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Applied Microbiology and Biotechnology (v.68, #3)

Animal cell cultures: recent achievements and perspectives in the production of biopharmaceuticals by Michael Butler (pp. 283-291).

There has been a rapid increase in the number and demand for approved biopharmaceuticals produced from animal cell culture processes over the last few years. In part, this has been due to the efficacy of several humanized monoclonal antibodies that are required at large doses for therapeutic use. There have also been several identifiable advances in animal cell technology that has enabled efficient biomanufacture of these products. Gene vector systems allow high specific protein expression and some minimize the undesirable process of gene silencing that may occur in prolonged culture. Characterization of cellular metabolism and physiology has enabled the design of fed-batch and perfusion bioreactor processes that has allowed a significant improvement in product yield, some of which are now approaching 5 g/L. Many of these processes are now being designed in serum-free and animal-component-free media to ensure that products are not contaminated with the adventitious agents found in bovine serum. There are several areas that can be identified that could lead to further improvement in cell culture systems. This includes the down-regulation of apoptosis to enable prolonged cell survival under potentially adverse conditions. The characterization of the critical parameters of glycosylation should enable process control to reduce the heterogeneity of glycoforms so that production processes are consistent. Further improvement may also be made by the identification of glycoforms with enhanced biological activity to enhance clinical efficacy. The ability to produce the ever-increasing number of biopharmaceuticals by animal cell culture is dependent on sufficient bioreactor capacity in the industry. A recent shortfall in available worldwide culture capacity has encouraged commercial activity in contract manufacturing operations. However, some analysts indicate that this still may not be enough and that future manufacturing demand may exceed production capacity as the number of approved biotherapeutics increases.

The use of genetically modified Saccharomyces cerevisiae strains in the wine industry by Dorit Schuller; Margarida Casal (pp. 292-304).

In recent decades, science and food technology have contributed at an accelerated rate to the introduction of new products to satisfy nutritional, socio-economic and quality requirements. With the emergence of modern molecular genetics, the industrial importance of Saccharomyces cerevisiae, is continuously extended. The demand for suitable genetically modified (GM) S. cerevisiae strains for the biofuel, bakery and beverage industries or for the production of biotechnological products (e.g. enzymes, pharmaceutical products) will continuously grow in the future. Numerous specialised S. cerevisiae wine strains were obtained in recent years, possessing a wide range of optimised or novel oenological properties, capable of satisfying the demanding nature of modern winemaking practise. The unlocking of transcriptome, proteome and metabolome complexities will contribute decisively to the knowledge about the genetic make-up of commercial yeast strains and will influence wine strain improvement via genetic engineering. The most relevant advances regarding the importance and implications of the use of GM yeast strains in the wine industry are discussed in this mini-review. In this work, various aspects are considered including the strategies used for the construction of strains with respect to current legislation requirements, the environmental risk evaluations concerning the deliberate release of genetically modified yeast strains, the methods for detection of recombinant DNA and protein that are currently under evaluation, and the reasons behind the critical public perception towards the application of such strains.

Morphological engineering of Streptomyces hygroscopicus var. geldanus: regulation of pellet morphology through manipulation of broth viscosity by C. O’Cleirigh; J. T. Casey; P. K. Walsh; D. G. O’Shea (pp. 305-310).

Actinomycetes, especially members of the genus Streptomyces, are responsible for producing the majority of known antibiotics. The production of antibiotics by filamentous organisms is often dependent on the morphology and size distribution of the pellet population within the culture. Particle interaction and subsequent pellet formation are primarily dependent on the rate of collision of particles in culture, which is in turn, a function of fluid turbulence. The microbial polysaccharide xanthan gum was used to artificially regulate the apparent viscosity (μ_a) of S. hygroscopicus fermentation broths with the aim of controlling particle interaction, aggregation and hence pellet formation. An increase in both pellet count and biomass concentration from approximately 2,000 to 8,000 pellets ml⁻¹ and 0.9–2.1 g l⁻¹ dry weight of biomass, as well a decrease in the mean pellet volume from 0.014 to 0.004 mm³ was observed in cultures supplemented with 3 g l⁻¹ xanthan gum. The addition of xanthan gum significantly alters fluid rheology by increasing the μ_a. Counter-intuitively, an increase in the μ_a within the experimental range examined resulted in an increase in the rate of gas–liquid mass transfer. This was attributed to the predominantly diffusive nature of oxygen transfer in shake flask cultures.

Biotransformation of 6,7-epoxygeraniol by fungi by Mirosław Anioł; Ewa Huszcza (pp. 311-315).

The biotransformation of 6,7-epoxygeraniol by resting cells of selected fungi was investigated. The main product obtained from the transformation in Rhodotorula glutinis and R. marina cultures was 6,7-epoxynerol (5–48% of chloroform extracts), whereas Saccharomyces cerevisiae, Candida parapsilosis and C. kefyr reduced this substrate to 6,7-epoxycitronellol (30–33% of chloroform extracts). Cultures of Yarrowia lipolytica, Botrytis cinerea and S. cerevisiae promoted the cyclisation of 6,7-epoxygeraniol to 2-methyl-2-(2-hydroxyethyl)-5-(2-hydroxyprop-2-yl)tetrahydrofuran (11–99% of chloroform extracts). The biotransformation of 6,7-epoxynerol was also investigated. However, none of the tested micro-organisms converted this compound.

An on-line technique for monitoring propionic acid fermentation by Houda Benjelloun; Alice Rochex; Didier Lecouturier; Siham Dechemi; Jean-Michel Lebeault (pp. 316-321).

An on-line technique, based on measuring the increase in pressure due to CO₂ release in a closed air-tight reactor, was used to evaluate the fermentation of lactate by propionibacteria. The method was applied to batch cultures of Propionibacterium shermanii grown in yeast extract/sodium lactate medium containing lactate as a carbon source under micro-aerophilic conditions. Gas pressure evolution was compared both with substrate consumption and metabolites production and with acidification and growth. Linear relationships were found between gas pressure variation, lactate consumption and propionate and acetate production. The technique also enabled the evaluation of total CO₂ produced, by taking account of pressure, oxygen and pH measurements. These results tend to show that this simple and rapid method could be useful to monitor propionic acid bacteria growth.

Modelling the production of nisin by Lactococcus lactis in fed-batch culture by Wenhua Lv; Xiaoyan Zhang; Wei Cong (pp. 322-326).

Nisin production in batch culture and fed-batch cultures (sucrose feeding rates were 6, 7, 8, and 10 g l⁻¹ h⁻¹, respectively) by Lactococcus lactis subsp. lactis ATCC 11454 was investigated. Nisin production showed primary metabolite kinetics, and could be improved apparently by altering the feeding strategy. The nisin titer reached its maximum, 4,185 IU ml⁻¹, by constant addition of sucrose at a feeding rate of 7 g l⁻¹ h⁻¹; an increase in 58% over that of the batch culture (2,658 IU ml⁻¹). Nisin biosynthesis was affected strongly by the residual sucrose concentration during the feeding. Finally, a mathematical model was developed to simulate the cell growth, sucrose consumption, lactic acid production and nisin production. The model was able to describe the fermentation process in all cases.

Comparison of cyanide-degrading nitrilases by Dakshina M. Jandhyala; Richard C. Willson; B. Trevor Sewell; Michael J. Benedik (pp. 327-335).

Recombinant forms of three cyanide-degrading nitrilases, CynD from Bacillus pumilus C1, CynD from Pseudomonas stutzeri, and CHT from Gloeocercospora sorghi, were prepared after their genes were cloned with C-terminal hexahistidine purification tags and expressed in Escherichia coli, and the enzymes purified using nickel-chelate affinity chromatography. The enzymes were compared with respect to their pH stability, thermostability, metal tolerance, and kinetic constants. The two bacterial genes, both cyanide dihydratases, were similar with respect to pH range, retaining greater than 50% activity between pH 5.2 and pH 8 and kinetic properties, having similar K_m (6–7 mM) and V_max (0.1 mmol min⁻¹ mg⁻¹). They also exhibited similar metal tolerances. However, the fungal CHT enzyme had notably higher K_m (90 mM) and V_max (4 mmol min⁻¹ mg⁻¹) values. Its pH range was slightly more alkaline (retaining nearly full activity above 8.5), but exhibited a lower thermal tolerance. CHT was less sensitive to Hg²⁺ and more sensitive to Pb²⁺ than the CynD enzymes. These data describe, in part, the current limits that exist for using nitrilases as agents in the bioremediation of cyanide-containing waste effluent, and may help serve to determine where and under what conditions these nitrilases may be applied.

Characterization of [3Fe-4S] ferredoxin DbfA3, which functions in the angular dioxygenase system of Terrabacter sp. strain DBF63 by Terufumi Takagi; Hiroshi Habe; Takako Yoshida; Hisakazu Yamane; Toshio Omori; Hideaki Nojiri (pp. 336-345).

Dibenzofuran 4,4a-dioxygenase (DFDO) from Terrabacter sp. strain DBF63 is comprised of three components, i.e., terminal oxygenase (DbfA1, DbfA2), putative [3Fe-4S] ferredoxin (ORF16b product), and unidentified ferredoxin reductase. We produced DbfA1 and DbfA2 using recombinant Escherichia coli BL21(DE3) cells as a native form and purified the complex to apparent homogeneity. We also produced and purified a putative [3Fe-4S] ferredoxin encoded by ORF16b, which is located 2.5 kb downstream of the dbfA1A2 genes, with E. coli as a histidine (His)-tagged form. The reconstructed DFDO system with three purified components, i.e., DbfA1A2, His-tagged ORF16b product, and His-tagged PhtA4 (which is a tentative reductase derived from the phthalate dioxygenase system of strain DBF63) could convert fluorene to 9-fluorenol (specific activity: 14.4 nmol min⁻¹ mg⁻¹) and convert dibenzofuran to 2,2′,3-trihydroxybiphenyl. This indicates that the ORF16b product can transport electrons to the DbfA1A2 complex; and therefore it was designated DbfA3. Based on spectroscopic UV-visible absorption characteristics and electron paramagnetic resonance spectra, DbfA3 was elucidated to contain a [3Fe-4S] cluster. Ferredoxin interchangeability analysis using several types of ferredoxins suggested that the redox partner of the DbfA1A2 complex may be rather specific to DbfA3.

Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region by Tao Liu; Jing-yu Chen; Zhong Zheng; Tian-hong Wang; Guo-Qiang Chen (pp. 346-354).

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P_vgb of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P_vgb was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P_vgb provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P_vgb, while a construct containing P_vgb, the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P_vgb, vgb operon) or pKVpPV (P_vgb, vgb operon, par) slightly attenuated (∼30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P_vgb, egfp) or pKVpP-E (P_vgb, par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P_vgb, vgb operon, par, tod) or pKVpP-T (P_vgb, par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.

Expression and characterization of a thermostable serine protease (TfpA) from Thermomonospora fusca YX in Pichia pastoris by Taewan Kim; Xin Gen Lei (pp. 355-359).

A serine protease produced by Thermomonospora fusca YX (TfpA) is heat-stable (up to 85°C) and has a broad pH activity range and strong resistance to detergents. The objective of this study was to determine if the methylotropic yeast Pichia pastoris could express TfpA extracellularly. A 1.0-kb DNA fragment (tfpA) encoding the pro-peptide and mature protein of TfpA was cloned into expression vectors pPICZαA (inducible) and pGAPZαA (constitutive) and introduced into P. pastoris by electroporation. Expression of r-TfpA was greater in the inducible system than in the constitutive one, producing 135 U ml⁻¹ medium supernatant 6 days after methanol induction. The r-TfpA was not glycosylated (21.7 kDa), and had pH and temperature optima of 8.5 and 80°C, respectively, using azocasein as a substrate. In conclusion, P. pastoris can be used as a host to produce extracellular r-TfpA, and expression efficiency may be improved by optimizing fermentation conditions and modifying factors related to protein expression and stability.

Genetically engineered termite gut bacteria (Enterobacter cloacae) deliver and spread foreign genes in termite colonies by Claudia Husseneder; J. Kenneth Grace (pp. 360-367).

Indigenous gut bacteria of the Formosan subterranean termite (Coptotermes formosanus Shiraki, Isoptera: Rhinotermitidae) were used as shuttle systems to deliver, express and spread foreign genes in termite colonies. The gut bacterium Enterobacter cloacae was transformed with a recombinant plasmid (pEGFP) containing genes encoding ampicillin resistance and green fluorescent protein (GFP). In laboratory experiments, termite workers and soldiers from three colonies were fed with filter paper inoculated with transformed bacteria. Transformed bacteria were detected in termite guts by growing the entire gut flora under selective conditions and checking the cultures visually for fluorescence. We demonstrated that (1) transformed bacteria were ingested within a few hours and the GFP gene was expressed in the termite gut; (2) transformed bacteria established a persistent population in the termite gut for up to 11 weeks; (3) transformed bacteria were efficiently transferred throughout a laboratory colony, even when the donor (termites initially fed with transformed bacteria) to recipient (not fed) ratio was low; (4) transformed E. cloacae were transferred into soil; however, they did not accumulate over time and the GFP plasmid was not transferred to other soil bacteria. In the future, transgenic bacteria may be used to shuttle detrimental genes into termite colonies for improved pest control.

The 18S rDNA sequence of Synchytrium endobioticum and its utility in microarrays for the simultaneous detection of fungal and viral pathogens of potato by Ismail Abdullahi; Marianne Koerbler; Hans Stachewicz; Stephan Winter (pp. 368-375).

Resting spores extracted from wart (Synchytrium endobioticum)-infected potato tubers were used for DNA extraction and amplification of 18S rDNA. Analysis of the cloned, sequenced fragment revealed high similarity to members of the Chytridiomycota. Using this information, specific oligonucleotide probes were designed and arrayed onto glass slides for detection of the pathogen. Viral sequence information available in the databank was retrieved, or new viral sequences were generated, and used to design probes for specific detection of important quarantine viruses of potato. To determine the sensitivity and specificity of the oligonucleotide probes, total RNA from infected plants was reverse transcribed, labelled with Cyanine 5, and hybridised with the microarray. A significant number of the oligonucleotide probes exhibited high specificity to S. endobioticum, Andean potato latent virus, Andean potato mottle virus, Potato black ringspot virus, and Potato spindle tuber viroid. Hybridisation signals of sub-arrays within slides were reproducible (r=0.79) with a high correlation coefficient of hybridisation repetitions (0.73). Our results demonstrate the potential of microarray-based hybridisation for identification of multiple pathogen targets, which will find application in quarantine laboratories, where parallel testing for diverse pathogens is essential.

Prediction of growth and biotransformation rates of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) in the presence of barium by Douglas M. Young; Karen Young; Kimberly L. Ogden (pp. 376-383).

The biotransformation of explosives has been investigated by many researchers. Bioremediation of soil and water contaminated with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is becoming the method of choice for clean-up of a variety of sites. In this study, we investigated biotransformation of RDX in the presence of barium. Ba is a metal commonly found in combination with RDX at sites requiring remediation. RDX was biotransformed by both a consortium of bacteria and an isolate from the consortium under anoxic conditions using a rich medium. However, Ba inhibited cell growth under both aerobic and anoxic conditions and slowed biotransformation rates by 40%. RDX and Ba inhibited growth of the isolate more than growth of the consortium. An additive inhibition model is proposed that accurately predicts the reduced growth rates observed.

Production of N-acetyl-β-d-glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme by J. H. Kuk; W. J. Jung; G. H. Jo; Y. C. Kim; K. Y. Kim; R. D. Park (pp. 384-389).

A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45°C, chitin was effectively hydrolyzed to N-acetyl-β-d-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50°C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45°C, the major reaction product was GlcNAc with a small amount of (GlcNAc)₂ and (GlcNAc)₃, whereas above 50°C the major product was (GlcNAc)₂. When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40°C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.

Carbon monoxide conversion by thermophilic sulfate-reducing bacteria in pure culture and in co-culture with Carboxydothermus hydrogenoformans by S. N. Parshina; S. Kijlstra; A. M. Henstra; J. Sipma; C. M. Plugge; A. J. M. Stams (pp. 390-396).

Biological sulfate (SO₄) reduction with carbon monoxide (CO) as electron donor was investigated. Four thermophilic SO₄-reducing bacteria, Desulfotomaculum thermoacetoxidans (DSM 5813), Thermodesulfovibrio yellowstonii (ATCC 51303), Desulfotomaculum kuznetsovii (DSM 6115; VKM B-1805), and Desulfotomaculum thermobenzoicum subsp. thermosyntrophicum (DSM 14055), were studied in pure culture and in co-culture with the thermophilic carboxydotrophic bacterium Carboxydothermus hydrogenoformans (DSM 6008). D. thermoacetoxidans and T. yellowstonii were extremely sensitive to CO: their growth on pyruvate was completely inhibited at CO concentrations above 2% in the gas phase. D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were less sensitive to CO. In pure culture, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum were able to grow on CO as the only electron donor and, in particular in the presence of hydrogen/carbon dioxide, at CO concentrations as high as 50–70%. The latter SO₄ reducers coupled CO oxidation to SO₄ reduction, but a large part of the CO was converted to acetate. In co-culture with C. hydrogenoformans, D. kuznetsovii and D. thermobenzoicum subsp. thermosyntrophicum could even grow with 100% CO (P_CO=120 kPa).

Experimental determination of viability loss of Penicillium bilaiae conidia during convective air-drying by Timothy Friesen; Gordon Hill; Todd Pugsley; Greg Holloway; Deanna Zimmerman (pp. 397-404).

A study was conducted on the drying of Penicillium bilaiae, a fungal micro-organism used to promote soil-bound phosphorous uptake in several crop species, such as wheat, canola and pulse crops. A wet pellet formed from a mixture of the inoculant and a starch-based carrier was air-dried to the appropriate water activity to extend the shelf-life of the viable fungal conidia. Convective air-drying was examined as a low-energy alternative to the more expensive freeze-drying technology that is currently in use. Experiments were conducted to measure the loss of conidia viability during drying in a fixed-bed, thin-layer convective dryer. The dryer air inlet temperature and relative humidity were controlled in experiments to determine the effect of thermal and dessicative stresses on conidial viability. The measured survivor fraction was determined to be dependent on solids temperature, moisture content and drying rate. Thermal stresses became significant for process temperatures above 30°C, while the survivor fraction fell sharply below a dry basis moisture ratio of 30%. Slower drying kinetics associated with high inlet air relative humidity were found to significantly improve the recovery of viable conidia. By minimising environmental stresses, survivor fractions of up to 75% could be achieved, but this result fell dramatically with the introduction of more severe conditions. A general linear statistical model is used to quantify experimental error and the significance level of each factor.

Identification of a homogentisate-1,2-dioxygenase gene in the fungus Exophiala lecanii-corni: analysis and implications by Claudia K. Gunsch; Qiang Cheng; Kerry A. Kinney; Paul J. Szaniszlo; Christian P. Whitman (pp. 405-411).

Exophiala lecanii-corni is a dimorphic fungus capable of degrading several volatile organic compounds (VOCs) including ethylbenzene, which has been classified as a hazardous air pollutant by the Environmental Protection Agency. In contrast to bacterial species, little is known about the mechanisms of fungal degradation of VOCs. The results described herein suggest a potential pathway for ethylbenzene degradation in E. lecanii-corni via styrene, phenylacetate and homogentisate. Consistent with this proposed pathway, a full-length homogentisate-1,2-dioxygenase gene (ElHDO) has been identified, cloned and sequenced. The nucleotide sequence of ElHDO consists of a 1,452-bp open reading frame encoding a protein with 484 amino acids. The expression of the gene product increases when grown on ethylbenzene, further suggesting that it could be involved in ethylbenzene degradation and may be responsible for the aromatic ring cleavage reaction. In addition, a 907-bp fragment isolated upstream from this gene shares 78% sequence identity at the amino acid level with the amino acid sequences of two fungal phenylacetate hydroxylase genes. This observation suggests that the genes responsible for ethylbenzene degradation may be clustered. This research constitutes the first step towards a better understanding of ethylbenzene degradation in E. lecanii-corni.

Synthesis of prebiotic galactooligosaccharides using whole cells of a novel strain, Bifidobacterium bifidum NCIMB 41171 by George Tzortzis; Athanasios K. Goulas; Glenn R. Gibson (pp. 412-416).

A novel strain of Bifidobacterium bifidum NCIMB 41171, isolated from a faecal sample from a healthy human volunteer and able to express β-galactosidase activity, was used in synthesis reactions for the production of galactooligosaccharide from lactose. The β-galactosidase activity of whole bifidobacterial cells showed an optimum activity at pH 6.8–7.0 and 40°C. The transgalactosylation activity of the B. bifidum cells from 50% (w/w) lactose resulted in a galactooligosaccharide mixture (20% w/w) comprising (w/w): 25% disaccharides, 35% trisaccharides, 25% tetrasaccharides and 15% pentasaccharides. Using different initial lactose concentrations, the conversion rate to galactooligosaccharides was maximum (35%) when 55% (w/w) lactose was used. In fermentation experiments, B. bifidum showed an increased preference towards the produced galactooligosaccharide mixture, displaying higher growth rate and short-chain fatty acid production when compared with commercially available oligosaccharides.

A microcosm study on bioremediation of p-nitrophenol-contaminated soil using Arthrobacter protophormiae RKJ100 by S. Labana; O. V. Singh; A. Basu; G. Pandey; R. K. Jain (pp. 417-424).

p-Nitrophenol (PNP), a toxic nitroaromatic compound, can build up in soils due to extensive usage of nitrophenolic pesticides and hence needs to be removed. Arthrobacter protophormiae RKJ100, a PNP-degrading organism, was used in this work to study factors affecting its growth, and then evaluated for its capacity to degrade PNP in soil microcosms. Molasses (10%) treated with 0.1% potassium hexacyanoferrate was found to be a suitable and cheap carbon source for inoculum preparation. Induction studies showed that PNP depletion was quicker when cells were induced by pre-exposure to PNP. The efficiency of PNP degradation in soil by strain RKJ100 was seen to be dependent on pH, temperature, initial PNP concentration and inoculum size. Microcosm studies performed with varying concentrations (1.4–210 ppm) of PNP-spiked soils showed that strain RKJ100 could effectively degrade PNP over the range 1.4–140 ppm. A cell density of 2×10⁸ colony forming units/g soil was found to be suitable for PNP degradation over a temperature range of 20–40°C and at a slightly alkaline pH (7.5). Our results indicate that strain RKJ100 has potential for use in in situ bioremediation of PNP-contaminated sites. This is a model study that could be used for decontamination of sites contaminated also with other compounds.

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Applied Microbiology and Biotechnology (v.68, #3)