Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture

Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D=0.05 h⁻¹) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive. Received: 24 February 1997 / Received revision: 22 May 1997 / Accepted: 25 May 1997     

Anaerobic biodegradation of pentachlorophenol in a contaminated soil inoculated with a methanogenic consortium or with Desulfitobacterium frappieri strain PCP-1

Anaerobic biodegradation of pentachlorophenol (PCP) in a contaminated soil from a wood-treating industrial site was studied in soil slurry microcosms inoculated with a PCP-degrading methanogenic consortium. When the microcosms containing 10%–40% (w/v) soil were inoculated with the consortium, more than 90% of the PCP was removed in less than 30 days at 29 °C. Less-chlorinated phenols, mainly 3-chlorophenol were slowly degraded and accumulated in the cultures. Addition of glucose and sodium formate to the microcosms was not necessary, suggesting that the organic compounds in the soil can sustain the dechlorinating activity. Inoculation of Desulfitobacterium frappieri strain PCP-1 along with a 3-chlorophenol-degrading consortium in the microcosms also resulted in the rapid dechlorination of PCP and the slow degradation of 3-chlorophenol. Competitive polymerase chain reaction experiments showed that PCP-1 was present at the same level throughout the 21-day biotreatment. D. frappieri, strain PCP-1, inoculated into the soil microcosms, was able to remove PCP from soil containing up to 200 mg PCP/kg soil. However, reinoculation of the strain was necessary to achieve more than 95% PCP removal with a concentration of 300 mg and 500 mg PCP/kg soil. These results demonstrate that D. frappieri strain PCP-1 can be used effectively to dechlorinate PCP to 3-chlorophenol in contaminated soils. Received: 14 November 1997 / Received revision: 29 January 1998 / Accepted: 24 February 1998     

Enhancing the mineralization of [U-14C]dibenzo-p-dioxin in three different soils by addition of organic substrate or inoculation with white-rot fungi

The potential for aerobic mineralization of [U-¹⁴C]dibenzo-p-dioxin (DD) was investigated in samples of three different agricultural soils already contaminated with polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by industrial activities. The influence of amendments, i.e. wheat straw and compost, and of soil treatment by inoculation with lignolytic fungi, grown on wheat straw substrate, was tested. All the soils tested contained an indigenous DD-mineralizing microflora. The soil characterized by the highest organic matter content and the highest content of soil microbial biomass displayed the best DD mineralization of 36.6% within 70 days, compared with the two organic-matter-poor soils with an endogenous DD mineralization of 19.5% and 23.3% respectively. Amendments with compost increased DD mineralization up to 28% in both soils with low organic matter and microbial biomass content, but did not affect mineralization in the organic-matter-rich soil. Addition of wheat straw had no constant influence on DD mineralization in the soils tested. The best DD mineralization resulted from inoculation with lignolytic white-rot fungi (Phanerochaete chrysosporium, Pleurotus sp. Florida, Dichomitus squalens) and with an unidentified lignolytic fungus, which was isolated originally from a long-term PCDD/F-contaminated soil. A mineralization of up to 50% within 70 days was reached by this treatment. The influence of inoculated fungi on mineralization differed between the soils investigated. Received: 14 April 1997 / Received revision: 24 June 1997 / Accepted: 29 June 1997     

Rapid method for detecting Desulfitobacterium frappieri strain PCP-1 in soil by the polymerase chain reaction

A rapid method was developed for detecting in soil Desulfitobacterium frappieri strain PCP-1, an anaerobic gram-positive bacterium, isolated from a methanogenic consortium degrading pentachlorophenol. The method involved the establishment of a protocol for extracting total DNA from soil with the least contamination, and the use of the polymerase chain reaction (PCR) to detect strain PCP-1 with primers targeted with PCP-1 16S rRNA. To optimize the DNA extraction conditions, a glass mill homogenizer and a low-salt buffer containing polyvinylpolypyrrolidone were used on a black soil rich in organic matter. Recovered DNA was further purified with phenol/chloroform extractions, ammonium acetate precipitation and a G200 Sephadex gel-filtration column. DNA was extracted from soil supplemented with different concentrations of PCP-1 cells. Detection of PCP-1 was by PCR. The limit of detection was 800 added PCP-1 cells/g dry soil. This level of detection was achieved when the T4 gene-32 protein and 1 μg soil DNA were added to the PCR mixture followed by a nested PCR. This method is quick, sensitive, and can process several samples at the same time. Received: 22 October 1996 / Received revision: 24 January 1997 / Accepted: 10 February 1997     

Isolation and characterization of a marine bacterium capable of utilizing 2-methylphenanthrene

A marine bacterium isolated from a coastal hydrocarbon-polluted sediment has been described and attributed on the basis of its phenotypic and genotypic characteristics to the genus Sphingomonas sp. This strain was capable of using an alkylated phenanthrene 2-methylphenanthrene, as sole source of carbon and energy. In experiments, 2-methylphenanthrene (0.2 g/l) was added as crystals to the culture medium. After 5 days of aerobic growth at 30 °C, 70% was degraded and the complete dissipation occurred after 20 days. Furthermore, the strain could degrade various kinds of polyaromatic compounds, but failed to grow on aliphatic hydrocarbons. Received: 27 December 1996 / Received last revision: 10 June 1997 / Accepted: 14 June 1997     

Isolation and characterization of dibenzofuran-degrading bacteria

Two bacterial strains capable of utilizing dibenzofuran (DF) as a sole carbon source were isolated from soil samples of reclaimed land. The strains designated HL1 and HL7 were identified as Klebsiella sp. and Sphingomonas sp., respectively, on the basis of biochemical characteristics and the sequences of the 16S ribosomal DNA. Sphingomonas sp. strain HL7 degraded non-, mono- and also dichlorinated DF and dibenzo-p-dioxin (DD). Klebsiella sp. strain HL1 was able to degrade non- and monochlorinated DFs and DDs, but not dichlorinated ones. The metabolites formed from DF by strains HL1 and HL7 were similar to those by dioxin-degrading bacteria Sphingomonas sp. strain RW1 except for salicylic acid and catechol. Strain HL7 had a gene homologous to that encoding the dioxin dioxygenase alpha-subunit (dxnA1) gene of Sphingomonas sp. strain RW1. However, Southern hybridization analysis showed that the size of an EcoRV-digested genomic fragment involving the dioxin dioxygenase gene of strain HL7 was smaller than that of strain RW1, and that strain HL1 did not have the homologous gene. Strains HL1 and HL7 provided useful information regarding the dioxygenase genes.     

Liquid fermentation to produce biomass of mycoherbicidal strains of Fusarium oxysporum

Conditions for optimizing spore production, especially chlamydospores, by host-specific mycoherbicidal strains of Fusarium oxysporum causing vascular wilts in coca (Erythroxylum coca) and poppy (Papaver somniferum) were studied in 2.5-1 fermentors. The fermentor dissolved oxygen and pH had significant effects on the growth characteristics of F. oxysporum strains. The effect of the fungal strain, however was not significant for most of the variables studied except for chlamydospore formation. After 14 days of fermentation, the spore types produced were microconidia and chlamydospores, with very little production of macroconidia. While the total viable counts were significantly higher under high than under low dissolved O₂, the chlamydospore counts were significantly higher under low than under high dissolved O₂. The percentage of chlamydospores obtained, as a proportion of total viable was significantly higher when the fermentor pH was increased, than when it was not. Scaling-up the liquid fermentation to 20 l, yielded log₁₀ c = 6.8 (where c = chlamydospores ml⁻¹) after 14 days' fermentation, with biomass viable counts of log₁₀ v∼8.0 (where v = viable counts g⁻¹ air-dried biomass). A single-step liquid fermentation reported in this study increased chlamydospore yields and reduced the time required for their production with techniques currently available from 5 weeks to less than 2 weeks. Received: 24 April 1997 / Received revision: 6 August 1997 / Accepted: 29 August 1997     

Optimization of compactin fermentation

A compactin producer Penicillium sp strain was isolated from soil in our screening program. The compactin-biosynthesising capacity of the strain was improved from 5 μg ml⁻¹ to 250 μg ml⁻¹ by mutation-selection method. We investigated the effect of the medium composition on compactin productivity. A central, orthogonal three-factor experimental design by Box and Wilson was used in the investigation. The results were analysed by non-linear regression analysis. The composition of the production medium was optimized according to the calculated mathematical model using the steepest ascent method. The compactin productivity was increased to 400 μg ml⁻¹ by applying this method. Received 08 October 1997/ Accepted in revised form 04 December 1997     

Diacetyl and acetoin production from the co-metabolism of citrate and xylose by Leuconostoc mesenteroides subsp. mesenteroides

The co-metabolism of citrate plus xylose by Leuconostoc mesenteroides subsp. mesenteroides results in a growth stimulation, an increase in d-lactate and acetate production and repression of ethanol production. This correlated well with the levels of key enzymes involved. A partial repression of alcohol dehydrogenase and a marked stimulation of acetate kinase were observed. High citrate bioconversion yields in diacetyl plus acetoin were obtained at pH 5.2 in batch (11.5%) or in chemostat (up to 17.4%) culture. In contrast, no diacetyl or acetoin was detected in citrate plus glucose fermentation. Received: 6 December 1996 / Received revision: 14 February 1997 / Accepted: 14 February 1997     

Effect of citrate on growth of Lactococcus lactis subsp. lactis in milk

The effect of citrate on the growth of Lactococcus lactis subsp. lactis var. diacetylactis in milk has been investigated. Five strains of Lactococcus lactis subsp. lactis var. diacetylactis were compared to their citrate-negative variants, which lack the plasmid coding for citrate permease. In most cases, acidification kinetics and the final bacterial concentration of pure cultures of parental and variant strains did not differ significantly. Co-cultures of parental and variant strains, however, systematically tended towards the predominance of parental strains. Citrate metabolism is responsible for this change, since the predominance of citrate-positive strains was not observed in the absence of citrate. Continuous culture in milk enabled the difference in growth rates between the parental strain Lactococcus lactis subsp. lactis var. diacetylactis CDI1 and its citrate-negative variant to be quantified by following changes in the populations of the two co-cultured strains. At 26 °C, the growth rate of the parental strain was 7% higher than that of its citrate-negative variant. These results show that citrate metabolism slightly stimulates the growth of lactococci in milk. Received: 18 February 1997 / Received revision: 2 May 1997 / Accepted: 4 May 1997     

Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in Aspergillus oryzae

For the development of an efficient gene expression system in a shoyu koji mold Aspergillus oryzae KBN616, the TEF1 gene, encoding translation-elongation factor 1α, was cloned from the same strain and used for expression of polygalacturonase genes. The TEF1 gene comprised 1647 bp with three introns. The TEF1-α protein consisted of 460 amino acids possessing high identity to other fungal TEF proteins. Two nucleotide sequences homologous to the upstream activation sequence, characterized for the ribosomal protein genes in Saccharomyces cerevisiae, as well as the pyrimidine-rich sequences were present in the TEF1 gene promoter region, suggesting that the A. oryzae TEF1 gene has a strong promoter activity. Two expression vectors, pTFGA300 and pTFGB200 for production of polygalacturonases A and B respectively, were constructed by using the TEF1 gene promoter. A polygalacturonase (PGB) gene cloned from the same strain comprised 1226 bp with two introns and encoded a protein of 367 amino acids with high similarity to other fungal polygalacturonases. PGA and PGB were secreted at approximately 100 mg/l in glucose medium and purified to homogeneity. PGA had a molecular mass of 41 kDa, a pH optimum of 5.0 and temperature optimum of 45 °C. PGB had a molecular mass of 39 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C. Received: 28 November 1997 / Received revision: 24 February 1998 / Accepted: 6 March 1998     

Bioremediation of atrazine-contaminated soil by repeated applications of atrazine-degrading bacteria

Bioaugmentation has previously been unreliable for the in situ clean-up of contaminated soils because of problems with poor survival and the rapid decline in activity of the bacterial inoculum. In an attempt to solve these problems, a 500-l batch fermenter was investigated for its ability to deliver inoculum repeatedly to contaminated soils via irrigation lines. In a field experiment, mesocosms were filled with 350 kg soil containing 100 mg kg⁻¹ atrazine, and inoculated one, four or eight times with an atrazine-degrading bacterial consortium that was produced in the fermenter. After 12 weeks, no significant degradation of atrazine had occurred in soil that was inoculated only once; whereas, mesocosms inoculated four and eight times mineralized 38% and 72% of the atrazine respectively. Similar results were obtained in a laboratory experiment using soil contaminated with 100 mg kg⁻¹ [¹⁴C]atrazine. After 35 days, soil that was inoculated once with 10⁸ cfu ml⁻¹ of the consortium or with the atrazine-degrading bacterium, Pseudomonas sp. strain ADP, mineralized 17% and 35% of the atrazine respectively. In comparison, microcosms inoculated every 3 days with the consortium or with Pseudomonas sp. (ADP) mineralized 64% or 90% of the atrazine over this same period. Results of these experiments suggest that repeated inoculation from an automated fermenter may provide a strategy for bioaugmentation of contaminated soil with xenobiotic-degrading bacteria. Received: 20 November 1998 / Received revision: 8 February 1999 / Accepted: 12 February 1999     

Bioconversion of 2-cyanopyrazine to 5-hydroxypyrazine-2-carboxylic acid with Agrobacterium sp. DSM 6336

5-Hydroxypyrazine-2-carboxylic acid, a versatile building block for the synthesis of new antituberculous agents, was prepared by whole-cell biotransformation from 2-cyanopyrazine via pyrazinecarboxylic acid using Agrobacterium sp. DSM 6336. By developing a fermentation process for this two-enzyme-step bioconversion, a product concentration of 286 mM (40 g/l) was obtained. After the isolation method had been optimized the total yield was 80%. Received: 28 February 1997 / Received revision: 28 April 1997 / Accepted: 4 May 1997     

Synthesis of dihydroxynaphthalene isomers by microbial oxidation of 1- and 2-naphthol

The mutant strain Pseudomonas fluorescens TTC1 (NCIMB 40605), derived from the naphthalene-degrading Pseudomonas fluorescens N3 (NCIMB 40530), was used for the oxidation of 1- and 2-naphthols to give different isomers of dihydroxynaphthalene. The oxidation reactions proceed through the formation of dihydrodiol intermediates, which are too unstable to be isolated, since they spontaneously eliminate water to give the fully aromatic dihydroxynaphthalenes. The high regioselectivity of the dehydration reaction was confirmed by the study of the acid-catalysed aromatization of a series of stable monosubstituted naphthalene cis-1,2-dihydrodiols. Received: 24 March 1997 / Received revision: 6 June 1997 / Accepted: 7 June 1997     

Biodegradation of azo and phthalocyanine dyes by Trametes versicolor and Bjerkandera adusta

Eighteen fungal strains, known for their ability to degrade lignocellulosic material or lignin derivatives, were screened for their potential to decolorize commercially used reactive textile dyes. Three azo dyes, Reactive Orange 96, Reactive Violet 5 and Reactive Black 5, and two phthalocyanine dyes, Reactive Blue 15 and Reactive Blue 38, were chosen as representatives of commercially used reactive dyes. From the 18 tested fungal strains only Bjerkandera adusta, Trametes versicolor and Phanerochaete chrysosporium were able to decolorize all the dyes tested. During degradation of the nickel-phthalocyanine complex, Reactive Blue 38, by B. adusta and T. versicolor respectively, the toxicity of this dye to Vibrio fischeri was significantly reduced. In the case of Reactive Violet 5, a far-reaching detoxification was achieved by treatment with B. adusta. Reactive Blue 38 and Reactive Violet 5 were decolorized by crude exoenzyme preparations from T. versicolor and B. adusta in a H₂O₂-dependent reaction. Specific activities of the exoenzyme preparations with the dyes were determined and compared to oxidation rates by commercial horseradish peroxidase. Received: 3 February 1997 / Received revision: 9 April 1997 / Accepted: 13 April 1997     

Rapid atrazine mineralisation in soil slurry and moist soil by inoculation of an atrazine-degrading Pseudomonas sp. strain

The evaluation of pesticide-mineralising microorganisms to clean-up contaminated soils was studied with the widely applied and easily detectable compound atrazine, which is rapidly mineralised by several microorganisms including the Pseudomonas sp. strain Yaya 6. The rate of atrazine removal was proportional to the water content of the soil and the amount of bacteria added to the soil. In soil slurry, 6 mg atrazine kg soil⁻¹ was eliminated within 1 day after application of 0.3 g dry weight inoculant biomass kg soil⁻¹ and within 5 days when 0.003 g kg soil⁻¹ was used. In partially saturated soil (60% of the maximal water-holding capacity) 15 mg atrazine kg soil⁻¹ was eliminated within 2 days by 1 g biomass kg soil⁻¹ and within 25 days when 0.01 g biomass kg soil⁻¹ was used. In unsaturated soil, about 60% [U-ring-¹⁴C]atrazine was converted to ¹⁴CO₂ within 14 days. Atrazine was very efficiently removed by the inoculant biomass, not only in soil that was freshly contaminated but also in soil aged with atrazine for up to 260 days. The bacteria exposed to atrazine in unsaturated sterile soil were still active after a starvation period of 240 days: 15 mg newly added atrazine kg soil⁻¹ was eliminated within 5 days. Received: 31 October 1997 / Received revision: 16 January 1998 / Accepted: 18 January 1998     

One-step transformation of the dimorphic yeast Yarrowia lipolytica

An efficient one-step transformation method for the dimorphic yeast Yarrowia lipolytica is described. Using cells grown overnight on agar plates, the whole process is carried out within 1 h. The transformant clones could be recovered on selective plates as early as 36–48 h after plating. The efficiency was better than 10⁵ transformants/μg replicative plasmid DNA. Effects of cell density, dithiothreitol, heat shock, poly(ethylene glycol) 4000 concentration and the wetness of selective plates were investigated. Received: 17 February 1997 / Received revision: 4 April 1997 / Accepted: 19 April 1997     

Biochemical basis for carbon monoxide tolerance and butanol production by Butyribacterium methylotrophicum

The biochemical mechanisms for growth tolerance to a 100% CO headspace in cultures, and butanol plus ethanol production from CO by Butyribacterium methylotrophicum were assessed in the wild-type and CO-adapted strains. The CO-adapted strain grew on glucose or CO under a 100% CO headspace, whereas, the growth of the wild-type strain was severely inhibited by 100% CO. The CO-adapted strain, unlike the wild-type, also produced butyrate, from either pyruvate or CO. The CO-adapted strain was a metabolic mutant having higher levels of ferredoxin–NAD oxidoreductase activity, which was not inhibited by NADH. Consequently, only the CO-adapted strain can grow on CO because CO oxidation generates reduced ferredoxin which, via the mutated ferredoxin–NAD reductase activity, forms reduced NADH required for catabolism. When the CO-adapted strain was grown at pH 6.0 it produced butanol (0.33 g/l) and ethanol (0.5 g/l) from CO and the cells contained the following NAD-linked enzyme activities (μmol min⁻¹ mg protein⁻¹): butyraldehyde dehydrogenase (227), butanol dehydrogenase (686), acetaldehyde dehydrogenase (82) and ethanol dehydrogenase (129). Received: 15 September 1998 / Received revision: 12 February 1999 / Accepted: 19 February 1999     

Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101

Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C. beijerinckii NCIMB 8052. C. beijerinckii BA101 demonstrated a greater increase in solvent production than C. beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium. In 1-l batch fermentations, C. beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose. To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture. Received: 29 September 1998 / Received revision: 13 February 1999 / Accepted: 26 February 1999     

Detection of Sphingomonas spp in soil by PCR and sphingolipid biomarker analysis

Sphingomonas spp possess unique abilities to degrade refractory contaminants and are found ubiquitously in the environment. We developed Sphingomonas genus-specific PCR primers (SPf-190 and SPr1-852) which showed specific amplification of a 627-bp 16S rDNA fragment from Sphingomonas spp. A PCR assay using these Sphingomonas specific primers was developed to detect Sphingomonas aromaticivorans B0695R in three texturally distinct soil types, showing detection limits between 1.3–2.2 × 10³ CFU g⁻¹ dry soil. A sphingolipid extraction protocol was also developed to monitor Sphingomonas populations in soil quantitatively. The detection limit of the assay was 20 pmol g⁻¹ dry soil, equivalent to about 3 × 10⁵ cells g⁻¹ dry soil. Survival of S. aromaticivorans B0695R was monitored in the three different soils by antibiotic selective plate counting, PCR and sphingolipid analysis. All three approaches showed that the B0695R cells persisted in the low biomass Sequatchie sub-soil at about 3–5 × 10⁷cells g⁻¹ dry soil. In comparison to the plate counting assay, both the PCR and sphingolipid analysis detected a significantly higher level of B0695R cells in the clay soil and Sequatchie top-soil, indicating the possibility of the presence of viable but non-culturable B0695R cells in the soils. The combination of PCR and sphingolipid analysis may provide a more realistic estimation of Sphingomonas population in the environment. Received 17 March 1999/ Accepted in revised form 07 April 1999