MRE, XRE, and CreA consensus sequences were identified in the lacA, lacB, and lacC promoter regions. Different from the TATA boxes of lacA and lacB at about −100, the TATA box of lacC is located at −172. For all the isozymes, copper ion is essential for laccase synthesis in Trametes sp. AH28-2. More interestingly, different aromatic compounds can selectively induce the production of distinct laccase isozymes, with o-toluidine inducing the expression of laccase A (LacA) while 3,5-dihydroxytoluene mainly stimulating the production of laccase B (LacB). Quantitative reverse transcriptase–polymerase chain reaction showed that the accumulation of laccase messenger RNA transcripts is accompanied by the increase of corresponding enzyme activity in cultures. The glucose-repression effect on laccase expression in Trametes sp. AH28-2 was also observed. Furthermore, lower Cu²⁺ concentration (lower than 0.5 mM) can induce LacA and a novel laccase (LacC), and the latter will disappear when Cu²⁺ concentration is increased up to 1−2 mM. Upon induction by 3,5-dihydroxytoluene, the ratio of LacA to LacB decreased in the later phase of induction.

Regioselective hydroxylation of adamantane by Streptomyces griseoplanus cells by Koichi Mitsukura; Yoshinori Kondo; Toyokazu Yoshida; Toru Nagasawa (pp. 502-504).

Hydroxylation of adamantane using whole cells of bacteria, actinomyces, and molds was examined. The structure of the product was determined using gas chromatography (GC), nuclear magnetic resonance (NMR), and mass spectroscopy (MS). Among 470 strains tested, Streptomyces griseoplanus was highly regioselective to give 1-adamantanol (0.096 mmol) from adamantane (0.3 mmol) in a 32% molar conversion yield after 72-h cultivation in the presence of 3% (v/v) Tween 60. A P450 inhibitor such as 0.5 mM 1-aminobenzotriazole or menadione significantly inhibited the hydroxylation activity. These results suggested that a P450 oxidation system might be involved in this hydroxylation reaction.

Symbiotic abilities of Sinorhizobium fredii with modified expression of purL by Bo Xie; Da-Song Chen; Kui Zhou; Yu-Qun Xie; You-Guo Li; Guo-Yuan Hu; Jun-Chu Zhou (pp. 505-514).

Previous reports showed that a transposon-induced PurL⁻ mutant of Sinorhizobium fredii induced pseudonodules on Glycine max and the addition of 5-aminoimidazole-4-carboxamide-riboside or adenine to the plant could not restore the mutant to establish effective symbiosis. To gain a better understanding of the impact of the purL gene on symbiosis formation, we measured the effect of modified expression of this gene on the symbiotic abilities of S. fredii on soybean (G. max). A 1.98-kb in-frame deletion mutant in the purL gene of S. fredii was constructed. Transcriptional modification of the purL gene was conducted using several promoters such as those of lac, nifH, nifQ, and fixN. It was found that reduced expression of purL gene or suitable symbiotic expression of purL (such as with the promoter nifH or nifQ) can efficiently establish symbiosis of S. fredii on G. max without the exogenous supplementation of any adenine or purine precursor; at least a minimal level of expression of purL is essential for effective symbiosis with soybean.

Presence of glucosylceramide in yeast and its relation to alkali tolerance of yeast by Katsuichi Saito; Naoya Takakuwa; Masao Ohnishi; Yuji Oda (pp. 515-521).

Glycosylceramide is a membrane lipid that has physiological functions in eukaryotic organisms. The presence of glucosylceramide has been confirmed in some yeast; however, the extent of the role of glucosylceramide in yeast is unknown. Thus, the extent of presence of glucosylceramide in yeast was surveyed using 90 strains of 24 genera. The strains were divided into two groups according to whether they had glucosylceramide (45 strains) or not (45 strains). The distribution of the ceramide glucosyltransferase gene (EC 2.4.1.80), which catalyzes glucosylation to a sphingoid lipid in glucosylceramide synthesis, and the phylogenetic classification of the strains were in agreement with those of glucosylceramide. Thus, the presence of glucosylceramide in yeast was caused by the presence of the gene involved in glucosylceramide synthesis and was closely associated with yeast evolution. Furthermore, the relationship between glucosylceramide presence and alkali tolerance of yeast was evaluated. The yeast with glucosylceramide tended to grow at higher pH, and a ceramide-glucosyltransferase-defective mutant from Kluyveromyces lactis did not grow at pH 8.5 even though the parent strain could grow under the same conditions. These results indicate that glucosylceramide in yeast might be a component that enables yeast to grow under alkali conditions.

Molecular characterization of cytochrome P450 genes in the polycyclic aromatic hydrocarbon degrading Mycobacterium vanbaalenii PYR-1 by Barbara Brezna; Ohgew Kweon; Robin L. Stingley; James P. Freeman; Ashraf A. Khan; Bystrik Polek; Richard C. Jones; Carl E. Cerniglia (pp. 522-532).

Mycobacterium vanbaalenii PYR-1 has the ability to degrade low- and high-molecular-weight polycyclic aromatic hydrocarbons (PAHs). In addition to dioxygenases, cytochrome P450 monooxygenases have been implicated in PAH degradation. Three cytochrome P450 genes, cyp151 (pipA), cyp150, and cyp51, were detected and amplified by polymerase chain reaction from M. vanbaalenii PYR-1. The complete sequence of these genes was determined. The translated putative proteins were ≥80% identical to other GenBank-listed mycobacterial CYP151, CYP150, and CYP51. Genes pipA and cyp150 were cloned, and the proteins partially expressed in Escherchia coli as soluble heme-containing cytochrome P450s that exhibited a characteristic peak at 450 nm in reduced carbon monoxide difference spectra. Monooxygenation metabolites of pyrene, dibenzothiophene, and 7-methylbenz[α]anthracene were detected in whole cell biotransformations, with E. coli expressing pipA or cyp150 when analyzed by gas chromatography/mass spectrometry. The cytochrome P450 inhibitor metyrapone strongly inhibited the S-oxidation of dibenzothiophene. Thirteen other Mycobacterium strains were screened for the presence of pipA, cyp150, and cyp51 genes, as well as the initial PAH dioxygenase (nidA and nidB). The results indicated that many of the Mycobacterium spp. surveyed contain both monooxygenases and dioxygenases to degrade PAHs. Our results provide further evidence for the diverse enzymatic capability of Mycobacterium spp. to metabolize polycylic aromatic hydrocarbons.

The effect of lactic acid on anaerobic carbon or nitrogen limited chemostat cultures of Saccharomyces cerevisiae by Elisabeth Thomsson; Christer Larsson (pp. 533-542).

Weak organic acids are well-known metabolic effectors in yeast and other micro-organisms. High concentrations of lactic acid due to infection of lactic acid bacteria often occurs in combination with growth under nutrient-limiting conditions in industrial yeast fermentations. The effects of lactic acid on growth and product formation of Saccharomyces cerevisiae were studied, with cells growing under carbon- or nitrogen-limiting conditions in anaerobic chemostat cultures (D=0.1 h⁻¹) at pH values 3.25 and 5. It was shown that lactic acid in industrially relevant concentrations had a rather limited effect on the metabolism of S. cerevisiae. However, there was an effect on the energetic status of the cells, i.e. lactic acid addition provoked a reduction in the adenosine triphosphate (ATP) content of the cells. The decrease in ATP was not accompanied by a significant increase in the adenosine monophosphate levels.

Investigation on bioremediation of oil-polluted wetland at Liaodong Bay in northeast China by S. H. Ye; L. C. Huang; Y. O. Li; M. Ding; Y. Y. Hu; D. W. Ding (pp. 543-548).

An investigation on the effect of various microbes on degradation was carried out as part of the study on bioremediation of oil-polluted wetland at LiaoDong Bay in northeast China. The method used involved direct inoculation of selected bacteria, which were capable of degrading oil, to the soil samples. The combination of various bacteria showed better results in terms of oil degradation than any single ones due to their synergetic effects. The operation conditions [pH 8.0, 25°C, C/N/P (40:5.6:1)] for these bacteria to degrade the oil content in the soil samples were also studied and optimized. Addition of appropriate surfactants was helpful for bacteria growth, thus favoring the oil degradation. For instance, after adding Tween 80 (300 mg/kg) for 8 days, the number of bacteria was amplified 6.22 times and the rate of oil degradation increased by 20%. Adequate amount of H₂O₂ was also beneficial for microbes to decompose oil. However, overdosage may cause the death of the bacteria. The addition of 400 mg/l H₂O₂ each time was suitable. Seven thousand milligrams of H₂O₂ was added entirely in 11 days, and the rate of oil degradation increased significantly from 27% (without H₂O₂) up to 67%. The study clearly demonstrated that the direct soil inoculation was an effective method for environmental bioremediation.

Degradation of toxaphene by Bjerkandera sp. strain BOL13 using waste biomass as a cosubstrate by Martha Lacayo Romero; Enrique Terrazas; Bert van Bavel; Bo Mattiasson (pp. 549-554).

The white-rot fungus Bjerkandera sp. strain BOL13 was capable of degrading toxaphene when supplied with wood chips, wheat husk or cane molasses as cosubstrates in batch culture experiments. Approximately 85% of toxaphene was removed when wheat husk was the main substrate. The production of lignin peroxidase was only stimulated when wheat husk was present in the liquid medium. Although xylanase was always detected, wheat husk supported the highest xylanase production. A negligible amount of β-glucosidase and cellulase were found in the batch culture medium. To the best of our knowledge, this is the first reported case of toxaphene degradation by white-rot fungi.

Long-term performance and microbial dynamics of an up-flow fixed bed reactor established for the biodegradation of fluorobenzene by M. F. Carvalho; R. Ferreira Jorge; C. C. Pacheco; P. De Marco; I. S. Henriques; A. Correia; P. M. L. Castro (pp. 555-562).

An up-flow fixed bed reactor (UFBR) was established to investigate the biodegradation of fluorobenzene (FB) under a number of operating conditions, which included variation in the concentration of FB in the feed stream (up to 180 mg l⁻¹) and temporary suspension of feeding. Degradation of FB was followed for a period of 8 months under a continuous flow regime. During the operation of the UFBR, FB was never detected in the reactor effluent, being biodegraded by the microbial biofilm or adsorbed to the granular activated carbon (GAC). Biodegradation of FB was observed from the beginning of the reactor operation, and overall, it accounted for 50% of the total amount fed to the bioreactor. High organic loads of FB (210–260 mg d⁻¹ dm⁻³) were found to affect the biological removal efficiency, possibly due to an inhibitory effect caused by the higher FB concentrations fed to the bioreactor (149–179 mg l⁻¹). When FB feeding was suspended for 1 month, biodegradation continued, indicating that the adsorbed FB became bioavailable. Biofilm bacterial dynamics were followed throughout the UFBR operation by denaturing gradient gel electrophoresis and plate-counting techniques, showing that a quite stable community was found in the bioreactor, and this was mainly attributed to the high selective pressure exerted by the presence of FB.

Growth and denitrifying activity of Xanthobacter autotrophicus CECT 7064 in the presence of selected pesticides by Florentina Sáez; Clementina Pozo; Miguel Angel Gómez; Maria Victoria Martínez-Toledo; Belén Rodelas; Jesús Gónzalez-López (pp. 563-567).

The effects of the application of nine pesticides used commonly in agriculture (aldrin, lindane, dimetoate, methylparathion, methidation, atrazine, simazine, captan and diflubenzuron) on growth, CO₂ production, denitrifying activity [as nitrous oxide (N₂O) released] and nitrite accumulation in the culture medium by Xanthobacter autotrophicus strain CECT 7064 (Spanish Type Culture Collection) (a micro-organism isolated from a submerged fixed-film) were studied. The herbicide atrazine and the insecticide dimetoate totally inhibited growth and biological activity of X. autotrophicus at 10 mg l⁻¹, while the rest of the tested pesticides delayed the growth of strain CECT 7064 but did not drastically affect the bacterial growth after 96 h of culture. The denitrifying activity of X. autotrophicus was negatively affected by the pesticides application with the exception of fungicide captan. The release of N₂O was strongly inhibited by several pesticides (aldrin, lindane, methylparathion, methidation and diflubenzuron), while dimetoate, atrazine and simazine inhibited totally the denitrifying activity of the strain. The effects of the pesticides on denitrifying submerged fixed-film reactor are discussed.

Novel method for aroma recovery from the bioconversion of lutein to β-ionone by Trichosporon asahii using a mesoporous silicate material by E. Rodríguez-Bustamante; G. Maldonado-Robledo; A. Sánchez-Contreras; T. Klimova; R. Arreguín-Espinosa; S. Sánchez (pp. 568-573).

In this work, we report on the synthesis and ability of the mesoporous material MCM-41 to adsorb the norisoprenoid β-ionone. This compound, with a violet aroma note, can be produced from lutein by the yeast Trichosporon asahii through a bioconversion process. We found that β-ionone inhibited the yeast growth and constrained aroma formation. Growth inhibition was overcome using silicate MCM-41 as sorbent device in a fermentation system that allowed product removal from the culture medium by headspace manipulation. Compared to a commercial silica gel, the mesoporous material exhibited a 4.5-fold higher β-ionone adsorption. Contrasting to cultures without the sorbent device, the presence of MCM-41 allowed a marked increase (14-fold) in β-ionone production. Our results suggested that confinement of the norisoprenoid into the sorbent material bypassed its toxicity which allowed a better β-ionone production. This study represents the first report on the use of MCM-41 to recover an aroma produced by fermentation and therefore, a novel application for a mesoporous material.

Development of a multifunctional and efficient conjugal plasmid for use in Streptomyces spp by H. B. Mo; X. Y. Chen; Y. Liu; F. Xia; K. Q. Yang (pp. 574-574).

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Applied Microbiology and Biotechnology (v.71, #4)

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Applied Microbiology and Biotechnology (v.71, #4)