Production of Surfactants by Rhodococcus erythropolis Strain EK-1, Grown on Hydrophilic and Hydrophobic Substrates

The ability of Rhodococcus erythropolis strain EK-1 to produce surfactants when grown on hydrophilic (ethanol and glucose) and hydrophobic (liquid paraffins and hexadecane) substrates was studied. The strain was found to produce surfactants with emulsifying and surface-active properties. The production of surfactants depended on the composition of the nutritive medium, nature and concentration of the sources of carbon and nitrogen, and duration of cultivation. Chemically, surfactants produced by Rhodococcus erythropolis EK-1 grown on ethanol are a complex of lipids with polysaccharide–proteinaceous substances. The lipids include glycolipids (trehalose mono- and dicorynomycolates) and common lipids (cetyl alcohol, palmitic acid, methyl n-pentadecanoate, triglycerides, and mycolic acids).     

Intracellular accumulation of mannosylerythritol lipids as storage materials by Candida antarctica

Summary Candida antarctica strain T-34, which was isolated as a biosurfactant producer, was found to produce organic acids and polyols extracellularly but not to produce biosurfactants, when grown on glucose or other carbohydrates as the sole carbon source. It was also observed microscopically that the strain contained oil globules within the cells. The intracellular lipids of the strain mainly consisted of triglycerides and mannosylerythritol lipids (MEL). The MEL content of the cells during the culture exceeded 10% of the dry cell weight, and the pattern of variation of the MEL content was very similar to that of triglycerides. All three stock strains of C. antarctica tested also accumulated a relatively large amount of MEL from glucose. These results suggested that these strains accumulated the MEL intracellularly as one of the storage materials together with triglycerides.Offprint requests to: D. Kitamoto     

Reproductive resting forms ofArthrobacter globiformis

Submerged cultures ofArthrobacter globiformis grown in media unbalanced with respect to carbon and nitrogen sources were found to contain cells exhibiting features typical of resting forms: long-term viability, specific ultrastructure, dormant metabolism, and thermoresistance. Such cells were produced not only in the collection strain VKM B-l 112, but also in the Aglobiformis strains isolated from 2-to 3-million-year-old permafrost sediments.     

Production of 2-ketobutyric acid from 1,2-butanediol by resting cells of Rhodococcus equi IFO 3730

Summary Various microorganisms were screened for their ability to produce 2-ketobutyric acid (2-KBA) from 1,2-butanediol (1,2-BD). Among them, Rhodococcus equi IFO 3730 was selected as the best strain. The various culture and reaction conditions were optimized using this strain. Limitation of thiamine in the growing medium was found to be effective. The resting cells of the strain grown on 1,2-propanediol as the carbon source yielded 15.7 g/l of 2-KBA from 20 g/l of 1,2-BD after 32 h incubation at 30 °C in the reaction mixture under optimal conditions.     

Discovery of Pseudozyma rugulosa NBRC 10877 as a novel producer of the glycolipid biosurfactants, mannosylerythritol lipids, based on rDNA sequence

The search for a novel producer of glycolipid biosurfactants, mannosylerythritol lipids (MEL) was undertaken based on the analysis of ribosomal DNA sequences on the yeast strains of the genus Pseudozyma. Pseudozyma rugulosa NBRC 10877 was found to produce a large amount of glycolipids from soybean oil. Fluorescence microscopic observation also demonstrated that the strain significantly accumulates polar lipids in the cells. The structure of the glycolipids produced by the strain was analyzed by ¹H and ¹³C nuclear magnetic resonance and gas chromatography–mass spectrometry methods, and was determined to be the same as MEL produced by Pseudozyma antarctica, a well-known MEL producer. The major fatty acids of the present MEL consisted of C8 and C10 acids. Based on high performance liquid chromatography, the composition of the produced MEL was as follows: MEL-A (68%), MEL-B (12%), and MEL-C (20%). To enhance the production of MEL by the novel strain, factors affecting the production, such as carbon and nitrogen sources, were further examined. Soybean oil and sodium nitrate were the best carbon and nitrogen sources, respectively. The supplementation of a MEL precursor, such as erythritol, drastically enhanced the production yield from soybean oil at a rate of 70 to 90%. Under the optimal conditions in a shake culture, a maximum yield, productivity, and yield coefficient (on a weight basis to soybean oil supplied) of 142 g l⁻¹, 5.0 g l⁻¹ day⁻¹, and 0.5 g g⁻¹ were achieved by intermittent feeding of soybean oil and erythritol using the yeast.     

Modification and Evolution of <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> for Enhanced Growth and Biotransformation Capabilities at Low Glucose Concentration

Gluconobacter oxydans is widely used in several biotechnological applications, where sorbitol or mannitol is commonly used as carbon source at high concentration. In this study, a membrane-bound glucose dehydrogenase-deficient strain (GDHK) was constructed to eliminate growth problems on glucose caused by direct oxidation of glucose in the medium. To achieve improved growth properties for the GDHK strain on glucose, a laboratory adaptive evolution experiment was performed with glucose as the sole carbon source. Results indicated evident, albeit modest, improvements in cell growth after a 50-day (about 430 generations) experimental evolution on glucose. The maximum specific growth rate and biomass yield of the resulting GDHE50 strain were increased around 1.35- to 1.4-fold compared with those of the GDHK strain. Meanwhile, two types of biotransformation reactions using resting cells of G. oxydans were investigated. Significant elevations in biotransformation performance of the GHDE50 strain were observed in comparison with that of the wild-type strain. In addition, resting cells of the GDHE50 strain grown on a relatively low concentration of glucose (10 g/l) could catalyze the biotransformation of glycerol to dihydroxyacetone and ethylene glycol to glycolic acid as efficient as the wild-type G. oxydans cultured on higher concentration of sorbitol or other carbon sources. These results suggest very favorable prospects of using glucose to lower production cost in many important industrial biocatalysis and biotransformation processes.     

Physiological differences in the formation of the glycolipid biosurfactants,mannosylerythritol lipids,between <Emphasis Type="Italic">Pseudozyma antarctica</Emphasis> and <Emphasis Type="Italic">Pseudozyma aphidis</Emphasis>

Vegetable oil is the usual carbon source for the production of biosurfactants (BS), mannosylerythritol lipids (MEL). To simplify the procedures of BS production and recovery, we investigated the extracellular production of MEL from water-soluble carbon sources instead of vegetable oils by using two representative yeast strains. The formation of extracellular MEL from glucose was confirmed by thin layer chromatography (TLC) and HPLC analysis. On glucose cultivation, pure MEL were easily prepared by only solvent extraction of the culture medium, different from the case of soybean oil cultivation. The fatty acid profile of the major MEL produced from glucose was similar to that produced from soybean oil based on GC–MS analysis. The resting cells of Pseudozyma antarctica T-34 produced MEL by feeding of glucose only and gave a yield of 12 g l⁻¹. In contrast, Pseudozyma aphidis ATCC 32657 gave no MEL from glucose. Moreover, the extracellular lipase activities were detected at high levels during the cultivation regardless of the carbon sources. These results indicate that all the biosynthesis pathways for MEL in P. antarctica T-34 should constitutively function. In conclusion, P. antarctica T-34 thus has potential for BS production from glucose.     

Production of enzymes and antimicrobial compounds by halophilic Antarctic <Emphasis Type="Italic">Nocardioides</Emphasis> sp. grown on different carbon sources

This study demonstrated the potential of microbial isolates from Antarctic soils to produce hydrolytic enzymes by using specific substrates. The results revealed potential of the strains to produce a broad spectrum of hydrolytic enzymes. Strain A-1 isolated from soil samples in Casey Station, Wilkes Land, was identified as Nocardioides sp. on the basis of morphological, biochemical, physiological observations and also chemotaxonomy analysis. Enzymatic and antimicrobial activities of the cell-free supernatants were explored after growth of strain A-1 in mineral salts medium supplemented with different carbon sources. It was found that the carbon sources favored the production of a broad spectrum of enzymes as well as compounds with antimicrobial activity against Gram-positive and Gram-negative bacteria, especially Staphylococcus aureus and Xanthomonas oryzae. Preliminary analysis showed that the compounds with antimicrobial activity produced by the strain A-1 are mainly glycolipids and/or lipopeptides depending on the used carbon source. The results revealed a great potential of the Antarctic Nocardioides sp. strain A-1 for biotechnological, biopharmaceutical and biocontrol applications as a source of industrially important enzymes and antimicrobial/antifungal compounds.     

Involvement of aeration in carbon source regulation of cephem antibiotic biosynthesis inStreptomyces clavuligerus

Summary The interference by glycerol and other carbon sources with production of cephem antibiotics by resting cells ofStreptomyces clawligerus was found to be related to aeration conditions. When a low cell density or increased aeration was used, carbon sources did not have any effect on the rate of production.     

A Novel Approach for Bioleaching of Sulfur,Iron, and Silica Impurities from Coal by Growing and Resting Cells of Rhodococcus spp

Coal is one of the most important sources of fossil energy on earth. However, direct combustion of coal with a high sulfur content can cause various environmental problems. Other constituents of coal that can cause environmental problems include iron oxide (hematite), iron hydroxide, and silica. In this study, growing and resting cells of Rhodococcus erythropolis strains PD1, R1, and FMF, and R. qingshengii were used in heterotrophic removal of sulfur and bioleaching of iron and silica from coal. All of the mentioned strains have an ability of dibenzothiophene (DBT) desulfurization via 4-S pathway. 2-hydroxybiphenyl, sulfate, and ferric ions (Fe³⁺) were assayed by Gibb’s test, barium chloride (BaCl₂), and thiocyanate ions (SCN^?), respectively. FTIR and XRF analyzer were used for detection of the coal bioleaching process by the selected strain of R. erythropolis (PD1). Results indicated that all strains have the ability to grow on coal as the sulfur source. Among them, strain PD1 produced the highest optical density and continued to grow even after 150-h incubation. In both growing- and resting-cells experiments, strain PD1 desulfurized coal most readily compared to other strains. Results of XRF showed that growing cells of strain PD1 had high desulfurizing ability of coal (46%) compared to resting cells in the absence of any carbon sources (24%). Growing cells of strain PD1 also leached 46% of the iron and 14% of the silicate after 7?days of incubation. Resting cells of PD1 leached 32% of the iron as determined by XRF analysis. Also, growing cells of PD1 removed most SiO₂ from coal as detected and confirmed by FTIR and XRF. To the best of our knowledge, this is the first report on bioleaching of iron and silica from coal by R. erythropolis strain PD1, making it a suitable candidate for coal bioremediation.     

Biodegradation of methyl parathion and <Emphasis Type="Italic">p</Emphasis>-nitrophenol: evidence for the presence of a <Emphasis Type="Italic">p</Emphasis>-nitrophenol 2-hydroxylase in a Gram-negative <Emphasis Type="Italic">Serratia</Emphasis> sp. strain DS001

A soil bacterium capable of utilizing methyl parathion as sole carbon and energy source was isolated by selective enrichment on minimal medium containing methyl parathion. The strain was identified as belonging to the genus Serratia based on a phylogram constructed using the complete sequence of the 16S rRNA. Serratia sp. strain DS001 utilized methyl parathion, p-nitrophenol, 4-nitrocatechol, and 1,2,4-benzenetriol as sole carbon and energy sources but could not grow using hydroquinone as a source of carbon. p-Nitrophenol and dimethylthiophosphoric acid were found to be the major degradation products of methyl parathion. Growth on p-nitrophenol led to release of stoichiometric amounts of nitrite and to the formation of 4-nitrocatechol and benzenetriol. When these catabolic intermediates of p-nitrophenol were added to resting cells of Serratia sp. strain DS001 oxygen consumption was detected whereas no oxygen consumption was apparent when hydroquinone was added to the resting cells suggesting that it is not part of the p-nitrophenol degradation pathway. Key enzymes involved in degradation of methyl parathion and in conversion of p-nitrophenol to 4-nitrocatechol, namely parathion hydrolase and p-nitrophenol hydroxylase component “A” were detected in the proteomes of the methyl parathion and p-nitrophenol grown cultures, respectively. These studies report for the first time the existence of a p-nitrophenol hydroxylase component “A”, typically found in Gram-positive bacteria, in a Gram-negative strain of the genus Serratia. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Production of 4,5-dihydro-4,5-dihydroxyphthalate from phthalate by a mutant strain of Pseudomonas testosteroni M4-1

Summary Pseudomonas testosteroni M4-1, capable of using phthalate as the sole carbon and energy source, was isolated. Tn5 mutagenesis using pSUP2021 yielded mutant strains of M4-1 that are defective in phthalate metabolism and produce a dihydrodiol compound. The dihydrodiol compound produced by mutant strain M4-122 was isolated and identified as 4,5-dihydro-4,5-dihydroxyphthalate (DDP) by elementary analysis, mass analysis and nuclear magnetic resonance. Various conditions to increase the yield of DDP from phthalate were examined for mutant strain M4-122. With resting cells 6 g DDP/1 were produced. The additional of ethanol to the resting-cell reaction mixture enhanced DDP production and 10 g DDP/1 was produced from 8.3 g/1 of phthalate. Offprint requests to: T. Omori     

Cellulase production on glucose-based media by the UV-irradiated mutants of Trichoderma reesei

From 22,791 mutants of a cellulase hyper-producing strain of Trichoderma reesei (Hypocrea jecorina), ATCC66589, as the parent, we selected two mutants, M2-1 and M3-1, that produce cellulases in media containing both cellulose and glucose. The mutation enabled the mutants to produce cellulases, which were measured as p-nitrophenyl β-d-lactopyranoside-hydrolyzing activities, in media with glucose as a sole carbon source, although M2-1 exhibited different sensitivities to glucose from M3-1. When the mutants were grown for 8 days on a medium with cellulose as a sole carbon source, the filter-paper-degrading activities (FPAs) per gram of cellulose were 257 and 281 U for M2-1 and M3-1, respectively, values that were 1.1–1.2 times higher than that of the parental strain. Cellulase production by M2-1 and M3-1 on a medium with a continuously fed mixture of glucose and cellobiose resulted in 214 and 210 U of FPA/gram carbon sources, respectively, whereas less efficient production (140 U of FPA/gram carbon source) was achieved by the parental strain. The improved cellulase productivity of the mutants allows us to use glucose as a carbon source for efficient on-site production of cellulases with quality/quantity-controlled feeding of soluble carbon sources and inducers.     

Cell wall peptidolipids of Mycobacterium avium: from genetic prediction to exact structure of a nonribosomal peptide

Mycobacteria have a complex cell wall structure that includes many lipids; however, even within a single subspecies of Mycobacterium avium these lipids can differ. Total lipids from an M. avium subsp. paratuberculosis (Map) ovine strain (S‐type) contained no identifiable glycopeptidolipids or lipopentapeptide (L5P), yet both lipids are present in other M. avium subspecies. We determined the genetic and phenotypic basis for this difference using sequence analysis as well as biochemical and physico‐chemical approaches. This strategy showed that a nonribosomal peptide synthase, encoded by mps1, contains three amino acid specifying modules in ovine strains, compared to five modules in bovine strains (C‐type). Sequence analysis predicted these modules would produce the tripeptide Phe‐N‐Methyl‐Val‐Ala with a lipid moiety, termed lipotripeptide (L3P). Comprehensive physico‐chemical analysis of Map S397 extracts confirmed the structural formula of the native L3P as D‐Phe‐N‐Methyl‐L‐Val‐L‐Ala‐OMe attached in N‐ter to a 20‐carbon fatty acid chain. These data demonstrate that S‐type strains, which are more adapted in sheep, produce a unique lipid. There is a dose‐dependent effect observed for L3P on upregulation of CD25+ CD8 T cells from infected cows, while L5P effects were static. In contrast, L5P demonstrated a significantly stronger induction of CD25+ B cells from infected animals compared to L3P.     

Media suitable for industrial production of Entomophthora virulenta zygospores

Entomophthora virulenta has been grown using inexpensive, commercial grade nutrient sources in order to produce a resting spore formulation available to agriculture. Solid media and liquid media in shake flasks and fermentors were examined. The best carbon sources for sporulation were dextrose and corn syrup, while the best nitrogen sources were yeast extract, soybean, and cottonseed flours. The optimal nutrient concentration (12%) produced 3 × 10⁶spores/ml with a 70% germination rate within five days.     

The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast

Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane. Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography. Their composition, after acid methanolysis, was analysed by gas-liquid chromatography. The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells. The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources. The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells. The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells. The ceramides contained an unusual long-chain base composition. In hexadecane-grown cells more than 60% of the long-chain bases were C₁₉ phytosphingosine. In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine. Received: 20 April 1998 / Received revision: 10 July 1998 / Accepted: 29 July 1998     

Overexpression of a diacylglycerol acyltransferase gene in Phaeodactylum tricornutum directs carbon towards lipid biosynthesis

Under nutrient deplete conditions, diatoms accumulate between 15% to 25% of their dry weight as lipids, primarily as triacylglycerols (TAGs). As in most eukaryotes, these organisms produce TAGs via the acyl‐CoA dependent Kennedy pathway. The last step in this pathway is catalyzed by diacylglycerol acyltransferase (DGAT) that acylates diacylglycerol (DAG) to produce TAG. To test our hypothesis that DGAT plays a major role in controlling the flux of carbon towards lipids, we overexpressed a specific type II DGAT gene, DGAT2D, in the model diatom Phaeodactylum tricornutum. The transformants had 50‐ to 100‐fold higher DGAT2D mRNA levels and the abundance of the enzyme increased 30‐ to 50‐fold. More important, these cells had a 2‐fold higher total lipid content and incorporated carbon into lipids more efficiently than the wild type (WT) while growing only 15% slower at light saturation. Based on a flux analysis using ¹³C as a tracer, we found that the increase in lipids was achieved via increased fluxes through pyruvate and acetyl‐CoA. Our results reveal overexpression of DAGT2D increases the flux of photosynthetically fixed carbon towards lipids, and leads to a higher lipid content than exponentially grown WT cells.     

Factors influencing the production of hydrogen by the purple non‐sulphur phototrophic bacterium Rhodopseudomonas acidophila KU001

Rhodopseudomonas acidophila KU001 was isolated from leather industry effluents and the effect of different cultural conditions on hydrogen production was studied. Anaerobic light induced more hydrogen production than anaerobic dark conditions. Growing cells produced more amounts of hydrogen between 96 and 144 h of incubation. Resting and growing cells preferred a pH of 6.0 ± 0.24 for hydrogen production. Succinate was the most preferred carbon source for the production of hydrogen while citrate was a poor source of carbon. Acetate and malate were also good carbon sources for hydrogen production under anaerobic light. Among the nitrogen sources, R. acidophila preferred ammonium chloride followed by urea for production of hydrogen_. L‐tyrosine was the least preferred nitrogen source by both growing and resting cells.     

Microbial metabolism of fluoranthene: isolation and identification of ring fission products

Summary The degradation of fluoranthene by pure cultures of Alcaligenes denitrificanss WW1, isolated from contaminated soil samples, was investigated. The strain showed maximum degradation rates of 0.3 mg fluoranthene/ml per day. A denitrificans was able to utilize also naphthalene, 1- and 2-methylnaphthalene, phenanthrene, and anthracene as sole carbon sources and to co-metabolize fuuorence, pyrene, and benzo(a)anthracene. During growth on fluoranthene in batch culture two metabolic products that were completely degraded before growth entered the stationary phase were detected in the culture fluid. Anslyses by UV, mass and NMR spectroscopy identified the products as acenaphthenone and 3-hydroxymethyl-4,5-benzocoumarine. Fluoranthene-grown resting cells of A. denitrificans showed degradative activity towards 2,3-dihydroxybenzoic acid, pyrogallol, salicylic acid, and catechol. The enzymatic activities in extracts of fluoranthene-induced cells indicate a meta ring fission involved in the degradation of fluoranthene. From these data new aspects of the biodegradative pathway of fluoranthene have been predicted.     

Substrate Specificity of Regiospecific Desaturation of Aliphatic Compounds by a Mutant Rhodococcus Strain

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.