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The distinctive flavours in hard cheeses are attributed largely to the activity of nonstarter lactic acid bacteria (NSLAB) which dominate the cheese matrix during maturation after lactose is consumed. Understanding how different strains of NSLAB survive, compete, and scavenge available nutrients is fundamental to selecting strains as potential adjunct starters which may influence product traits. Three Lacticaseibacillus paracasei isolates which dominated at different stages over 63-week maturation periods of Australian Cheddar cheeses had the same molecular biotype. They shared many phenotypic traits, including salt tolerance, optimum growth temperature, growth on N-acetylglucosamine and N-acetylgalactosamine plus delayed growth on D-ribose, carbon sources likely present in cheese due to bacterial autolysis. However, strains 124 and 163 (later named GCRL163) survived longer at low pH and grew on D-tagatose and D-mannitol, differentiating this phenotype from strain 122. When cultured on growth-limiting lactose (0.2%, wt/vol) in the presence of high concentrations of L-leucine and other amino acids, GCRL163 produced, and subsequently consumed lactate, forming acetic and formic acids, and demonstrated temporal accumulation of intermediates in pyruvate metabolism in long-term cultures. Strain GCRL163 grew in Tween 80-tryptone broths, a trait not shared by all L. casei-group dairy isolates screened in this study. Including citrate in this medium stimulated growth of GCRL163 above citrate alone, suggesting cometabolism of citrate and Tween 80. Proteomic analysis of cytosolic proteins indicated that growth in Tween 80 produced a higher stress state and increased relative abundance of three cell envelope proteinases (CEPs) (including PrtP and Dumpy), amongst over 230 differentially expressed proteins.

In addition to cell membrane phospholipids, Actinobacteria in the order Corynebacteriales possess a waxy cell envelope containing mycolic acids (MA). In optimized culture condition, some species can also accumulate high concentrations of intracellular triacylglycerols (TAG), which are a potential source of biodiesel. Bacterial lipid classes and composition alter in response to environmental stresses, including nutrient availability, thus understanding carbon flow into different lipid classes is important when optimizing TAG synthesis. Quantitative and qualitative analysis of lipid classes normally requires combinations of different extraction, derivatization, chromatographic and detection methods. In this study, a single-step thin-layer chromatography-flame ionization detection (TLC-FID) technique was applied to quantify lipid classes in six sub-Antarctic Corynebacteriales strains identified as Rhodococcus and Williamsia species. A hexane:diethyl-ether:acetic acid solvent system separated the total cellular lipids extracted from cells lysed by bead beating, which released more bound and unbound MA than sonication. Typical profiles included a major broad non-polar lipid peak, TAG and phospholipids, although trehalose dimycolates, when present, co-eluted with phospholipids. Ultra-performance liquid chromatography-tandem mass-spectrometry and nuclear magnetic resonance spectroscopy detected MA signatures in the non-polar lipid peak and indicated that these lipids were likely bound, at least in part, to sugars from cell wall arabinogalactan. Waxy esters were not detected. The single-solvent TLC-FID procedure provides a useful platform for the quantitation and preliminary screening of cellular lipid classes when testing the impacts of growth conditions on TAG synthesis.

Bacteria containing mycolic acids in their cell envelope are often recalcitrant to cell lysis, so extracting DNA of sufficient quality for third-generation sequencing and high-fidelity genome assembly requires optimization, even when using commercial kits with protocols for hard-to-lyse bacteria. We benchmarked three spin-column-based kits against a classical DNA extraction method employing lysozyme, proteinase K and SDS for six lysozyme-resistant, sub-Antarctic strains of Corynebaceriales. Prior cultivation in broths containing glycine at highly growth-inhibitory concentrations (4.0–4.5%) improved cell lysis using both classical and kit methods. The classical method produced DNA with average fragment sizes of 27–59 Kbp and tight fragment size ranges, meeting quality standards for genome sequencing, assembly and phylogenomic analyses. By 16S rRNA gene sequencing, we classified two strains as Williamsia and four strains as Rhodococcus species. Pairwise comparison of average nucleotide identity (ANI) and alignment fraction (AF), plus genome clustering analysis, confirmed Rhodococcus sp. 1163 and 1168 and Williamsia sp. 1135 and 1138 as novel species. Phylogenetic, lipidomic and biochemical analyses classified psychrotrophic strains 1139 and 1159 as R. qingshengii and R. erythropolis, respectively, using ANI similarity of &gt, 98% and AF &gt, 60% for species delineation. On this basis, some members of the R. erythropolis genome cluster groups, including strains currently named as R. enclensis, R. baikonurensis, R. opacus and R. rhodochrous, would be reclassified either as R. erythropolis or R. qingshengii.

Bacterial cell wall hydrolases, including amidases and peptidases, play a critical role in peptidoglycan turnover during growth, impacting daughter cell separation, and cell death, through autolysis. When exploring the regulation of protein expression across the growth cycle of an acid-resistant strain of Lactobacillus paracasei, GCRL 46, we observed temporal up-regulation of proteins in the 40&ndash, 45 kDa molecular weight range for whole-cell extracts when culturing in fermenters at a controlled pH of 4.0 versus optimum growth pH of 6.3. Up-regulation of proteins in this size range was not detected in SDS-PAGE gels of the cytosolic fraction, but was routinely detected following growth at low pH in whole cells and cell debris obtained after bead beating and centrifugation, indicating a cell surface location. N-terminal sequencing and in silico analyses showed sequence similarity with proteins in the L. casei group (L. casei, L. paracasei and L. rhamnosus) which were variously annotated as uncharacterized proteins, surface antigens, possible TrsG proteins, CHAP (cysteine, histidine-dependent amidohydrolases/peptidases)-domain proteins or putative peptidoglycan d,l-endopeptidase due to the presence of a CwlO domain. This protein is a homologue of the p40 (Msp2) secreted protein of L. rhamnosus LGG, which is linked to probiotic functionality in this species, and is phylogenetically related to structurally-similar proteins found in Enterococcus, Streptococcus and Bifidobacterium species, including the glucan-binding (GbpB), surface antigen (SagA) proteins detected in pathogenic group A streptococci species as secreted, immunoglobulin-binding (SibA) proteins (also named PcsB). Three-dimensional (3D) modelling predicted structural similarities in the CHAP proteins from the L. casei group and streptococcal species, indicating retention of overall architecture despite sequence divergence, and an implied retention of function during evolution. A phylogenetically-related hydrolase also contained the CwlO domain with a NLPC_P60 domain, and showed similar overall but distinct architecture to the CHAP proteins. We concluded that the surface-located, CHAP protein in L. casei is up-regulated during long-term exposure to acidic conditions during growth but not during acid shock.

Zinc (Zn) occurs in high concentrations in heavy metal polluted ecosystems which are colonized by bacterial populations. Some members of these communities are attractive candidates for phytoremediation of Zn-contaminated soils as they are able to convert insoluble forms of Zn into soluble forms, making Zn available to plants as a micronutrient. However, mechanisms of bacterial Zn solubilization and homeostasis are often not well understood. In the present study, two Gram positive rhizosphere strains, Bacillus sp. ZM20 and Bacillus subtilis ZM63, which were shown previously to solubilize phosphate and produce indole acetic acid plus siderophores, and two laboratory strains selected as controls (Bacillus cereus and Paenibacillus polymyxa), were tested for their ability to solubilize zinc oxide (ZnO). All of the strains tested could solubilize Zn to some degree, as they produced acids that lowered the medium pH. Two strains, Bacillus sp. ZM20 and B. cereus, were more Zn and acid resistant. When cultured aerobically in a minimum salt medium with glucose as carbon source plus 0.1% (w/v) Zn as ZnO, Bacillus sp. ZM20 and B. cereus both produced significantly higher concentrations of lactic and acetic acids than detected in control broths lacking Zn, with formic, citric, succinic, isobutyric and isovaleric acids detected as minor end-products. In in vitro tests, mixtures and individual acids at concentrations seen in broth cultures were shown to solubilize ZnO. However, the less acid- and Zn-resistant strains showed lower Zn solubility efficiency at higher concentrations of ZnO, as their growth was impaired, and higher concentrations of acids were not induced to the same extent by culture in ZnO. Induction of lactic, acetic, succinic, formic, isobutyric and isovaleric acids during culture correlated with ZnO solubilization, which has not been shown before for Gram positive species.

Prolonged heat stress is one of the harsh conditions Lactobacillus casei strains encounter as non-starter lactic acid bacteria in dairy product manufacture. To understand the physiological and molecular mechanisms through which Lb. casei GCRL163 adapts to persistent elevated temperature, label-free quantitative proteomics of cell-free extracts was used to characterize the global responses of the strain cultured anaerobically in bioreactors at 30 to 45°C, pH 6.5, together with GC-MS for fatty acid methyl ester analysis at different growth phases. At higher growth temperatures, repression of energy-consuming metabolic pathways, such as fatty acid, nucleotide and amino acid biosynthesis, was observed, while PTS- and ABC-type transporter systems associated with uptake of nitrogen and carbon sources were up-regulated. Alkaline shock protein Asp23_2 was only detected at 45°C, expressed at high abundance, and presumptive α-L-fucosidase only at 40 and 45°C, with highly increased abundance (log2-fold change of 7) at 45°C. We identified a novel SecB homolog as a protein export chaperone putatively involved in posttranslational translocation systems, which was down-regulated as growth temperature increased and where the modelled 3D-structure shared architectural similarities with the Escherichia coli SecB protein. Membrane lipid analyses revealed temporal changes in fatty acid composition, cyclization of oleic acid to cyclopropane and novel cyclopentenyl moieties, and reduced synthesis of vaccenic acid, at higher temperatures. An 18kDa α-crystallin domain, Hsp20 family heat shock protein was more highly up-regulated in response to heat stress compared to other molecular chaperones, suggesting this protein could be a useful biomarker of prolonged heat stress in Lb. casei GCRL163.

In this research, changes were observed in cell surface proteins of a typical strain of the Lactobacillus casei was investigated in response to acidic growth conditions. Two dimensional electrophoresis and Western blot analyses were carried out to detect changes in relative abundance of proteins at the cell surface. The identity of the differentially expressed proteins extracted by LiCl, a chemical routinely used to extract surface proteins of lactic acid bacteria, was determined by MALDI-TOF/TOF MS. Many enzymes involved in glycolysis were up-regulated in the cell surface fraction following growth at low pH, including enolase, lactate dehydrogenase, and glyceraldehyde-3-phosphate dehydrogenase. Several of these proteins were also related to adhesion and generalized stress responses. It is demonstrated that growth of L. casei under acidic conditions caused molecular changes at the cell surface to develop an adaptive strategy corresponding to slower growth at low pH.

Physiological and metabolic traits of Lactobacillus casei strain GCRL163 were studied in a buffered semi-defined medium supplemented with different concentrations of lactose. The extent of growth (maximum OD 600 reached) was limited by concentrations of ≤1% lactose in this medium. Survival of the strain during extended incubation periods (up to 30 days) in cultures without lactose was found to be better than in cultures supplemented with lactose. Important metabolites detected in lactose-starved (0% lactose) cultures were acetate, as a major end product, as well as ethanol, diacetyl, 3-methyl-1-butanol, benzyl alcohol, 1-butanol, isopropanol, 3-(methylthio)-propanoic acid and other compounds. Viability of recovered cells declined but metabolic products continued accumulating, when the pH was kept constant at pH 5.2. It was proposed that cells entered a non-culturable state but remained metabolically active. Results indicated the potential of this strain, as a possible non-starter lactic acid bacteria adjunct, to survive and utilize alternative available substrates as energy source in cheese curd.

To investigate impediments to plasmid transformation inBrevibacterium flavum BF4 andB. lactofermentum BL1, cell surface barriers were determined by measuring growth inhibition whilst enzymatic barriers were determined by comparing DNA methylation properties.B. lactofermentum was more sensitive to growth inhibition by glycine thanB. flavum. Release of cellular proteins during sonication was more rapid forB. lactofermentum than forB. flavum. Plasmid DNA (pCSL17) isolated fromB. flavum transformed recipient McrBC+ strains ofEscherichia coli with lower efficiency than McrBC−.McrBC digestion of this DNA confirmed thatB. flavum contain methylated cytidines in the target sequence ofMcrBC sequences butB. lactofermentum contained a different methylation pattern. DNA derived from theB. lactofermentum transformed recipient EcoKR+ strains ofE. coli with lower efficiency than EcoKR−, indicating the presence of methylated adenosines in the target sequence of EcoK sequences. The present data describe the differences in the physical and enzymatic barriers between two species of corynebacteria and also provide some insight into the successful foreign gene expression in corynebacteria.

The ability of Stenotrophomonas maltophilia strain VUN 10,003 to degrade and detoxify high molecular weight polycyclic aromatic hydrocarbons (PAHs) was evaluated in a basal liquid medium. Using high cell density inocula of strain VUN 10,003, the concentration of pyrene, fluoranthene, benz[a]anthracene, benzo[a]pyrene, dibenz[a, h]anthracene and coronene decreased by 98, 45, 26, 22, 22 and 55% over periods ranging from 5 to 42 d. When a PAH mixture containing three- to seven-ring compounds was used, degradation of both low and high molecular weight compounds occurred concurrently. Mutagenicity assays (Ames Test) demonstrated a decrease in the mutagenic potential of dichloromethane culture extracts from all cultures containing single PAH over the incubation period, corresponding to the decrease in the concentration of the PAH. These observations indicate that strain VUN 10,003 could be used for the detoxification of PAH-contaminated wastes.

Creosote was evaluated as an inexpensive carbon source for growing inocula of a polycyclic aromatic hydrocarbon (PAH)-degrading bacterial community (community five). Creosote was a poor growth substrate when provided as sole carbon source in a basal salts solution (BSM). Alternatively, peptone, yeast extract or glucose in BSM supported high growth rates, but community five could not subsequently degrade pyrene. A combination of creosote and yeast extract in BSM (CYEM) supported growth and maintained the pyrene-degrading capacity of community five. Optimum pyrene-degrading activity occurred when the inocula were grown in creosote and yeast extract concentrations of 2 ml L−1 and 1 g L−1 respectively: concentrations outside these values resulted in either low biomass yields or loss of PAH-degrading activity. CYEM-grown community five inocula degraded 250 mg L−1 of pyrene in BSM at a rate comparable to cultures inoculated with community five grown in BSM-pyrene. However, the CYEM-grown community showed a 40% lower rate of PAH degradation in a synthetic PAH mixture compared with pyrene-grown cells and there was an increase in the lag period before the onset of PAH degradation. This appears to reflect a weaker induction of PAH catabolism by CYEM compared to BSM-pyrene. Journal of Industrial Microbiology & Biotechnology (2000) 24, 277–284.

The objectives of this study were to isolate and evaluate microorganisms with the ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) in the presence of synthetic surfactants. Stenotrophomonas maltophilia VUN 10,010, isolated from PAH-contaminated soil, utilized pyrene as a sole carbon and energy source and also degraded other high molecular weight PAHs containing up to seven benzene rings. Various synthetic surfactants were tested for their ability to improve the PAH degradation rate of strain VUN 10,010. Anionic and cationic surfactants were highly toxic to this strain, and the Tween series was used as a growth substrate. Five nonionic surfactants (Brij 35, Igepal CA-630, Triton X-100, Tergitol NP-10, and Tyloxapol) were not utilized by, and were less toxic to, strain VUN 10,010. MSR and log Km values were determined for fluoranthene, pyrene, and benzo[a]pyrene in the presence of these nonionic surfactants and their apparent solubility was increased by a minimum of 250-fold in the presence of 10 g L−1 of all surfactants. The rate of pyrene degradation by strain VUN 10,010 was enhanced by the addition of four of the nonionic surfactants (5–10 g L−1); however, 5 g L−1 Igepal CA-630 inhibited pyrene degradation and microbial growth. The specific growth rate of VUN 10,010 on pyrene was increased by 67% in the presence of 10 g L−1 Brij 35 or Tergitol NP-10. The addition of Brij 35 and Tergitol NP-10 to media containing a single high molecular weight PAH (four and five benzene rings) as the sole carbon source increased the maximum specific PAH degradation rate and decreased the lag period normally seen for PAH degradation. The addition of Tergitol NP-10 to VUN 10,010 cultures which contained a PAH mixture (three to seven benzene rings) substantially improved the overall degradation rate of each PAH and increased the specific growth rate of VUN 10,010 by 30%. Evaluation of the use of VUN 10,010 for degrading high molecular weight PAHs in leachates from surfactant-flushed, weathered, PAH-contaminated sites is warranted. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 59:482–494, 1998.

Auxotrophic mutants of Corynebacterium glutamicum strain ATCC 13059 (parent of AS019, a rifampicin-resistant variant), which were morphologically distinct from the parent and formed protoplasts more readily, had been isolated previously. Mutants MLB130-133 and MLB194 were more sensitive to growth inhibition by isonicotinic acid hydrazide (INH) and glycine, which caused branching and budding. Fatty acid and mycolic acid (MA) profiles were determined after growth in LBG (Luria broth plus glucose), LBG-glycine (LBG- and LBG-INH (LBG-I). The fatty acid profiles of all strains were similar, except that mutant MLB133 showed some increase in stearic acid (C18:0), normally a minor component, late in the growth cycle and oleic acid proportionately decreased. All strains had five major types of MAs (C32:0, C34:0, C34:1, C36:1, C36:2) but the relative proportion of each varied with the strain, age of culture and medium composition. Mutants MLB133 and MLB194 showed slightly higher levels of non-covalentiy bound MAs than the parent and normally showed a higher proportion of longer-chained, unsaturated MAs. The proportion of extracellular MAs increased with culture age for these mutants. Typically, by late stationary phase, mycolic acids in culture fluids increased to 6.5% of the total MAs for MLB194 and 7.9% for MLB133 compared with 3.5% for the parent strain grown in LBG. The main effect of glycine (2%, w/v) addition was to increase the proportion of mycolic acids found in extracellular fluids (16.1 % for AS019 and 31% for MLB133). The most significant effects of INH were seen when strains were cultured in LBG with 8 mg INH ml−1. When harvested at late stationary phase, strains MLB133 and MLB194 had 18.8% and 21.2% extracellular mycolic acids respectively, with a significant increase in the relative proportion of unsaturated mycolic acids. This effect was not as marked for AS019, which also showed a similar decrease in C32:0 relative to increases in the proportion of C34:1 and C36:2 plus a corresponding increase in the overall proportion of unsaturated mycolic acids and increased extracellular mycolates (8.5%). These results suggest that the mutations in strains MLB133 and MLB194 are associated with synthesis of specific mycolic acids (e.g. C32:0) and attachment of mycolic acids to the cell surface, both of which are likely target sites for glycine and INH action for cell-surface modifications. In addition to previously reported targeting of the peptidoglycan cross-linking, these results show that glycine affects mycolic acid attachment to the cell surface of C. glutamicum.

Microbiological analysis of soils from a polycyclic aromatic hydrocarbon (PAH)-contaminated site resulted in the enrichment of five microbial communities capable of utilizing pyrene as a sole carbon and energy source. Communities 4 and 5 rapidly degraded a number of different PAH compounds. Three pure cultures were isolated from community 5 using a spray plate method with pyrene as the sole carbon source. The cultures were identified as strains of Burkholderia (Pseudomonas) cepacia on the basis of biochemical and growth tests. The pure cultures (VUN 10 001, VUN 10 002 and VUN 10 003) were capable of degrading fluorene, phenanthrene and pyrene (100 mg l−1) to undetectable levels within 7–10 d in standard serum bottle cultures. Pyrene degradation was observed at concentrations up to 1000 mg l−1. The three isolates were also able to degrade other PAHs including fluoranthene, benz[a]anthracene and dibenz[a,h]anthracene as sole carbon and energy sources. Stimulation of dibenz[a,h]anthracene and benzo[a]pyrene degradation was achieved by the addition of small quantities of phenanthrene to cultures containing these compounds. Substrate utilization tests revealed that these micro-organisms could also grow on n-alkanes, chlorinated- and nitro-aromatic compounds.

The global proteomic response of the nonstarter lactic acid bacteria Lactobacillus casei strain GCRL163 under carbohydrate depletion was investigated to understand aspects of its survival following cessation of fermentation. The proteome of L. casei GCRL163 was analyzed quantitatively after growth in modified MRS (with and without Tween 80) with different levels of lactose (0% lactose, starvation; 0.2% lactose, growth limiting; 1% lactose, non-growth-limited control) using gel-free proteomics. Results revealed that carbohydrate starvation lead to suppression of lactose and galactose catabolic pathways as well as pathways for nucleotide and protein synthesis. Enzymes of the glycolysis/gluconeogenesis pathway, amino acid synthesis, and pyruvate and citrate metabolism become more abundant as well as other carbohydrate catabolic pathways, suggesting increased optimization of intermediary metabolism and scavenging. Tween 80 did not affect growth yield; however, proteins related to fatty acid biosynthesis were repressed in the presence of Tween 80. The data suggest that L. casei adeptly switches to a scavenging mode, using both citrate and Tween 80, and efficiently adjusts energetic requirements when carbohydrate starved and thus can sustain survival for weeks to months. Explaining the adaptation of L. casei during lactose starvation will assist efforts to maintain viability of L. casei and extend its utility as a beneficial dietary adjunct and fermentation processing aid.

Photobacterium phosphoreum is a marine bacterium which is used extensively as a bioluminescent indicator of pollutants, where the presence of toxicants diminishes light output. To evaluate the utility of cell immobilisation in continuous toxicity testing, the sensitivity of P. phosphoreum to five gelling agents was evaluated relative to the retention of bioluminescence in 3% NaCl-glycerol suspensions. Following storage at 4°C, the control cultures retained light output for up to 2 weeks before significant decline; alginate-glycerol suspensions were stable for up to 4 weeks and bioluminescence was detectable for up to 6 weeks. Cells stored in agar were no more stable than the control, whereas cells gelled in agarose and low-melting point agarose showed a significant decline in bioluminescence within 2 weeks of storage. Bioluminescence was totally retained in alginate-glycerol suspensions stored at −80°C for up to 12 weeks. P. phosphoreum was successfully immobilised in strontium alginate and showed a dose-related response to four of the five heavy metal ions, SDS and pentachlorophenol tested when responses were followed over a time-course. A flow-through system for Sr-alginate immobilised cells was developed and conditions for operation were optimised. When cells were exposed to a pulse of 4-nitrophenol or salicylate then the nutrient feed continued, bioluminescence declined in response (pulse of 4–6 min) to these pollutants then recovered to a new stable rate of decline which was faster than the pre-exposure rate. These results demonstrate the potential of using immobilised P. phosphoreum in a continuous flow-through system for real-time environmental monitoring of water quality.

Plasmid DNA (pCSL17) isolated from Corynebacterium glutamicum transformed recipient McrBC+ strains of Escherichia coli with lower efficiency than McrBC- strains, confirming a previous report by Tauch et al. (FEMS Microbiol. Lett. 123 (1994) 343-348) which inferred that C. glutamicum DNA contains methylcytidine. Analysis of nucleotides in C. glutamicum-derived chromosomal and plasmid DNA failed to detect significant levels of methylated adenosine, but methylated cytidine was readily detected. Restriction enzymes which are inhibited by the presence of methylcytidine in their recognition sequence failed to cut pCSL17 from C. glutamicum, whereas enzymes which require methylation at adenosine in GATC sequences failed to cut. Failure of HaeIII to cut two specific sites of C. glutamicum-derived pCSL17 identified the first cytidine in the sequence GGCCGC as one target of methylation in this species, which contains the methyltransferase recognition sequence. Although Brevibacterium lactofermentum-derived DNA showed a similar methylation pattern by HPLC analysis, HaeIII cleaved these GGCCGC sites, suggesting differences in the specificity of methylation between these two species. Results for all analyses of B. flavum DNA were identical to those for C. glutamicum.

SUMMARY: A rapid and efficient method of transforming Corynebacterium glutamicum by electroporation is described. A number of factors are important in determining the level of transformation obtained. Cells grown in the presence of glycine and isonicotinic acid hydrazide and harvested in early exponential growth phase were much easier to transform. The recovery medium on which transformants were isolated also had a significant effect on the number of transformants obtained because cells were osmotically or electrochemically sensitive following electroporation. Transformation efficiencies of up to 5 x 105 transformants per μg plasmid DNA with homologously derived DNA and 2 x 103 transformants per μg of DNA derived from Escherichia coli were obtained.

Large inocula of Stenotrophomonas maltophilia VUN 10,003 were used to investigate bacterial degradation of benzo[a]pyrene and dibenz[a,h]anthracene. Although strain VUN 10,003 was capable of degrading 10-15 mg (-1) of the five-ring compounds in the presence of pyrene after 63 days, further addition of pyrene after degradation of the five-ring polycyclic aromatic hydrocarbons (PAHs) ceased did not stimulate significant decreases in the concentration of benzo[a]pyrene or dibenz[a,h]anthracene. However, pyrene was degraded to undetectable levels 21 days after its addition. The amount of benzo[a]pyrene and dibenz[a,h]anthracene degraded by strain VUN 10,003 was not affected by the initial concentration of the compounds when tested at 25-100 mg l(-1), by the accumulation of by-products from pyrene catabolism or a loss of ability by the cells to catabolise benzo[a]pyrene or dibenz[a,h]anthracene. Metabolite or by-product repression was suspected to be responsible for the inhibition: By-products from the degradation of the five-ring compounds inhibited their further degradation.

This study investigated the biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs) in liquid media and soil by bacteria ( Stenotrophomonas maltophilia VUN 10,010 and bacterial consortium VUN 10,009) and a fungus ( Penicillium janthinellum VUO 10,201) that were isolated from separate creosote- and manufactured-gas plant-contaminated soils. The bacteria could use pyrene as their sole carbon and energy source in a basal salts medium (BSM) and mineralized significant amounts of benzo[ a ]pyrene cometabolically when pyrene was also present in BSM. P. janthinellum VUO 10,201 could not utilize any high-molecular-weight PAH as sole carbon and energy source but could partially degrade these if cultured in a nutrient broth. Although small amounts of chrysene, benz[ a ]anthracene, benzo[ a ]pyrene, and dibenz[ a , h ]anthracene were degraded by axenic cultures of these isolates in BSM containing a single PAH, such conditions did not support significant microbial growth or PAH mineralization. However, significant degradation of, and microbial growth on, pyrene, chrysene, benz[ a ]anthracene, benzo[ a ]pyrene, and dibenz[ a , h ]anthracene, each as a single PAH in BSM, occurred when P. janthinellum VUO 10,201 and either bacterial consortium VUN 10,009 or S. maltophilia VUN 10,010 were combined in the one culture, i.e., fungal-bacterial cocultures: 25% of the benzo[ a ]pyrene was mineralized to CO 2 by these cocultures over 49 days, accompanied by transient accumulation and disappearance of intermediates detected by high-pressure liquid chromatography. Inoculation of fungal-bacterial cocultures into PAH-contaminated soil resulted in significantly improved degradation of high-molecular-weight PAHs, benzo[ a ]pyrene mineralization (53% of added [ 14 C]benzo[ a ]pyrene was recovered as 14 CO 2 in 100 days), and reduction in the mutagenicity of organic soil extracts, compared with the indigenous microbes and soil amended with only axenic inocula.

Twenty-five lead-tolerant strains of Paecilomyces lilacinus which had been isolated from a lead-contaminated site in Melbourne, Australia, were screened for lead biosorption capacity using biomass collected following culture in a basal salts medium supplemented with 0.5 gN l−1 as either ammonium or nitrate. All of these strains showed similar patterns of biosorption and lead uptake by dried biomass was always superior to freshly-harvested biomass and nitrate-grown biomass was superior to ammonium-grown biomass. Six strains were selected for detailed analysis of lead biosorption following growth in media with different C:N ratios and in lead solutions of pH between 3 and 7, comparing lead uptake for the lead-tolerant strains with the type strain of P. lilacinus (FRR 895), Aspergillus niger and Penicillium fumigatus. In comparison to the latter reference strains, all strains of P. lilacinus showed better biosorptive capacity following growth in media supplemented with high levels of nitrate and lead sorption was poor for all strains if biomass was exposed to lead in low pH solutions. Growth in high levels of ammonium (0.5 gNl−1) was accompanied by decreases in medium pH and relatively poor biosorption by dried biomass. Supplying low levels of ammonium (0.1 gN l−1) improved lead biosorption but controlling pH at 6–7 for media containing higher levels of ammonium improved the normally poor biosorption capacity. Biosorption isotherms for ammonium-grown biomass fitted the Freundlich model, but nitrate-grown biomass did not. Maximum biosorption values were 9–12% w/w and most of the lead-tolerant strains had higher Qmax values than the type strain, for nitrate-grown biomass. The lead-resistant strains could initiate growth in media containing 5,500 μg Pb ml−1, taking up lead into or onto cells (insensitive to EDTA treatment) early in growth, after which cell-associated lead declined as growth progressed. If lead was added later in growth, uptake was lower, suggesting that lead was excluded. Under the same growth conditions, the type strain FRR 895 was completely inhibited. Cells grown in lower concentrations of lead bound less lead but growth was still impaired.

A Burkholderia cepacia strain (VUN 10,001) isolated from manufacturing gas plant soil was selected for its ability to grow on and degrade pyrene as a sole source of carbon and energy. VUN 10,001 was able to grow on fluorene, phenanthrene benz[ a ]anthracene, fluoranthene and pyrene, but not benzo[ a ]pyrene and dibenz[ a , h ]anthracene when tested using inocula with low cell numbers. However, a small decrease in the concentrations of these latter compounds was observed. When media were inoculated at a high cell density, VUN 10,001 degraded the high molecular weight PAHs benzo[ a ]pyrene, dibenz[ a , h ]anthracene and coronene when these were present as individual substrates or as components of a complex mixture. Degradation of the high molecular weight PAHs was found to increase in the presence of the lower molecular weight compounds.

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous environmental pollutants which have been detected in numerous aquatic and terrestrial ecosystems (Cemiglia, 1992). There is concern about the presence of these compounds in the environment as they have been shown to exhibit toxic, mutagenic and carcinogenic effects (Mersch-Sundermann et al., 1992; Nylund et al., 1992). PAHs may enter the environment via many mechanisms; however, anthropogenic processes such as the combustion of fossil fuels, the accidental spilling of hydrocarbons and oils, coal gasification and liquefaction, wood treatment processes, open burning and the incineration of wastes are the major sources of PAHs in the environment (Guerin and Jones, 1988; Freeman and Cattell, 1990).

When inoculated at high cell densities, three strains of Pseudomonas cepacia degraded the polycyclic aromatic hydrocarbons (PAHs) benzo[a]pyrene, dibenz[a,h]anthracene and coronene as sole carbon and energy sources. After 63 days incubation, there was a 20 to 30% decrease in the concentration of benzo[a]pyrene and dibenz[a,h]anthracene and a 65 to 70% decrease in coronene concentration. The three strains were also able to degrade all the PAHs simultaneously in a PAH substrate mixture containing three-, four-, five- and seven-benzene ring compounds. Furthermore, improved degradation of the five- and seven-ring PAHs was observed when low molecular weight PAHs were present.

Cultured tissue slices from normal immature rabbit articular cartilage released latent neutral metal‐loproteinases into serum‐free medium. On activation with 4‐aminophenylmercuric acetate, these metalloproteinases could degrade collagen, proteoglycan, and gelatin. Also produced were an acid proteinase with the properties of cathepsin D and an inhibitor of the neutral metalloproteinases. The appearance of both the proteinases and the inhibitor in the culture medium could be prevented by incubation of cultures with cycloheximide. The active and latent forms of the proteinases were characterized using Ultrogel AcA 54 chromatography. Copyright © 1983 American College of Rheumatology

Cells from mid-exponential cultures of Brevibacterium lactofermentum were successfully transformed with pUL340 plasmid DNA using a standard (BioRad) electroporation protocol. However, growth in the presence of glycine plus tween 80 greatly increased the transformation frequencies and selecting transformants on an osmotically-protective medium resulted in further improvements. Under these optimal conditions, 4 × 107 transformants/μg DNA were isolated per 109 cells electroporated. Similar transformation frequencies were obtained for Corynebacterium glutamicum when tween 80 was added to the growth medium in addition to glycine and isonicotinic acid hydrazide, conditions which had been found previously to be essential for obtaining significant electrotransformation levels in this species.

Auxotrophic mutants of Corynebacterium glutamicum strain ATCC 13 059 showed considerable variation in their ability to form protoplasts when treated with lysozyme and ethylene-diaminetetraacetic acid (EDTA) following growth in nutrient medium containing up to 2% glycine. The proportion of protoplasts formed by the parent strain was normally 10 to 15% whereas certain amino-acid auxotrophs formed protoplasts at much higher frequencies (>95%). There was no obvious correlation between the presence of specific auxotrophic markers or the number of rounds of mutagenesis and protoplasting ability. Strains which were most readily protoplasted were morphologically distinct from other auxotrophs and the parent strain but were similar to the parent strain in their sensitivity to lysozyme. However, isoleucine auxotrophs were more sensitive to penicillin G. All strains produced osmotically sensitive cells (lysed by dilution in water) after growth in glycine-containing media and lysozyme-EDTA treatment. These damaged cells could be distinguished from true protoplasts by their ability to recover on osmotically non-protective media if diluted in high osmotic pressure buffers. Protoplasts were regenerated on an osmotically protective medium (ET) in 48 to 72 h, at frequencies averaging 50–60%.