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Properties of some enzymes involved in l-glutamine biosynthesis in an l-glutamine-producing mutant of Flavobacterium rigense were examined. Glutamate-oxaloacetate transaminase in the mutant was nearly at the same level as that in the parent strain and was the most active among the enzymes participating in glutamate biosynthesis from alpha-ketoglutarate. Glutamine synthetase formation in the mutant was enhanced by increasing the concentration of (NH(4))(2)-fumarate in the medium, but the activity of this enzyme in the parent strain was very low, and its formation was not influenced by the concentration of (NH(4))(2)-fumarate. Glutaminase formation by both strains was similar and was not influenced by the levels of (NH(4))(2)-fumarate. Glutaminase activity of the mutant was inhibited by ammonia and fumarate. Intracellular amino acids and extracellular free amino acids in the mutant were compared with those of the parent strain. It seems reasonable to conclude that l-glutamine leaks out specifically through the cell membrane of strain 703 and that this specific excretion of l-glutamine probably allows a continuous conversion of l-glutamate to l-glutamine inside the cell. |
Aureobasidium pullulans (de Bary) Arnaud isolated from the phylloplane of sycamore exposed to heavy atmospheric pollution oxidized S to S(2)O(3), S(4)O(6), and SO(4) in vitro. The intermediates S(2)O(3) and S(4)O(6) were also oxidized to SO(4). Cell-free extracts of A. pullulans also oxidized reduced forms of S, the oxidation increasing linearly with increasing protein concentration, showing that the process is enzymatic. The possible role of fungi in S oxidation in soils is discussed. |
Whole cells of Escherichia coli containing aspartase activity were immobilized by mixing a cell suspension with a liquid isocyanate-capped polyurethane prepolymer (Hypol). The immobilized cell preparation was used to convert ammonium fumarate to l-aspartic acid. Properties of the immobilized E. coli cells containing aspartase were investigated with a batch reactor. A 1.67-fold increase in the l-aspartic acid production rate was observed at 37 degrees C as compared to 25 degrees C operating temperature. The pH optimum was broad, ranging from 8.5 to 9.2. Increasing the concentration of ammonium fumarate to 1.5 M from 1.0 M negatively affected the reaction rate. l-Aspartic acid was produced at an average rate of 2.18 x 10 mol/min per g (wet weight) of immobilized E. coli cells with a 37 degrees C substrate solution consisting of 1.0 M ammonium fumarate with 1 mM Mg (pH 9.0). |
Bacteria associated with the marine wood-boring isopod Limnoria lignorum were enumerated by acridine orange epifluorescence microscopy and by plate counts on several media; the plate-viable bacteria were isolated and identified. Similar procedures were followed to enumerate and identify bacteria associated with the wood substrate from which the isopods were collected and with the surrounding water from the isopod habitat. Approximately 1.4 x 10 bacterial cells were associated with each individual L. lignorum. Aeromonas hydrophila, Pseudomonas, and Vibrio were the most common genera in the isopod microflora. Wood from L. lignorum burrows had an associated bacterial flora of 1.6 x 10 cells per mg (damp weight). A. hydrophila also predominated in the wood microflora. The water from which the isopod population was collected contained 2.3 x 10 bacteria per ml. Pseudomonas and Vibrio species were very common in the water microflora, but A. hydrophila was not detected. Interactions between the isopod, its associated microorganisms, and the microorganisms within the wood substrate are discussed in the light of the known absence of a resident digestive tract microflora in these animals. |
An enzymatic method using l-phenylalanine ammonia-lyase (EC 4.3.1.5) for the rapid conversion of trans-cinnamic acid to l-phenylalanine has been investigated. With Rhodotorula glutinis, enzyme activity as high as 0.3 U/ml of culture broth was obtained. The enzyme activity was kept stable for a relatively long time during cultivation by the addition of l-isoleucine. Optimization of the parameters of the conversion reaction resulted in accumulation of 18 mg of l-phenylalanine per ml of reaction mixture. The conversion yield from trans-cinnamic acid was about 70%. The method may provide a rapid and practical way to produce l-phenylalanine useful as an essential amino acid. |
A rumen isolate, Coprococcus, sp. Pe(1)5, was found to carry phloroglucinol reductase, which catalyzed the initial step in the breakdown of phloroglucinol. The organism uses phloroglucinol as the sole source of carbon and energy when grown in the absence of oxygen. Induced levels of enzyme were detected in cells grown either on phloroglucinol or on other carbon sources in the presence of limiting quantities of phloroglucinol. Although the organism is a strict anaerobe, the enzyme from anaerobically grown cells was insensitive to air. The partially purified enzyme required reduced nicotinamide adenine dinucleotide phosphate as an electron donor and was specific for phloroglucinol. However, partial enzyme activity (14 to 17%) was also detected in the presence of 2-methyl-1,4-naphthoquinone but not in the presence of several other phenolic compounds. The enzyme exhibited a higher affinity for phloroglucinol than for reduced nicotinamide adenine dinucleotide phosphate, with K(m) values of 3.0 x 10 M and 29.0 x 10 M, respectively. The optimum pH for maximal enzyme activity was 7.4, and the molecular weight of the native protein was about 130,000, as determined by the Sephadex gel filtration technique. |
The antibiotic protein synthesis inhibitor chloramphenicol specifically blocked the incorporation of [S]sulfate into the residue protein of two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus. Simultaneous inhibition of total protein synthesis occurred, but incorporation of S into low-molecular-weight organic compounds continued. A. luteo-violaceus rapidly autolyzed, with similar reduction in cell counts, total culture protein and cellular sulfur, whereas P. halodurans remained viable. Treatment with chloramphenicol, growth during nitrogen and carbon limitation, and the carbon and energy sources used for growth did not alter the sulfur content of P. halodurans protein. The mean value (1.09%, by weight), representing a wide variety of environmentally relevant growth conditions, was in agreement with model protein composition. The variability of cellular composition of P. halodurans and A. luteo-violaceus is discussed with respect to the measurement of bacterial growth in natural environments. Total carbon and nitrogen per cell varied greatly (coefficient of variation, ca. 100%) depending on growth conditions. Variation in total sulfur and protein per cell was much less (coefficient of variation, <50%), but the least variation was found for sulfate incorporation into residue protein (coefficient of variation, ca. 15%). Thus, sulfate incorporation into residue protein can be used as an accurate measurement of de novo protein synthesis in these bacteria. |
The sulfur content of residue protein was determined for pure cultures of Nitrosococcus oceanus, Desulfovibrio salexigens, 4 mixed populations of fermentative bacteria, 22 samples from mixed natural population enrichments, and 11 nutritionally and morphologically distinct isolates from enrichments of Sargasso Sea water. The average 1.09 +/- 0.14% (by weight) S in protein for 13 pure cultures agrees with the 1.1% calculated from average protein composition. An operational value encompassing all mixed population and pure culture measurements has a coefficient of variation of only 15.1% (n = 41). Short-term [S]sulfate incorporation kinetics by Pseudomonas halodurans and Alteromonas luteoviolaceus demonstrated a rapid appearance of S in the residue protein fraction which was well modelled by a simple exponential uptake equation. This indicates that little error in protein synthesis determination results from isotope dilution by endogenous pools of sulfur-containing compounds. Methionine effectively competed with sulfate for protein synthesis in P. halodurans at high concentrations (10 muM), but had much less influence at 1 muM. Cystine competed less effectively with sulfate, and glutathione did not detectably reduce sulfate-S incorporation into protein. [S]sulfate incorporation was compared with [C]glucose assimilation in a eutrophic brackish-water environment. Both tracers yielded similar results for the first 8 h of incubation, but a secondary growth phase was observed only with S. Redistribution of C from low-molecular-weight materials into residue protein indicated additional protein synthesis. [S]sulfate incorporation into residue protein by marine bacteria can be used to quantitatively measure bacterial protein synthesis in unenriched mixed populations of marine bacteria. |
Psychrotrophic Kanagawa-positive marine vibrios were isolated from soft-shelled clams (Mya arenaria) collected in Yaquina Bay, Oreg. The 235 vibrio isolates obtained were screened for Gram reaction and morphology, Kanagawa reaction on Wagastsuma agar, and response to selected biochemical tests. The vibrio selected for further study was grown in broth, and the hemolysin was precipitated from a cleared supernatant with solid ammonium sulfate. The hemolytic substance was partially purified by DEAE-cellulose and Sephadex G-100 column chromatography. The hemolysin contained protein essential for activity, was thermolabile, and was more active against rabbit erythrocytes at 37 degrees C than at lower temperatures. The molecular weight was estimated at 55,000 by using a Sephadex G-100 column. Hemolytic activity was partially inactivated by gangliosides and lowered against horse erythrocytes. The hemolysin did not react with antibody prepared against vibriolysin from Vibrio parahaemolyticus WP-1 by the Ouchterlony method. The hemolysin was high in aspartic and glutamic acids and low in arginine and histidine. Electrophoresis on a sodium dodecyl sulfate-polyacrylamide gel gave three major bands. The hemolysin from a psychrotrophic vibrio and the hemolytic exotoxin of V. parahaemolyticus had some similar and dissimilar characteristics. The possibility that a Vibrio sp. other than V. parahaemolyticus might serve as the reservoir for the Kanagawa phenotype is discussed. |
A method for the study of microbial biofilms in flowing-water systems was developed with special reference to the flow conditions in electrochemical concentration cells. Seawater was circulated in a semiclosed flow system through biofilm reactors (3 cm s) with microscope cover slips arranged in lamellar piles parallel with the flow. At fixed time intervals cover slips with their biofilm were removed from the pile, stained with crystal violet, and mounted on microscope slides. The absorbances of the slides were measured at 590 nm and plotted against time to give microbial biofilm development. From calibration experiments a staining time of 1 min and a rinse time of 10 min in a tap water flow (3 cm s) were considered sufficient. When an analysis of variance was performed on biofilm development data, 78% of the total variance was found to be due to random natural effects; the rest could be explained by experimental effects. The absorbance values correlated well with protein N, dry weight, and organic weight in two biofilm experiments, one with a biofilm with a high (75%) and one with a low ( approximately 25%, normal) inorganic content. Comparisons of regression lines revealed that the absorbance of the stained biofilms was an estimate closely related to biofilm dry weight. |
Two isolates of Neodiprion sertifer (Geoffr.) nuclear polyhedrosis virus from Britain and North America were compared using three biochemical techniques. Alkaline protease assays of polyhedra revealed the presence of endogenous enzyme activity in the British isolate but not in the North American isolate. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of virus particle structural polypeptides revealed only minor differences, with the exception that the North American virus was contaminated with polyhedral protein. The restriction endonucleases SalI, HindIII, and HpaII were used as a definitive method of distinguishing the two variants, with all endonucleases achieving this to a greater or lesser extent. The possible significance of all of these observations is discussed in terms of their possible influence on the registration and field application of this virus. |
The production of maltase, an inducible and repressible catabolic enzyme in Saccharomyces italicus, was studied and compared in batch, fed-batch, and continuous fermentations. Tight genetic controls on maltase synthesis limited the effect of environmental manipulations such as fed-batch or continuous culture in enhancement of maltase synthesis, and neither approach was able to improve the performance above the batch process for maltase production. S. italicus was mutated, and a constitutive producer of maltase was isolated. The mutant was detected by its ability to grow on sucrose, which is a noninducing substrate that is hydrolyzed by maltase; S. italicus does not possess invertase and will not normally grow on sucrose. Maltase production by this mutant was studied during growth on sucrose in batch and continuous cultures and marked improvement in enzyme productivity was observed. The specific activity of maltase produced by this mutant was more than twice that of the parent wild type: 2,210 and 1,370 U/g of cells for the mutant versus 890 and 510 U/g of cells for the wild type in batch and continuous cultures, respectively. Maltase specific productivity was increased from 74 to 288 U/g of cells per h by switching from batch growth of the wild type to continuous cultivation of the mutant. |
The osmotolerance of Saccharomyces rouxii 48-28 was confirmed with both NaCl- and KCl-fortified growth media, with more tolerance being exhibited for the potassium salt. Washed and buffered cells from unfortified medium were challenged with a variety of compounds (and also with physical treatments) that potentially would elicit membrane perturbations. The efficacy of these brief treatments was judged primarily by monitoring subsequent viability. Change in the degree of expression of beta-fructofuranosidase (EC 3.2.1.26), which is cryptic in young cells of S. rouxii, was a second criterion. There was a linear correlation between cell death and enzyme expression for treatments with polyenes, detergents, some organic solvents which did not denature the enzyme, and various freeze-thaw regimens in graded amounts of glycerol. The species is relatively insensitive to polyene antimycotics, the order of decreasing effect being filipin, nystatin, and amphotericin B. S. rouxii was found to be less sensitive to osmotic shock than is Saccharomyces cerevisiae, but in neither species is beta-fructofuranosidase released to the medium. The sensitivity of S. rouxii to ionic detergents, but not to nonionic detergents, was rationalized as being due to cell wall discrimination against larger micelles for the nonionic examples. This was confirmed by showing that protoplasts were sensitive to both classes. In cultures older than 5 days the normal agreement between colony-forming units and methylene blue exclusion (another test of viability) no longer held. Delayed fermentation of sucrose by S. rouxii, which is a diagnostic feature of the species, is explained by death of some cells, expression of their beta-fructofuranosidase, and utilization of the monosaccharides by the surviving cells. |
The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45 degrees to 47 degrees C, maximum protein yield at 45 degrees C, and no growth at 25 degrees C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45 degrees C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10 to 10 per animal) injected by various routes into normal and gamma-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections. |
Volvariella volvacea, commonly known as the straw or paddy mushroom, had the following growth characteristics: minimum temperature, 25 degrees C; optimal temperature, 37 degrees C; maximal temperature, 40 degrees C; pH optimum 6.0. Optimal pH for cellulase production was 5.5. The optimal initial pH for cellulase production and mycelial growth was found to be 6.0. The pH and temperature optima for cellulolytic activity were 5.0 and 50 degrees C, respectively. Maximal cellulolytic activity was obtained within 5 days in shake-flask culture. The cellulases were found to be partly cell free and partly cell bound during growth on microcrystalline cellulose. The endoglucanase activity was primarily extracellular, and beta-glucosidase activity was found exclusively extracellularly. Weak cellulase activity was detected when cells were grown on cellobiose and lactose. V. volvacea could not digest the lignin portion of newspaper in shake-flask cultivation. Phenol oxidase, an important enzyme in lignin biodegradation, also was lacking in the cell-free filtrate. However, the organism oxidized phenolic compounds when it was cultured on agar plates containing commercial lignin. |
Phytophthora palmivora, P. cinnamomi, P. drechsleri, and P. cactorum were readily separated on the basis of the aminopeptidase substrate specificities of their mycelial suspensions. Variability among isolates of the same species was not significant with most substrates and isolates tested, regardless of source. Both qualitative and quantitative differences in enzyme activity were useful for species identification. Differentiation of these four species was possible with comparative reactions of l-alanyl-, l-arginyl-, l-benzoylarginyl, l-gamma-glutamyl-, l-glycyl-, l-hydroxyprolyl-, l-leucyl-, l-lysyl-, 4-methoxyleucyl-, l-prolyl-, and 4-methoxyalanyl-beta-naphthylamides. Variation in peptidase activity was usually detectable after 4 h of incubation, with increased activity sometimes manifest after 24 h of incubation. P. palmivora exhibited the lowest, and P. drechsleri exhibited the highest, overall peptidase activity. This fluorescent aminopeptidase profile procedure provides a relatively rapid method to supplement other taxonomic criteria for identification of these species of Phytophthora. |
The bioconversion of waste paper to single-cell protein at pH <1 by Scytalidium acidophilum is described. Waste paper pretreated with 72% H(2)SO(4) at 4 degrees C was diluted with water to a pH of <0.1 and hydrolyzed. This yielded an adequate sugar-containing substrate for the growth of the fungus. A total of 97% of the sugars (glucose, galactose, mannose, xylose, arabinose) in the hydrolysates were converted to cell biomass. Microbial contamination was not observed. Based on the sugars consumed, S. acidophilum produced higher yields in shake cultures than many other Fungi Imperfecti. In aerated cultures, productivity increased, and yields of 43 to 46% containing 44 to 47% crude protein were obtained. This compares favorably with Candida utilis, a yeast used commercially to produce single-cell protein. The chemical constituents and the essential amino acids of the fungal cells were similar to those of other fungi. The nucleic acid content was characteristic of microbes containing low levels of nucleic acid. The advantages of using S. acidophilum for single-cell protein production are discussed. |
We investigated protease formation by Cephalosporium sp. strain KM388, which produced trypsin inhibitor in the same cultures, in medium containing polypeptone, meat extract, and glucose (natural medium) and in medium containing NaNO(3), glucose, and yeast extract (semisynthetic medium). In natural medium, protease was secreted into the culture broth after cessation of growth caused by consumption of the polypeptone, the growth-limiting substrate. Enzyme formation in the stationary growth phase was due to de novo and so-called preferential synthesis, because cycloheximide immediately inhibited enzyme formation. In semisynthetic medium, protease was produced in parallel with mycelial growth, but production was repressed by the addition of polypeptone to the medium; protease production began after the added polypeptone was consumed. On the other hand, if glucose was eliminated from natural medium, the lag period of initiation of enzyme production was reduced until the late exponential phase. The addition of phosphate up to a concentration of 1.0% to natural medium also shortened the lag period and damped the pH change of the broth during cultivation. |
Bacteria were isolated that could utilize quantitatively the s-triazine herbicide prometryne [N,N' -bis(1-methylethyl)-6-(methylthio)-1,3,5-triazine-2,4-diamine] or ametryne [N-ethyl-N'-(1-methylethyl)-6-(methylthio)-1,3,5-triazine- 2,4-diamine], or both, as a sole source of sulfur for growth. The success of enrichments depended on previous exposure of the soil inoculum to s-triazine herbicides. Deaminoethylametryne [4-(1-methylethyl)amino-6-(methylthio)-1,3,5-triazine-2-(1H)-one], methylsulfonic acid, and sodium sulfate could also be used as sulfur sources. Utilization of a compound was quantified as the growth yield per mole of sulfur supplied. Yields were about 6 kg of protein per mol of sulfur. The product of the desulfuration of an s-triazine was identified as the corresponding hydroxy-derivative. This is the first substantiated report of the utilization of these s-triazines as sulfur sources by bacteria. |
Dissimilatory reduction of NO(2) to N(2)O and NH(4) by a soil Citrobacter sp. was studied in an attempt to elucidate the physiological and ecological significance of N(2)O production by this mechanism. In batch cultures with defined media, NO(2) reduction to NH(4) was favored by high glucose and low NO(3) concentrations. Nitrous oxide production was greatest at high glucose and intermediate NO(3) concentrations. With succinate as the energy source, little or no NO(2) was reduced to NH(4) but N(2)O was produced. Resting cell suspensions reduced NO(2) simultaneously to N(2)O and free extracellular NH(4). Chloramphenicol prevented the induction of N(2)O-producing activity. The K(m) for NO(2) reduction to N(2)O was estimated to be 0.9 mM NO(2), yet the apparent K(m) for overall NO(2) reduction was considerably lower, no greater than 0.04 mM NO(2). Activities for N(2)O and NH(4) production increased markedly after depletion of NO(3) from the media. Amendment with NO(3) inhibited N(2)O and NH(4) production by molybdate-grown cells but not by tungstate-grown cells. Sulfite inhibited production of NH(4) but not of N(2)O. In a related experiment, three Escherichia coli mutants lacking NADH-dependent nitrite reductase produced N(2)O at rates equal to the wild type. These observations suggest that N(2)O is produced enzymatically but not by the same enzyme system responsible for dissimilatory reduction of NO(2) to NH(4). |
Microbial transformations of C-labeled substrates (sodium glutamate, Casamino Acids, glucose, and sodium acetate) were measured in undecompressed seawater samples collected from depths of 1,800 to 6,000 m, during 14- to 21-day incubation periods at in situ temperature (3 degrees C). Each substrate was tested at two concentrations (ca. 0.5 and 5.0 mug/ml) and two in situ pressures. The data were compared to 1-atmosphere (ca. 1.013 x 10 kPa) controls. The rates of C incorporation and CO(2) production as well as the amounts of total substrate utilization were generally lower at pressure than in the decompressed controls but were significantly different for each of the four substrates used. The utilization of acetate was the least affected by pressure; rates were similar to those measured at 1 atmosphere in two out of four experiments. In contrast, transformation rates of the amino acids at pressure averaged to only 38% of those in the controls. A single but reproducible "barophilic" response was observed with glucose as a substrate in samples collected from a depth of 4,500 m at a specific area in the northwestern Atlantic Ocean. Except for this latter set of experiments, the transformation of all substrates showed an increased lag period at pressure as compared to the 1-atmosphere controls. |
True cellulase activity has been demonstrated in cell-free preparations from the thermophilic anaerobe Clostridium thermocellum. Such activity depends upon the presence of Ca and a thiol-reducing agent of which dithiothreitol is the most promising. Under these conditions, native (cotton) and derived forms of cellulose (Avicel and filter paper) were all extensively solubilized at rates comparable with cellulase from Trichoderma reesei. Maximum activity of the Clostridium cellulase was displayed at 70 degrees C and at pH 5.7 and 6.1 on Avicel and carboxymethylcellulose, respectively. In the absence of substrate at temperatures up to 70 degrees C, carboxymethylcellulase was much more unstable than the Avicel-hydrolyzing activity. |
The rates of mineralization of phenol, benzoate, benzylamine, p-nitrophenol, and di(2-ethylhexyl) phthalate added to lake water at concentrations ranging from a few picograms to nanograms per milliliter were directly proportional to chemical concentration. The rates were still linear at levels of <1 pg of phenol or p-nitrophenol per ml, but it was less than the predicted value at 1.53 pg of 2,4-dichlorophenoxyacetate per ml. Mineralization of 2,4-dichlorophenoxyacetate was not detected in samples of lake water containing 200 ng of the chemical per ml. The slope of a plot of the rate of phenol mineralization in samples of three lakes as a function of its initial concentration was lower at levels of 1 to 100 mug/ml than at higher concentrations. In lake water and sewage supplemented with <60 ng of C-labeled benzoate or phenylacetate per ml, 95 to 99% of the radioactivity disappeared from solution, indicating that the microflora assimilated little or none of the carbon. The extent of mineralization of some compounds in samples of two lakes and sewage was least in the water with the lowest nutrient levels. No mineralization of 2,4-dichlorophenoxyacetate and the phthalate ester was observed in samples of an oligotrophic lake. These data suggest that mineralization of some chemicals at concentrations of <1 mug/ml is the result of activities of organisms different from those functioning at higher concentrations or of organisms that metabolize the chemicals at low concentrations but assimilate little or none of the substrate carbon. |
A sensitive and rapid method was developed to measure the mineralization of C-labeled organic compounds at picogram-per-milliliter or lower levels in samples of natural waters and sewage. Mineralization was considered to be equivalent to the loss of radioactivity from solutions. From 93 to 98% of benzoate, benzylamine, aniline, phenol, and 2,4-dichlorophenoxyacetate at one or more concentrations below 300 ng/ml was mineralized in samples of lake waters and sewage, indicating little or no incorporation of carbon into microbial cells. Assimilation of C by the cells mineralizing benzylamine in lake water was not detected. Mineralization in lake waters was linear with time for aniline at 5.7 pg to 500 ng/ml, benzylamine at 310 ng/ml, phenol at 102 fg to 10 mg/ml, 2,4-dichlorophenoxyacetate at 1.5 pg/ml, and di-(2-ethylhexyl) phthalate at 21 pg to 200 ng/ml, but it was exponential at several p-nitrophenol concentrations. The rate of mineralization of 50 and 500 ng of aniline per ml and 200 pg and 2.0 ng of the phthalate per ml increased with time in lake waters. The phthalate and 2,4-dichlorophenoxyacetate were mineralized in samples from a eutrophic but not an oligotrophic lake. Addition to eutrophic lake water of a benzoate-utilizing bacterium did not increase the rate of benzoate mineralization at 34 and 350 pg/ml but did so at 5 and 50 ng/ml. Glucose and phenol reduced the percentage of p-nitrophenol mineralized at p-nitrophenol concentrations of 200 ng/ml but not at 22.6 pg/ml and inhibited the rates of mineralization at both concentrations. These results show that the kinetics of mineralization, the capacity of the aquatic community to assimilate carbon from the substrate or the extent of assimilation, and the sensitivity of the mineralizing populations to organic compounds are different at trace levels than at higher concentrations of organic compounds. |
Plate (or slope) cultures of endomycin-producing Streptomyces endus (KCC S-0213) showed spontaneously developing pocks which increased in number during subculturing. Neither spore formation nor typical aerial hyphae formation was observed in the pocks, whereas formation of substrate hyphase was not inhibited. Almost all of the hyphae were broken or lysed in the pocks, and many phage tail tiplike particles were observed in the pocks. No self-replication activity was associated with the particles. The particles often formed a hexagonal crystal or a large crystal mass. The production of these particles did not occur in the liquid culture or in young or normal plate cultures having no pocks. These results were similar to those obtained from the plaque-making phenomenon, except for active phage production, in thiostrepton-producing Streptomyces azureus (ATCC 14921), which has been described previously. |
The contribution of attached and free-floating bacteria to the bacterial numbers and heterotrophic uptake in 44 diverse aquatic environments was studied. A factor analysis reduced the variability of the raw data base to three major factors explaining 53.6% of total variance. These factors were (i) salinity, (ii) heterotrophic uptake, and (iii) particulate load. A cluster analysis categorized the 44 habitats into five distinct environmental types based on these three factors. There was no significant pattern in the distribution of attached versus free-floating bacteria when assessed by epifluorescent microscopy. However the contribution of attached bacteria to the uptake of an amino acids mix was reduced in marine waters. Heavy particulate loads resulted in an increased percentage uptake of amino acids and glucose from the attached bacteria. Uptake response was found to be substrate specific especially in oliogotrophic freshwater. Amino acid uptake was more associated with the attached fraction, whereas glucose uptake was mediated more by the free-floating fraction. |
Cleared lysates of a proteolytic (Prt) strain and a naturally occurring non-proteolytic (Prt) variant of Streptococcus cremoris Wg2 contain equal amounts of covalently closed circular plasmid DNA. An analysis of this plasmid DNA by agarose gel electrophoresis revealed the presence of at least five different plasmid species in the Prt strain and only three plasmid species in the Prt variant. Curing studies with acriflavine indicated that a 16-megadalton plasmid determined proteolytic activity in the Prt strain. In energy-limited chemostats inoculated with both strains it was observed that the Prt strain was replaced by the Prt variant. This effect was most apparent when the pH of the culture was fixed at a value above 6.3. No selection for the Prt variant was observed at pH 5.9. Since the two types of organisms contain equal amounts of plasmid DNA, it was concluded that the energy gain of the Prt variants at pH values above 6.0 probably has to be found in protein synthesis rather than in plasmid DNA synthesis. |
Hyphal budding bacteria were observed by electron microscopy in thin sections of surface material from Baltic Sea ferromanganese concretions. Similar bacteria were also observed in and isolated from enrichment cultures prepared from the same concretion material. Three morphologically similar strains of Mn-Fe-depositing budding bacteria were isolated from the enrichment cultures. Strain B-4 possessed extracellular anionic polymers that accumulated Mn oxides. Mn deposition by B-4 was inhibited by elevated concentrations of Mn, 0.05% glutaraldehyde, 0.1 mM HgCl(2), and heating at 93 degrees C for 15 min, suggesting the participation of an enzyme protein in the Mn-depositing activity. |
A bubble contact angle method was used to determine interfacial free-energy characteristics of polystyrene substrata in the presence and absence of potential surface-conditioning proteins (bovine glycoprotein, bovine serum albumin, fatty acid-free bovine serum albumin), a bacterial culture supernatant, and a bacterial exopolymer. Clean petri dish substrata gave a contact angle of 90 degrees , but tissue culture dish substrata were more hydrophilic, giving an angle of 29 degrees or less. Bubble contact angles at the surfaces exposed to the macromolecular solutions varied with the composition and concentration of the solution. Modification by pronase enzymes of the conditioning effect of proteins depended on the nature of both the substratum and the protein, as well as the time of addition of the enzyme relative to the conditioning of the substratum. The effects of dissolved and substratum-adsorbed proteins on the attachment of Pseudomonas sp. strain NCMB 2021 to petri dishes and tissue culture dishes were consistent with changes in bubble contact angles (except when proteins were adsorbed to tissue culture dishes before attachment) as were alterations in protein-induced inhibition of bacterial attachment to petri dishes by treatment with pronase. Differences between the attachment of pseudomonads to petri dishes and tissue culture dishes suggested that different mechanisms of adhesion are involved at the surfaces of these two substrata. |
We report that two species of basidiomycete fungi (Polyporus versicolor and Poria monticola) grow in minimal liquid or solid medium when supplemented with crushed lignite coal. The fungi also grow directly on crushed lignite coal. The growth of both fungi was observed qualitatively as the production and extension of hyphae. No fungal growth occurred in minimal agar medium without coal. The fungi degraded solid lignite coal to a black liquid product which never appeared in cultures unless fungi and coal were present together. Apparently, lignite coal can serve as the principal substrate for the growth of the fungi. Infrared analyses of the liquid products of lignite degradation showed both similarities to and differences from the original lignite. |
Pyruvic acid and O-acetyl groups are the major noncarbohydrate substituents in exopolysaccharides (EPS) produced by fast-growing species of Rhizobium. EPS substituent variations were observed among strains of the same species. The amounts of these substituents also varied with culture age; pyruvic acid increased in the EPS of all four species, whereas O-acetyl increased in Rhizobium trifolii and R. leguminosarum EPS, decreased in R. meliloti EPS, and remained constant in R. phaseoli EPS. The use of glycerol as a substrate for R. meliloti significantly increased EPS yields, whereas mannitol increased those of the other three Rhizobium species. |
Strains tentatively identified as Bacillus sp. were isolated from sewage sludge and soil and shown to elaborate extracellular enzymes that degrade the extracellular polysaccharide (xanthan gum, polysaccharide B-1459) of Xanthomonas campestris NRRL B-1459. Enzyme production by one strain was greatly enhanced when the strain was incubated in a mixed culture. Products of degradation were identified as d-glucuronic acid, d-mannose, pyruvylated mannose, 6-O-acetyl d-mannose, and a (1-->4)-linked glucan. These products correlate with the known structure of the gum. The complexity of the product mixture indicated that the xanthanase was a mixture of carbohydrases. The xanthanase complexes were similar to one another in temperature stability, pH and temperature optima, degree of substrate degradation, and enzymolysis products. Differences in pH stability, salt tolerance, recoverability, and yields of enzyme were observed. |
I examined the role of aerobic microbial populations in cellulose digestion by two sympatric species of desert millipedes, Orthoporus ornatus and Comanchelus sp. High numbers of bacteria able to grow on media containing cellulose, carboxymethyl cellulose, or cellobiose as the substrate were found in the alimentary tracts of the millipedes. Enzyme assays indicated that most cellulose and hemicellulose degradation occurred in the midgut, whereas the hindgut was an important site for pectin degradation. Hemicellulase and beta-glucosidase in both species and possibly C(x)-cellulase and pectinase in O. ornatus were of possible microbial origin. Degradation of [C]cellulose by millipedes whose gut floras were reduced by antibiotic treatment and starvation demonstrated a reduction in CO(2) release and C assimilation and an increase in C excretion over values for controls. It appears that the millipede-bacterium association is mutualistic and makes available to millipedes an otherwise mostly unutilizable substrate. Such an association may be an important pathway for decomposition in desert ecosystems. |
The kinetics of formate metabolism in Methanobacterium formicicum and Methanospirillum hungatei were studied with log-phase formate-grown cultures. The progress of formate degradation was followed by the formyltetrahydrofolate synthetase assay for formate and fitted to the integrated form of the Michaelis-Menten equation. The K(m) and V(max) values for Methanobacterium formicicum were 0.58 mM formate and 0.037 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanobacterium formicicum was 26 muM. The K(m) and V(max) values for Methanospirillum hungatei were 0.22 mM and 0.044 mol of formate h g (dry weight), respectively. The lowest concentration of formate metabolized by Methanospirillum hungatei was 15 muM. The apparent K(m) for formate by formate dehydrogenase in cell-free extracts of Methanospirillum hungatei was 0.11 mM. The K(m) for H(2) uptake by cultures of Methanobacterium formicicum was 6 muM dissolved H(2). Formate and H(2) were equivalent electron donors for methanogenesis when both substrates were above saturation; however, H(2) uptake was severely depressed when formate was above saturation and the dissolved H(2) was below 6 muM. Formate-grown cultures of Methanobacterium formicicum that were substrate limited for 57 h showed an immediate increase in growth and methanogenesis when formate was added to above saturation. |
Mutants of an industrial-type strain of Saccharomyces cerevisiae which rapidly and completely fermented equimolar mixtures of glucose and galactose to ethanol were isolated. These mutants fell into two general phenotypic classes based upon their fermentation kinetics and enzyme induction patterns. One class apparently specifically effects the utilization of galactose and allows sequential utilization of first glucose and then galactose in an anaerobic fermentation. The second class of mutants was resistant to general catabolite repression and produced maltase, invertase, and galactokinase in the presence of repressive levels of glucose. These mutants were completely dominant and appear to represent an as yet undescribed class of mutant. |
Montmorillonite-benzylamine complexes were formed immediately upon addition of 20 pg to 20 mug of amine per ml of suspensions containing the clay. The extent of amine sorbed was a linear function of equilibrium amine concentration in lake water. Increases in the clay concentration decreased the percentage of the organic compound that was mineralized at amine levels of 20 pg to 200 ng, but not at 20 mug/ml. A larger percentage of the chemical was released from the complex during mineralization in the presence of high clay concentrations than in the presence of low clay concentrations. The rates of desorption and mineralization increased linearly with benzylamine levels up to 200 ng/ml. Montmorillonite did not enhance mineralization rates at amine levels of 200 ng/ml or lower, but it was stimulatory at 20 mug/ml. Except at high amine and clay concentrations, mineralization was more rapid than desorption during the early periods of decomposition when the amine concentration in solution was relatively high. However, relative to the microbial demand, desorption was more rapid during later periods of decomposition when the amine level in solution was very low. Mineralization of benzoate was not usually affected by montmorillonite, kaolinite, or glass beads. More than 90% of the carbon from benzylamine and benzoate was often mineralized when the substrate concentration was 250 ng/ml or less. After incubation of the chemical in lake water, none of the radioactivity from benzylamine was in the particulate fraction containing natural sediment and microbial cells. The data indicate that clay may have a significant effect on the microbial decomposition of low concentrations of certain organic compounds. |
Propionibacterium shermanii and Lactobacillus acidophilus were grown in batch mixed culture in a 5-liter fermenter under controlled conditions of pH 5.8 and 35 degrees C on a semisynthetic medium with glucose as an energy source. Cellular efficiencies and fermentation balances were developed for this pair and compared with P. shermanii grown in pure culture on glucose, lactate, and a mixture of these substrates and with L. acidophilus grown on glucose. P. shermanii had ATP yield coefficient values of 17 for each substrate alone but had an average value of 30 for substrate mixtures. Growth rates were similar for P. shermanii on glucose or lactate but higher cell yields were observed for glucose. P. shermanii used both lactate and glucose in mixed substrate until lactate was exhausted, and growth rates slowed thereafter. L. acidophilus had a similar ATP yield coefficient of 15 but produced lower cell yields than did P. shermanii on glucose. Mixed culture of both microorganisms on glucose resulted in much faster and nearly equal growth rates for both and no lactate accumulation in the medium. Acetic acid production rates per generation were lower in mixed culture, suggesting use by the growing culture. The cause of the synergistic effect was not determined but may be due to the rapid production and removal of lactate or CO(2) enhancement in mixed culture. |
A new starch hydrolysis detection method which does not rely on iodine staining or the use of color-complexed starch is described. A linear relationship was obtained with agar-starch plates when net clearing zones around colonies of yeasts were plotted against enzyme levels (semilogarithm scale) produced by the same yeast strains in liquid medium. A similar relationship between starch clearing zones and alpha-amylase levels from three different sources was observed. These observations suggest that the method is useful in mutant isolations, strain improvement programs, and the prediction of alpha-amylase activities in culture filtrates or column effluents. |
The bioconversion of sugars present in wood hemicellulose to 2,3-butanediol (hereafter referred to as butanediol) by Klebsiella pneumoniae grown on high initial concentrations (up to 10%) of sugars was investigated. Initial fermentation studies with a chemically defined medium suggested that sugar levels in excess of 2% could not be utlized even when a higher inoculum size (5 to 10%) was used. The addition of nutrient supplements, viz., yeast extract, urea, ammonium sulfate, and trace elements resulted in a 10 to 50% increase in butanediol yields, although sugar utilization remained incomplete. The concentration of end products normally found at the termination of fermentation was shown to be noninhibitory to growth and substrate utilization. Acetic acid was inhibitory at concentrations above 1%, although growth and butanediol yield were stimulated in cultures supplemented with lower levels of acetic acid. The efficient utilization of 4% substrate concentrations of d-glucose and d-xylose was achieved, resulting in butanediol yields of 19.6 and 22.0 g/liter, respectively. |
Under nitrogen (ammonia)-limited continuous culture conditions, the ruminal anaerobe Selenomonas ruminantium was grown at various dilution rates (D). The proportion of the population that was viable increased with D, being 91% at D = 0.5 h. Washed cell suspensions were subjected to long-term nutrient starvation at 39 degrees C. All populations exhibited logarithmic linear declines in viability that were related to the growth rate. Cells grown at D = 0.05, 0.20, and 0.50 lost about 50% viability after 8.1, 4.6, and 3.6 h, respectively. The linear rates of decline in total cell numbers were dramatically less and constant regardless of dilution rate. All major cell constituents declined during starvation, with the rates of decline being greatest with RNA, followed by DNA, carbohydrate, cell dry weight, and protein. The rates of RNA loss increased with cells grown at higher D values, whereas the opposite was observed for rates of carbohydrate losses. The majority of the degraded RNA was not catabolized but was excreted into the suspending buffer. At all D values, S. ruminantium produced mainly lactate and lesser amounts of acetate, propionate, and succinate during growth. With starvation, only small amounts of acetate were produced. Addition of glucose, vitamins, or both to the suspending buffer or starvation in the spent culture medium resulted in greater losses of viability than in buffer alone. Examination of extracts made from starving cells indicated that fructose diphosphate aldolase and lactate dehydrogenase activities remained relatively constant. Both urease and glutamate dehydrogenase activities declined gradually during starvation, whereas glutamine synthetase activity increased slightly. The data indicate that nitrogen (ammonia)-limited S. ruminantium cells have limited survival capacity, but this capacity is greater than that found previously with energy (glucose)-limited cells. Apparently no one cellular constituent serves as a catabolic substrate for endogenous metabolism. Relative to losses in viability, cellular enzymes are stable, indicating that nonviable cells maintain potential metabolic activity and that generalized, nonspecific enzyme degradation is not a major factor contributing to viability loss. |
Metabolic characteristics of a heterotrophic, nitrifier-denitrifier Alcaligenes sp. isolated from soil were further characterized. Pyruvic oxime and hydroxylamine were oxidized to nitrite aerobically by nitrification-adapted cells with specific activities (V(max)) of 0.066 and 0.003 mumol of N x min x mg of protein, respectively, at 22 degrees C. K(m) values were 15 and 42 muM for pyruvic oxime and hydroxylamine, respectively. The greater pyruvic oxime oxidation activity relative to hydroxylamine oxidation activity indicates that pyruvic oxime was a specific substrate and was not oxidized appreciably via its hydrolysis product, hydroxylamine. When grown as a denitrifier on nitrate, the bacterium could not aerobically oxidize pyruvic oxime or hydroxylamine to nitrite. However, hydroxylamine was converted to nearly equimolar amounts of ammonium ion and nitrous oxide, and the nature of this reaction is discussed. Cells grown as heterotrophic nitrifiers on pyruvic oxime contained two enzymes of denitrification, nitrate reductase and nitric oxide reductase. The nitrate reductase was the dissimilatory type, as evidenced by its extreme sensitivity to inhibition by azide and by its ability to be reversibly inhibited by oxygen. Cells grown aerobically on organic carbon sources other than pyruvic oxime contained none of the denitrifying enzymes surveyed but were able to oxidize pyruvic oxime to nitrite and reduce hydroxylamine to ammonium ion. |
Comparative studies of the fermentation of cane molasses into ethanol by Saccharomyces cerevisiae in the presence or absence of fungal invertase were performed. When cane molasses was fermented by the yeast at 30 degrees C and pH 5.0, the presence of the enzyme had no effect on ethanol production. At pH 3.5, ethanol production was increased by the addition of invertase. At 40 degrees C, the addition of invertase increased ethanol production by 5.5% at pH 5.0 and by 20.9% at pH 3.5. |
In the course of exploring new microbial sources of extracellular beta-d-galactosidase (EC. 3.2.1.23), Alternaria alternata was found to excrete elevated quantities of a thermostable form of the enzyme when cultivated in whey growth medium. Optimum cultural conditions for maximum enzyme production were a whey lactose concentration of 6%, supplementation of the medium with 0.050 M (NH(4))(2)SO(4), an inoculum size of 10 conidia per ml, and a cultivation time at 28 to 30 degrees C of 5 days. The fungus utilized whey lactose for the production of the enzyme most efficiently, and the observed maximum yield, 280 nanokatals of hydrolyzed o-nitrophenyl-beta-d-galactopyranoside per g of whey lactose, was comparable to maximum yields reported for certain commercial fungi. The optimum pH and temperature of the enzymatic reaction were 4.5 to 5.5 and 60 to 70 degrees C, respectively, and the enzyme lost half of its activity when heated at 65 degrees C for 84 min. These properties make the enzyme particularly suitable for processing acid and less-acid (pH 5 to 6) dairy products and by-products. |
Five strains of Rhizobium trifolii were evaluated in competition with indigenous populations in nodulating red clover (Trifolium pratense L.) cv. Kenland in two different soils in Mississippi. Double antibiotic resistance acquisition was used to measure the proportion of nodules occupied by the introduced mutant strains. In vertisol soil, strains RP113-7, 162BB1, LM1, and 162P17 were recovered in at least 94% of the assayed nodules, whereas TA1 was found in 83.8% of the nodules. At an ultisol location, significant differences were detected within the introduced rhizobia. Strain RP113-7 was recovered at very high rates (99.2% of the assayed nodules), whereas strains 162BB1, LM1, 162P17, and TA1 were all found in 84.9 to 96.0% of the nodules sampled. Forage yield and percent crude protein levels were lower with the less effective but competitive strain (TA1) at both locations. Results indicated that more effective strains of R. trifolii can increase red clover production and symbiotic nitrogen fixation under different environmental conditions in Mississippi. |
The functional response to and recovery from coal-coking waste effluent was evaluated for sediment microbial communities. Twenty estimates of microbial population density, biomass, and activity were measured five times during a 15-month period. Significant effects on microbial communities were observed in response to both wastewater contamination and diversion of the wastewater. Multivariate analysis of variance and discriminant analysis indicated that accurate differentiation between uncontaminated and contaminated sediments required a minimum of nine estimates of community response. Total viable population density, ATP, alkaline phosphatase, naphthalene, and phenanthrene mineralization rates were found to be highly weighted variables in site discrimination. Lipid and glucose mineralization, nitrogen fixation, and sediment protein also contributed significantly to explaining variation among sites. Estimates of anaerobic population densities and rates of methane production contributed little to discrimination among sites in the environment examined. In general, total viable population density, ATP, and alkaline phosphatase activity were significantly depressed in contaminated sediments. However, after removal of this contamination, the previously affected sites demonstrated greater temporal variability but a closer approximation of the mean response at the control site. Naphthalene and phenanthrene mineralization did not follow the general trend and were elevated at the contaminated sites throughout the investigation. Results of the investigation supported the hypothesis that multiple functional measures of microbial community response are required to evaluate the effect of and recovery from environmental contamination. In addition, when long-term effects are evaluated, select physiological traits, i.e., polyaromatic hydrocarbon mineralization, may not reflect population and biomass estimates of community response. |
A strain of the starch-converting yeast Lipomyces kononenkoae produced, when grown on starch, a debranching enzyme that proved to be an isoamylase (glycogen 6-glucanohydrolase; E.C. 3.2.1.68). So far, only bacteria have been found to produce extracellular isoamylases. The yeast isoamylase enhanced beta-amylolysis of amylopectin and glycogen and completely hydrolyzed these substrates into maltose when combined with a beta-amylase but had no action on dextran or pullulan. By isopropanol precipitation and carboxymethyl cellulose chromatography, L. kononenkoae isoamylase was partially purified from the supernatant of cultures grown on a mineral medium with soluble starch. Optimum temperature and pH for activity of the isoamylase were 30 degrees C and 5.6. The molecular weight was around 65,000, and the pI was at pH 4.7 to 4.8. The K(m) (30 degrees C, pH 5.5) for soluble starch was 9 g liter. |
Selective enrichment of yellow-orange-pigmented, gram-negative bacteria related to Cytophaga johnsonae from lake sediment was dependent on low temperatures (ca. 5 degrees C). However, this temperature effect was abolished when excessive amounts of dissolved organic carbon (10 mM N-acetylglucosamine) were added. A psychrotrophic freshwater isolate of C. johnsonae was used to study the physiological versatility of this group. Exponential growth rates were found to be dependent on the temperature to which the cells used as the inocula were acclimated. Glucose incorporation and respiration were also dependent upon the acclimation temperature of the inocula. Patterns of CO(2) evolution obtained from position-labeled [C]glucose indicated that glucose was predominantly metabolized via the Embden-Meyerhof-Parnas pathway, which, however, was greatly reduced at 25 degrees C when the concentration of glucose was as low as 5 muM/liter. Transport, respiration, and incorporation of glucose (0.2- to 20,000-muM/liter concentrations) into macromolecular cellular compounds were characterized by multiple K(m) values which were a function of substrate concentration and temperature. It appeared possible that these multiple K(m) values reflected the changing participation of the Embden-Meyerhof-Parnas pathway in glucose metabolism. These results may provide a physiological explanation for the selective enrichment of psychrotrophic freshwater cytophagas. Moreover, they exhibit the limits of interpreting kinetic data based on conventional heterotrophic potential measurements, especially when some complications may arise from temperature and substrate adaptations of the more versatile members of the chemoorganotrophic microflora such as C. johnsonae. |
A beta-glucosidase (EC 3.2.1.21) from the fungus Aspergillus terreus was purified to homogeneity as indicated by disc acrylamide gel electrophoresis. Optimal activity was observed at pH 4.8 and 50 degrees C. The beta-glucosidase had K(m) values of 0.78 and 0.40 mM for p-nitrophenyl-beta-d-glucopyranoside and cellobiose, respectively. Glucose was a competitive inhibitor, with a K(i) of 3.5 mM when p-nitrophenyl-beta-d-glucopyranoside was used as the substrate. The specific activity of the enzyme was found to be 210 IU and 215 U per mg of protein on p-nitrophenyl-beta-d-glucopyranoside and cellobiose substrates, respectively. Cations, proteases, and enzyme inhibitors had little or no effect on the enzyme activity. The beta-glucosidase was found to be a glycoprotein containing 65% carbohydrate by weight. It had a Stokes radius of 5.9 nm and an approximate molecular weight of 275,000. The affinity and specific activity that the isolated beta-glucosidase exhibited for cellobiose compared favorably with the values obtained for beta-glucosidases from other organisms being studied for use in industrial cellulose saccharification. |
The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and C-labeled tracers. Time course analyses of the loss of [H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-C]glucose uptake and C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H(2) to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments. |
Michaelis-Menten kinetic parameters for H(2) consumption by three methanogenic habitats were determined from progress curve and initial velocity experiments. The influences of mass transfer resistance, endogenous H(2) production, and growth on apparent parameter estimates were also investigated. Kinetic parameters could not be determined for undiluted rumen fluid and some digestor sludge from gas-phase measurements of H(2), since mass transfer of H(2) across the gas-liquid interface was rate limiting. However, accurate values were obtained once the samples were diluted. H(2) consumption by digestor sludge with a long retention time and by hypereutrophic lake sediment was not phase transfer limited. The K(m) values for H(2) uptake by these habitats were similar, with means of 5.8, 6.0, and 7.1 muM for rumen fluid, digestor sludge, and sediment, respectively. V(max) estimates suggested a ratio of activity of approximately 100 (rumen fluid):10 (sludge):1 (sediment); their ranges were as follows: rumen fluid, 14 to 28 mM h; Holt sludge, 0.7 to 4.3 mM h; and Wintergreen sediment, 0.13 to 0.49 mM h. The principles of phase transfer limitation, studied here for H(2), are the same for all gaseous substrates and products. The limitations and errors associated with gas phase determination of kinetic parameters were evaluated with a mathematical model that combined mass transport and Michaelis-Menten kinetics. Three criteria are described which can be used to evaluate the possibility that a phase transfer limitation exists. If it does not exist, (i) substrate consumption curves are Michaelis-Menten and not first order, (ii) the K(m) is independent of initial substrate concentration, and (iii) the K(m) is independent of biomass (V(max)) and remains constant with dilution of sample. Errors in the Michaelis-Menten kinetic parameters are caused by endogenously produced H(2), but they were <15% for rumen fluid and 10% for lake sediment and digestor sludge. Increases in V(max) during the course of progress curve experiments were not great enough to produce systematic deviations from Michaelis-Menten kinetics. |
The ability of microorganisms to ferment waste from cattle fed monensin, lasalocid, or salinomycin to methane was determined. Continuously mixed anaerobic fermentors with 3-liter working volumes at 55 degrees C were used; fermentors were fed once per day. Initially, all fermentors were fed waste without antibiotics at 6% volatile solids (VSs, organic matter) and a 20-day retention time (RT) for 60 days. Waste from animals fed monensin, lasalocid, or salinomycin at 29, 20, and 16.5 mg per kg of feed, respectively, was added to duplicate fermentors at the above VSs, and RT. Avoparcin (5 to 45 mg/liter) was not fed to animals but was added directly to duplicate fermentors. Lasalocid and salinomycin had minimal effects on the rate of methane production at RTs of 20 days and later at 6.5 days. Avoparcin caused an increase in organic acids from 599 to 1,672 mg/liter (as acetate) after 4 weeks, but by 6 weeks, acid concentrations declined and the rate of methane production was similar to controls at a 6.5-day RT. The monensin fermentors stopped producing methane 3 weeks after antibiotic addition. However, after a 6-month acclimation period, the microorganisms apparently adapted, and methane production rates of 1.65 and 2.51 liters per liter of fermentor volume per day were obtained with 6% VSs, and RTs of 10 and 6.5 days, respectively. This compares with 1.78 and 2.62 liters/liter per day for controls (P > 0.05). All fermentors that were fed waste containing antibiotics had lower pH values and ammonia and alkalinity concentrations, suggesting less buffering capacity and protein catabolism than in controls. Acclimation results obtained with fermentors at 35 degrees C were similar to those for fermentors at 55 degrees C. These studies indicate that waste from cattle fed these selected growth-promoting antibiotics can be thermophilically fermented to methane at RTs of 6.5 days or longer and VS concentrations of 6%, at rates comparable to waste without antibiotics. |
Hemicellulose-derived sugars were obtained from a variety of pretreated wood substrates such as water-soluble fractions from steam-exploded aspen, solvent-extracted aspen, and commercial xylan. These fractions were enzymatically hydrolyzed by commercial enzyme preparations and by the culture filtrates of eight highly cellulolytic fungi. The sugars released were assayed by high-pressure liquid chromatography. Over 30% of the hemicellulose fractions, at a 10% substrate concentration, could be hydrolyzed to monosaccharides. These hemicellulose hydrolysates were used as the substrates for growth of Clostridium acetobutylicum and Klebsiella pneumoniae. Comparatively low butanol values were obtained with C. acetobutylicum, although over 50% of the hemicellulose fraction, at a 1% substrate concentration, could be converted to 2,3-butanediol, ethanol, and acetic acid by K. pneumoniae. |
A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5'-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg and Ca, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50 degrees C on DNA and at 60 degrees C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5'-mononucleotides. |
Several different kinds of substrate were used to investigate the proteolytic activity of rumen bacteria and of proteases released from rumen bacteria by blending ("coat proteases"). These substrates included diazotized feed proteins and diazotized soluble and insoluble pure proteins. It was concluded that, while solubility was an important factor, the secondary and tertiary structure of a protein had a major influence on its rate of digestion. The resistance of elastin congo red to digestion indicated that similar fibrous proteins in plant material might resist proteolytic attack by rumen bacteria. Coat proteases had a broad specificity, including several exo- and endopeptidase activities, as determined by using synthetic peptide substrates. |
Metabolism of linalyl acetate by Pseudomonas incognita isolated by enrichment culture on the acyclic monoterpene alcohol linalool was studied. Biodegradation of linalyl acetate by this strain resulted in the formation of linalool, linalool-8-carboxylic acid, oleuropeic acid, and Delta-4-acetoxy-4-methyl hexenoic acid. Cells adapted to linalyl acetate metabolized linalyl acetate-8-aldehyde to linalool-8-carboxylic acid, linalyl acetate-8-carboxylic acid, Delta-4-acetoxy-4-methyl hexenoic acid, and geraniol-8-carboxylic acid. Resting cell suspensions previously grown with linalyl acetate oxidized linalyl acetate-8-aldehyde to linalyl acetate-8-carboxylic acid, Delta-4-acetoxy-4-methyl hexenoic acid, and pyruvic acid. The crude cell-free extract (10,000 g of supernatant), obtained from the sonicate of linalyl acetate-grown cells, was shown to contain enzyme systems responsible for the formation of linalyl acetate-8-carboxylic acid and linalool-8-carboxylic acid from linalyl acetate. The same supernatant contained NAD-linked alcohol and aldehyde dehydrogenases involved in the formation of linalyl acetate-8-aldehyde and linalyl acetate-8-carboxylic acid, respectively. On the basis of various metabolites isolated from the culture medium, resting cell experiments, growth and manometric studies carried out with the isolated metabolites as well as related synthetic analogs, and the preliminary enzymatic studies performed with the cell-free extract, a probable pathway for the microbial degradation of linalyl acetate with the acetoxy group intact is suggested. |
The binding of viable Escherichia coli cells to an immobilized ligand of a surface receptor for maltodextrins has recently been demonstrated (T. Ferenci and K. S. Lee, J. Mol. Biol. 160:431-444, 1982). The interaction of bacteria and ligand immobilized in a chromatographic column was investigated over a wide range of applied cell densities, temperatures, eluant pH values, osmotic concentrations, and flow rates. Over 95% retention of bacteria applied to starch-Sepharose was found at cell densities up to 10 per ml of matrix, between pH 5.5 and 8.0, between 8 and 55 degrees C, in the presence of 0 to 0.5 M NaCl, and at elution flow rates up to 37 column volumes per h. The catalytic capability and stability of affinity-immobilized cells was demonstrated with the cytoplasmic beta-galactosidase activity of starch-bound cells. Intact immobilized bacteria exhibited slowly increasing beta-galactosidase activity over several days with a plateau after 6 days. Bacteria made permeable by treatment with toluene were also bound to starch-Sepharose but showed maximum beta-galactosidase activity within 1 day and exhibited no loss of enzyme activity in 8 days of continuous elution at ambient temperatures. |
Growing and nongrowing cells of Clostridium sporogenes fermented betaine with l-alanine, l-valine, l-leucine, and l-isoleucine as electron donors in a coupled oxidation-reduction reaction (Stickland reaction). For the substrate combinations betaine and l-alanine and betaine and l-valine balance studies were performed; the results were in agreement with the following fermentation equation: 1 R- CH(NH(2))-COOH + 2 betaine + 2 H(2)O --> 1 R-COOH + 1 CO(2) + 1 NH(3) + 2 trimethylamine + 2 acetate. Growth and production of trimethylamine were strictly dependent on the presence of selenite in the medium. With cell suspensions it was shown that C. sporogenes was unable to catabolize betaine as a single substrate. Betaine, however, was reduced to trimethylamine and acetate under an atmosphere of molecular hydrogen. For the reduction of betaine by cell extracts of C. sporogenes, dimercaptans such as 1,4-dithiothreitol could serve as electron donors. No betaine reductase activity was detected in cells grown in a complex medium without betaine. The pH optimum of betaine reductase was at pH 7.3. When C. sporogenes was cocultured with Methanosarcina barkeri strain Fusaro on betaine together with l-alanine, an almost complete conversion of the two substrates to CH(4), NH(3), and presumably CO(2) was observed. |
A comprehensive view of the diazotrophic bacterial flora of plants requires that attention be paid to the appropriate carbon and oxygen requirements during isolation of the bacteria. Twenty compounds (monosaccharides, disaccharides, polyols, and organic acids) were therefore examined as carbon and energy sources for nitrogenase activity in semisolid stab cultures at pO(2) values of 0.21, 0.02, and </=0.002 with 12 strains of diazotrophic root-associated bacteria. With the facultatively anaerobic bacteria of the genera Klebsiella and Enterobacter, the best substrate was sucrose, followed by fructose and mannitol, whereas among the organic acids, only malic and fumaric acids supported any activity. With the obligately aerobic bacteria of the genera Azospirillum and Pseudomonas, disaccharides were not utilized for nitrogen fixation, but several organic acids were accepted in addition to monosaccharides and polyols; malate and glucose were the best substrates. The patterns of the carbon sources utilized for nitrogen fixation were coherent within the species, with the exception of one Klebsiella pneumoniae and one Enterobacter agglomerans strain, both isolated from the same individual grass plant, which were unable to utilize lactose. Anaerobic conditions (pO(2) value of </=0.002) were required for maximum nitrogenase activity with the facultatively anaerobic bacteria, with the exception of one strain of E. agglomerans, which required atmospheric oxygen (pO(2) value of 0.21). Also, the obligately aerobic diazotrophs required atmospheric oxygen for maximum nitrogenase activity. The maximum specific nitrogenase activities (expressed as micromoles of C(2)H(4) . milligram of bacterial protein . hour) noted during the exponential growth phase of the bacteria were the following: 2.68 with Azospirillum lipoferum on malate, 2.41 with K. pneumoniae and 1.58 with E. agglomerans on sucrose, and 0.95 with Pseudomonas sp. on malate. |
The dissolution and degradation of dagger-endotoxin (crystal) of Bacillus thuringiensis subsp. kurstaki strain HD-1 were investigated. Crystals were dissolved in 0.1 M phosphate-carbonate-NaOH buffer at pH > 12. Swelling of crystals occurred in the buffer between pH 10 and 11, and crystals dissolved in the same buffer supplemented with gut juice protease of the silkworm Bombyx mori. The proteolytic dissolution of crystals occurred after a time lag of several minutes in 0.1 M carbonate-NaOH buffer, pH 10.2. The time lag was not observed when crystals were suspended in the buffer for 30 min before the addition of protease. After the dissolution of the crystals and further degradation of the solubilized protein, the appearance of a toxic protein with a molecular weight of 59,000, designated P-59, was observed. Lower-molecular-weight peptides (less than 40,000) showed no toxicity to the silkworm larvae on feeding. Digestion of the 120,000-dalton subunit of the crystal by gut juice protease also produced P-59. These observations suggest the occurrence of a similar process in vivo, i.e., the swelling of crystals due to the alkalinity of gut juice and the production of P-59, dependent on the hydrolysis of swollen crystals by gut juice protease. |
Accurate measurement of the toxic protein crystal produced during deep-tank fermentation of Bacillus thuringiensis is critical for optimum process yield. The currently accepted method is a bioassay that requires more time to generate data than to complete the fermentation itself. A noncompetitive enzyme-linked immunosorbent assay has been developed with purified B. thuringiensis crystals to generate rabbit antiserum. This technique gives a quantitative crystal protein value with a colorimetric endpoint for either liquids or powders within 4 h of sampling. Reproducibility of this enzyme-linked immunosorbent assay satisfies criteria for use in a commercial process. |
Using Candida tenuis, a yeast isolated from the digestive tube of the larva of Phoracantha semipunctata (Cerambycidae, Coleoptera), we were able to demonstrate the bioconversion of citronellal to citronellol. Response surface methodology was used to achieve the optimization of the experimental conditions for that bioconversion process. To study the proposed second-order polynomial model, we used a central composite experimental design with multiple linear regression to estimate the model coefficients of the five selected factors believed to influence the bioconversion process. Only four were demonstrated to be predominant: the incubation pH, temperature, time, and the amount of substrate. The best reduction yields (close to 90%) were obtained with alkaline pH conditions (pH 7.5), a low temperature (25 degrees C), a small amount of substrate (15 mul), and short incubation time (16 h). This methodology was very efficient: only 36 experiments were necessary to assess these conditions, and model adequacy was very satisfactory as the coefficient of determination was 0.9411. |
Of 10 strains of the purple non-sulfur bacterium Rhodopseudomonas sphaeroides, 8 acquired the ability to grow on d-(-)-tartrate; however, growth occurred only after extended lag phases ranging from 2 to 14 days. These lag phases occurred because only a small number of inoculum cells were able to grow by forming the enzyme d-(-)-tartrate dehydratase [d-(-)-tartrate hydro-lyase; EC number not yet available]. Once cells had grown on d-(-)-tartrate, d-(-)-tartrate dehydratase was formed constitutively. Therefore, mass cultivation of R. sphaeroides for production of large quantities of enzyme was possible on substrates much cheaper than d-(-)-tartrate. When 0.38 mol of dl-malate was used as a substrate in a chemotrophic fed-batch culture, a final biomass of 15 g (dry weight) liter and 1,500 U of d-(-)-tartrate dehydratase liter of culture were formed. The enzyme can be used for selective cleavage of racemic tartaric acid and for quantitative determination of d-(-)-tartrate. |
Levels of DNA, RNA, protein, ATP, glutathione, and radioactivity associated with [S]methionine-labeled cellular protein were estimated at various times during the starvation-survival process of a marine psychrophilic heterotrophic Vibrio sp., Ant-300. Values for the macromolecules were analyzed in terms of total, viable, and respiring cells. Electron micrographs (thin sections) were made on log-phase and 5.5-week-starved cells. On a per-cell basis, the levels of protein and DNA rapidly decreased until a constant level was attained. A second method in which radioactive sulfur was used for monitoring protein demonstrated that the cellular protein level decreased for approximately 2.5 weeks and then remained constant. An initial decrease in the RNA level with starvation was noted, but with time the RNA (orcinol-positive material) level increased to 2.5 times the minimum level. After 6 weeks of starvation, 45 to 60% of the cells remained capable of respiration, as determined by iodonitrotetrazolium violet-formazan granule production. Potential respiration and endogenous respiration levels fell, with an intervening 1-week peak, until at 2 weeks no endogenous respiration could be measured; respiratory potential remained high. The cell glutathione level fell during starvation, but when the cells were starved in the presence of the appropriate amino acids, glutathione was resynthesized to its original level, beginning after 1 week of starvation. The cells used much of their stored products and became ultramicrocells during the 6-week starvation-survival process. Ant-300 underwent many physiological changes in the first week of starvation that relate to the utilization or production of ATP. After that period, a stable pattern for long-term starvation was demonstrated. |
Thirty-five nontarget host cell lines, 23 of human and 12 of nonhuman vertebrate origin, were exposed to Autographa californica nuclear polyhedrosis virus preparations derived from four different sources: polyhedra, hemolymph, cell culture medium, and cultured cells. The virus and cells were incubated together at two different temperatures, 28 or 37 degrees C, for four different lengths of time, 16, 40, 64, or 168 h, and the cells were assayed for the presence of virus by a peroxidase-antiperoxidase detection method. The estimated sensitivity of the assay as routinely conducted was 0.98 ng of alkali-liberated viral protein and 1.95 ng of budded viral protein per mm. No evidence of frank replication was obtained in any of the 35 cell lines tested, although virus uptake appeared to be quite common. Virus uptake was confirmed in some cases by electron microscopy. The degree of virus uptake appeared to be dependent on cell type, time and temperature of incubation, and viral phenotype. Virus purified from polyhedra was generally taken up more readily than were the other forms tested. |
Varying the amount of labeled substrate or the amount of leaf material resulted in significant nonlinear changes in lignocellulose mineralization, as measured with natural [C]lignin-labeled lignocellulose. The use of periodic rather than continuous aeration was found not to have significant effects on measured mineralization. |
Thirty-three bacterial strains were isolated from soil, utilizing optically asymmetric degradation of dl-2-hydroxy-4-methylpentanoic acid (dl-HMPA) as the screening probe. Those strains were distributed in the following group and genera: Coryneform and Bacillus, Pseudomonas, and Streptomyces. Among them, the most potent strains, Bacillus freudenreichii NRS-137KH20B and Brevibacterium albidum NRS-130KH20B, could perform the resolution of more than 30 g of dl-HMPA per liter within 4 to 5 days of fermentation. Optically pure l- and d-HMPA enantiomers were obtained in more than 80% theoretical yield, whereas the transformed enantiomer was almost quantitatively recovered as 2-oxo-4-methyl-pentanoic acid in the culture broth. The enantiospecific dehydrogenation responsible for this resolution reaction had a rather wide substrate specificity on straight or branched aliphatic C(4) to C(16) 2-hydroxy acids, exhibiting the optima at chain lengths of either C(7) or C(5), although the enantiospecificity was not changed by chain length. The process was thus successfully extended to the preparation of optically pure C(5) to C(9) 2-hydroxy acids. |
alpha-Amylase (EC 3.2.1.1) was excreted by Calvatia gigantea in liquid growth media containing different sources of starch. Among the factors affecting enzyme production in shake flasks were the type and the concentration of starch and the nitrogen source supplied. Optimum cultural conditions for maximum enzyme production were: soluble starch concentration, 5%; inoculum size, 3.75 x 10 conidia per ml; 5-day cultivation time at 28 to 30 degrees C. The observed maximum yield of 81.3 U of saccharifying enzyme activity per ml of growth medium was the highest ever reported in the literature for submerged cultures. Partially purified enzyme functioned optimally at pH 4.5 to 5.5 and 53 to 58 degrees C. The activation energy of enzymic hydrolysis of starch in the range of 20 to 40 degrees C was 8,125 cal/mol (ca. 3.41 x 10 J). The apparent K(m) value of the enzyme at 25 degrees C was 7.68 x 10 g/ml. Some of the properties of the enzyme under investigation were similar to those of alpha-amylases excreted from molds producing large amounts of the enzyme. |
The distribution, activity, and generic diversity of nitrifying bacteria in a stream receiving geothermal inputs of ammonium were studied. The high estimated rates of benthic nitrate flux (33 to 75 mg of N . m . h) were a result of the activity of nitrifiers located in the sediment. Nitrifying potentials and ammonium oxidizer most probable numbers in the sediments were at least one order of magnitude higher than those in the waters. Nitrifiers in the oxygenated surface (0 to 2 cm) sediments were limited by suboptimal temperature, pH, and substrate level. Nitrifiers in deep (nonsurface) oxygenated sediments did not contribute significantly to the changes measured in the levels of inorganic nitrogen species in the overlying waters and presumably derived their ammonium supply from ammonification within the sediment. Ammonium-oxidizing isolates obtained by a most-probable number nonenrichment procedure were species of either Nitrosospira or Nitrosomonas, whereas all those obtained by an enrichment procedure (i.e., selective culture) were Nitrosomonas spp. The efficiency of the most-probable-number method for enumerating ammonium oxidizers was calculated to be between 0.05 and 2.0%, suggesting that measurements of nitrifying potentials provide a better estimate of nitrifying populations. |
A cell-free extract of Daphnia magna was found to lyse Escherichia coli cells as shown by leakage of the enzymes alkaline phosphatase and beta-galactosidase from the bacteria. The cell-free extract was separated on Sephadex G-200, and the fractions showing an ability to lyse E. coli cels were isolated. The factor which was responsible for the lysis of the bacterial cells was probably a protein with a molecular weight of several thousands. Mg and Ca ions augmented the activity of the Daphnia extract on E. coli cells. |
The interactions between colorless sulfur bacteria and the chemical microgradients at the oxygen-sulfide interface were studied in Beggiatoa mats from marine sediments and in Thiovulum veils developing above the sediments. The gradients of O(2), H(2)S, and pH were measured by microelectrodes at depth increments of 50 mum. An unstirred boundary layer in the water surrounding the mats and veils prevented microturbulent or convective mixing of O(2) and H(2)S. The two substrates reached the bacteria only by molecular diffusion through the boundary layer. The bacteria lived as microaerophiles or anaerobes even under stirred, oxic water. Oxygen and sulfide zones overlapped by 50 mum in the bacterial layers. Both compounds had concentrations in the range of 0 to 10 mumol liter and residence times of 0.1 to 0.6 s in the overlapping zone. The sulfide oxidation was purely biological. Diffusion calculations showed that formation of mats on solid substrates or of veils in the water represented optimal strategies for the bacteria to achieve a stable microenvironment, a high substrate supply, and an efficient competition with chemical sulfide oxidation. The continuous gliding movement of Beggiatoa cells in mats or the flickering motion of Thiovulum cells in veils were important for the availability of both O(2) and H(2)S for the individual bacteria. |
Anabaena sp. strain 7120 appeared more responsive to nitrogen control than A. cylindrica. Growth in the presence of nitrate strongly repressed the differentiation of heterocysts and fixation of dinitrogen in Anabaena sp. strain 7120, but only weakly in A. cylindrica. Nitrate assimilation by ammonium-grown cultures was strongly repressed in Anabaena sp. strain 7120, but less so in A. cylindrica. The repressive effect of nitrate on dinitrogen assimilation in Anabaena sp. strain 7120, compared to A. cylindrica, did not correlate with a greater rate of nitrate transport, reduction to ammonium, assimilation into amino acids, or growth. Although both species grew at similar rates with dinitrogen, A. cylindrica grew faster with nitrate, incorporated more NO(3) into amino acids, and assimilated (transported) nitrate at the same rate as Anabaena sp. strain 7120. Full expression of nitrate assimilation in the two species occurred within 2.5 h (10 to 14% of their generation times) after transfer to nitrate medium. The induction and continued expression of nitrate assimilation was dependent on protein synthesis. The half-saturation constants for nitrate assimilation and for nitrate and ammonium repression of dinitrogen assimilation have ecological significance with respect to nitrogen-dependent growth and competitiveness of the two Anabaena species. |
Pseudomonas putida cooxidized norcamphor and pericyclocamphanone to hydroxylated and lactonized products during growth on camphor. Norcamphor was hydroxylated at the 5 position, similar to the corresponding process in camphor, but pericyclocamphanone was oxidized at the 6 position. We conclude that the regiochemistry of the hydroxylation may be substrate controlled. |
The kinetic parameters associated with the microbial dehalogenation of 3-chlorobenzoate, 3,5-dichlorobenzoate, and 4-amino-3,5-dichlorobenzoate were measured in anoxic sediment slurries and in an enriched methanogenic culture grown on 3-chlorobenzoate. The initial dehalogenation of the substrates exhibited Michaelis-Menten kinetics. The apparent K(m) values for the above substrates ranged from 30 to 67 muM. The pattern of degradation, however, was unusual. The enrichment culture accumulated partially dehalogenated intermediates to 72 and 98% of that possible when incubated with either 3,5-dichloro- or 4-amino-3,5-dichlorobenzoate, respectively, but did not accumulate significant amounts of benzoate when 3-chlorobenzoate was the sole carbon and energy source. The accumulated intermediates were rapidly metabolized only after the parent substrate concentrations were nearly depleted (<5 muM). A sequential Michaelis-Menten model was developed to account for the observed pattern of biodegradation. Using this model, we found that relative differences in the K(m) and V(max) parameters for substrate and intermediate dehalogenations alone were insufficient to explain the transitory accumulation of intermediates. However, by inserting a competitive inhibition term, with the primary substrate as the inhibitor, the observed pattern of degradation was simulated. Apparently, the dichlorinated substrates competitively inhibit the dehalogenation of the monochlorinated substrates. Similar kinetic patterns were noted for sediments, although the rates were slower than in the enrichment culture. |
Eighteen strains of Agrobacterium tumefaciens isolated from crown galls were tested for agrocin production. Of six agrocin-producing strains, one (D286) produced a broad-host-range agrocin active against strains carrying nopaline, octopine, and agropine type Ti plasmids. Sensitivity to agrocin D286 was found to map in the 11- to 18-megadalton region of the nopaline Ti plasmid pTiC58. The agrocin was partially purified, and its physical characteristics were consistent with its being a nucleotide, as is agrocin 84. Agrocin D286 was shown to inhibit DNA, RNA, and protein syntheses. Strain D286 spontaneously lost its pathogenicity, and its potential for use in the biological control of crown gall is discussed. |
Vibrio gazogenes ATCC 29988 growth and prodigiosin synthesis were studied in batch culture on complex and defined media and in chemostat cultures on defined medium. In batch culture on complex medium, a maximum growth rate of 0.75 h and a maximum prodigiosin concentration of 80 ng of prodigiosin . mg of cell protein were observed. In batch culture on defined medium, maximum growth rates were lower (maximum growth rate, 0.40 h), and maximum prodigiosin concentrations were higher (1,500 ng . mg of protein). In batch culture on either complex or defined medium, growth was characterized by a period of logarithmic growth followed by a period of linear growth; on either medium, prodigiosin biosynthesis was maximum during linear growth. In batch culture on defined medium, the initial concentration of glucose optimal for growth and pigment production was 3.0%; higher levels of glucose suppressed synthesis of the pigment. V. gazogenes had an absolute requirement for Na; optimal growth occurred in the presence of 100 mM NaCl. Increases in the concentration of Na up to 600 mM resulted in further increases in the concentration of pigment in the broth. Prodigiosin was synthesized at a maximum level in the presence of inorganic phosphate concentrations suboptimal for growth. Concentrations of KH(2)PO(4) above 0.4 mM caused decreased pigment synthesis, whereas maximum cell growth occurred at 1.0 mM. Optimal growth and pigment production occurred in the presence of 8 to 16 mg of ferric ion . liter, with higher concentrations proving inhibitory to both growth and pigment production. Both growth and pigment production were found to decrease with increased concentrations of p-aminobenzoic acid. The highest specific concentration of prodigiosin (3,480 ng . mg protein) was observed in chemostat cultures at a dilution rate of 0.057 h. The specific rate of prodigiosin production at this dilution rate was approximately 80% greater than that observed in batch culture on defined medium. At dilution rates greater than 0.057 h, the concentration of cells decreased with increasing dilution rate, resulting in a profile comparable to that expected for linear growth kinetics. No explanation could be found for the linear growth profiles obtained for both batch and chemostat cultures. |
The polymeric dyes Poly B-411, Poly R-481, and Poly Y-606 were examined as possible alternatives to the radiolabeled lignin previously used as a substrate in lignin biodegradation assays. Like lignin degradation, the decolorization of these dyes by the white rot basidiomycete Phanerochaete chrysosporium occurred during secondary metabolism, was suppressed in cultures grown in the presence of high levels of nitrogen, and was strongly dependent on the oxygen concentration in the cultures. A variety of inhibitors of lignin degradation, including thiourea, azide, and 4'-O-methylisoeugenol, also inhibited dye decolorization. A pleiotropic mutant of P. chrysosporium, 104-2, lacking phenol oxidase and ligninolytic activity was also not able to decolorize the polymeric dyes, whereas a phenotypic revertant strain, 424-2, regained this capacity. All of these results suggest that the ligninolytic degradation activity of the fungus was responsible for the decolorization of these dyes. |
Fingerprint protein patterns were produced by two-dimensional polyacrylamide electrophoresis on lysed cells of a Vibrio sp., Ant-300, which were prepared from growing and starved cultures. The cells were labeled with [S]methionine during growth and subsequently starved for up to 30 days. Samples were taken at selected time points representing stages in the starvation-survival process. Unlabeled starved cells were allowed to recover in the presence of [S]methionine to determine protein changes associated with the recovery from starvation. All growth conditions produced similar protein fingerprints; however, some protein spots disappeared, whereas others were seen only during starvation. |
The relative heterotrophic activity of marine microorganisms was determined at two sites by the heterotrophic uptake technique throughout the water column, the sediment-water interface, and the surface layer of sediment. In the water column, uptake was greatest at the surface and steadily decreased with depth. The percentage of the substrate that was respired also decreased with depth from 69 to 56%. The activity of the sediment-water interface was several orders of magnitude greater than that of the overlying water and twice that of the sediment immediately below. Hand-collected water samples carefully taken as close as 1 cm from the sediment-water interface had the same characteristically low activity as the bottom few meters of water. Microautoradiography with H-labeled glucose, glutamic acid, or thymidine revealed a general decrease in the percentage of active cells with depth from 35 to <1%. The number of active cells in the interface and sediment averaged <10% of the total population. The data indicate that the sediment-water interface is the most active region in this system due to an increased number of active cells rather than an increased percentage of active cells or increased per-cell activity. |
The sediment-water interface in Halifax Harbor supports a microbial population of 6.95 x 10 cells per g (dry weight). As determined by the standard technique of suspending subsamples in filtered seawater, the uptake of added glutamic acid by this population is 113.5 ng g (dry weight) h. An alternate technique was developed to measure the heterotrophic activity of the interface over longer periods of time, using undisturbed cores with the sediment-water interface intact. Under these conditions, the microbes in the water column and the interface increased exponentially in number, with mean doubling times of 9.6 and 4.5 days, respectively. The uptake of glutamic acid by the microbial population of the interface was determined to be 12.7 ng g (dry weight) h, almost an order of magnitude less than the uptake determined by the previous method. This indicates that substrate diffusion and competition for substrate by the microbes in the water column are important factors when considering the heterotrophic activity of the sediment microbial population. After 48 h of incubation, uptake and respiration ceased, probably due to the exhaustion of labeled substrate. Additional substrate added after 48 h of incubation was taken up at a rate similar to that measured after the first addition. It appears that the microbial population of the interface is able to respond quickly and repeatedly to relatively large nutrient additions. After 10 days of incubation, the number of "viable" cells as determined by autoradiography was much smaller than the increase in numbers as determined by direct counts. Apparently a large part of the viable population is unaffected by nutrient addition. |
Conditions for the production of tryptophanase from Achromobacter liquidum and for the conversion of l-serine and indole to l-tryptophan were studied. The enzyme could be produced in amounts as great as 0.750 U/ml (degradation) and 0.294 U/ml (synthesis) by shaking cultures at 30 degrees C in a medium containing dextrin, yeast extract, l-tryptophan, and l-glutamic acid. l-Tryptophan was produced most efficiently by shaking the cells at 37 degrees C in a reaction mixture containing 60 mg of l-serine per ml, 60 mg of indole per ml, and 0.5 mM pyridoxal phosphate. After 3 days, 96 mg of l-tryptophan per ml was formed, and l-tryptophan was easily isolated to 85.4% yield by concentration of the reaction mixture. |
Cellobiase (beta-glucosidase) production was compared for two streptomycetes: Streptomyces flavogriseus, a known producer of cellulase complex, and Streptomyces sp. strain CB-12, a strain isolated for its rapid growth on cellobiose. The optimal conditions for enzyme activity were established in relation to pH, temperature, enzyme stability, and substrate affinity. The production of beta-glucosidase by the two strains depended on the carbon substrate in the medium. Cellobiose was found to repress the biosynthesis of the enzyme in S. flavogriseus and to stimulate its production in strain CB-12. The biosynthesis of the enzyme correlated well with the accumulation of glucose in the culture filtrates. The combined action of the beta-glucosidases produced by the two Streptomyces strains might allow a better utilization of the reaction products which arise during the biodegradation of cellulose. |
Sixteen new cultures of propane-utilizing bacteria were isolated from lake water from Warinanco Park, Linden, N.J. and from lake and soil samples from Bayway Refinery, Linden, N.J. In addition, 19 known cultures obtained from culture collections were also found to be able to grow on propane as the sole carbon and energy source. In addition to their ability to oxidize n-alkanes, resting-cell suspensions of both new cultures and known cultures grown on propane oxidize short-chain alkenes to their corresponding 1,2-epoxides. Among the substrate alkenes, propylene was oxidized at the highest rate. In contrast to the case with methylotrophic bacteria, the product epoxides are further metabolized. Propane and other gaseous n-alkanes inhibit the epoxidation of propylene. The optimum conditions for in vivo epoxidation are described. Results from inhibition studies indicate that a propane monooxygenase system catalyzes both the epoxidation and hydroxylation reactions. Experiments with cell-free extracts show that both hydroxylation and epoxidation activities are located in the soluble fraction obtained after 80,000 x g centrifugation. |
Nineteen new C(2) to C(4)n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C(2) to C(4)n-alkanes. Cell suspensions of these C(2) to C(4)n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60 degrees C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes. |
POL-88, a mutant of the white-rot fungus Phanerochaete chrysosporium, was selected for diminished phenol-oxidizing enzyme activity. A wide variety of phenolic compounds were degraded by ligninolytic cultures of this mutant. With several o-diphenolic substrates, degradation intermediates were produced that had UV spectra consistent with muconic acids. Extensive spectrophotometric and polarographic assays failed to detect classical ring-cleaving dioxygenases in cell homogenates or in extracts from ligninolytic cultures. Even so, a sensitive carrier-trapping assay showed that intact cultures degraded [U-C]catechol to [C]muconic acid, establishing the presence of a system capable of 1,2-intradiol fission. Significant accumulation of [C]muconic acid into carrier occurred only when evolution of CO(2) from [C]catechol was inhibited by treating cultures with excess nutrient nitrogen (e.g., l-glutamic acid) or with cycloheximide. l-Glutamic acid is known from past work to repress the ligninolytic system in P. chrysosporium and to mimic the effect of cycloheximide. The results here indicate, therefore, that the enzyme system responsible for degrading ring-cleavage products to CO(2) turns over faster than does the system responsible for ring cleavage. |
Stream fungi have the capacity to degrade leaf litter and, through their activities, to transform it into a more palatable food source for invertebrate detritivores. The objectives of the present study were to characterize various aspects of fungal modification of the leaf substrate and to examine the effects these changes have on leaf palatability to detritivores. Fungal species were grown on aspen leaves for two incubation times. Leaves were analyzed to determine the weight loss, the degree of softening of the leaf matrix, and the concentrations of ATP and nitrogen associated with leaves. The activities of a protease and 10 polysaccharide-degrading enzymes produced by each fungus were also determined. Most fungi caused similar changes in physicochemical characteristics of the leaves. All fungi exhibited the capability to depolymerize pectin, xylan, and cellulose. Differences among fungi were found in their capabilities to produce protease and certain glycosidases. Leaf palatability was assessed by offering leaves of all treatments to larvae of two caddisfly shredders (Trichoptera). Feeding preferences exhibited by the shredders were similar and indicated that they perceived distinct differences among fungi. Two fungal species were highly consumed, some moderately and others only slightly. No relationships were found between any of the fungal characteristics measured and detritivore feeding preferences. Apparently, interspecific differences among fungi other than parameters associated with biomass or degradation of structural polysaccharides influence fungal palatability to caddisfly detritivores. |
3-Hydroxypropionaldehyde is a precursor to acrolein, which can be used as an intermediate for making acrylic acid and a variety of other useful industrial chemicals. Conversion of glycerol, a renewable resource, to 3-hydroxypropionaldehyde was attempted via action of glycerol dehydrase isolated from Lactobacillus sp. strain NRRL B-1720. This method, however, was unsatisfactory because enzyme activity was lost within 60 to 90 min after the reaction initiation. Fermentation of glycerol by whole cells of Klebsiella pneumoniae NRRL B-199 in the presence of optimal semicarbazide hydrochloride proved more effective. Using this technique, glycerol solutions of 30 g/liter yielded 3-hydroxypropionaldehyde solutions of 13.1 g/liter. Thus, a conversion efficiency equal to 55% of the theoretical maximum was realized. |
Characterization of the proteins and nucleic acid of the gypsy moth nuclear polyhedrosis virus isolated in Ithaca, N.Y. (LdNPV-IT) is presented. A total of 29 viral structural proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis when the virus was isolated in the absence of alkaline protease activity. Fourteen surface envelope viral proteins were identified by lactoperoxidase iodination. Eleven proteins were associated with nucleocapsids prepared by Nonidet P-40 detergent treatment. Distinct alterations of viral proteins were documented when virions were purified in the presence of occlusion body-associated alkaline protease(s). Restriction enzyme digests of viral DNA indicated that this isolate was composed of a large number of genetic variants. On the basis of the major molar fragments resulting from EcoRI, BamHI, BglII, and HindIII digests, the molecular weight of the LdNPV genome was approximately 88 x 10. |
Quantitation and detection of xylem-limited bacteria with an enzyme-linked immunosorbent assay with a peroxidase conjugate is described. The use of the Trinder reagent (4-aminoantipyrine) allows the determination of extremely small quantities of peroxidase with no precipitate formation or inactivation of the enzyme by H(2)O(2). Comparison of the enzyme-linked immunosorbent assay method with microscopic and histochemical tests for the presence of the phony peach disease bacterium in 9-year-old "June Gold" peach trees gave comparable results. The peroxidase conjugate with the Trinder reagent is more sensitive than the alkaline phosphatase conjugate typically used for enzyme-linked immunosorbent assay quantitation. |
alpha-Amylase produced by Bacillus licheniformis CUMC305 was purified 212-fold with a 42% yield through a series of four steps. The purified enzyme was homogeneous as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and discontinuous gel electrophoresis. The purified enzyme showed maximal activity at 90 degrees C and pH 9.0, and 91% of this activity remained at 100 degrees C. The enzyme retained 91, 79, and 71% maximal activity after 3 h of treatment at 60 degrees C, 3 h at 70 degrees C, and 90 min at 80 degrees C, respectively, in the absence of substrate. On the contrary, in the presence of substrate (soluble starch), the alpha-amylase enzyme was fully stable after a 4-h incubation at 100 degrees C. The enzyme showed 100% stability in the pH range 7 to 9; 95% stability at pH 10; and 84, 74, 68, and 50% stability at pH values of 6, 5, 4, and 3, respectively, after 18 h of treatment. The activation energy for this enzyme was calculated as 5.1 x 10 J/mol. The molecular weight was estimated to be 28,000 by sodium dodecyl sulfate-gel electrophoresis. The relative rates of hydrolysis of soluble starch, amylose, amylopectin, and glycogen were 1.27, 1.8, 1.94, and 2.28 mg/ml, respectively. V(max) values for hydrolysis of these substrates were calculated as 0.738, 1.08, 0.8, and 0.5 mg of maltose/ml per min, respectively. Of the cations, Na, Ca, and Mg, showed stimulatory effect, whereas Hg, Cu, Ni, Zn, Ag, Fe, Co, Cd, Al, and Mn were inhibitory. Of the anions, azide, F, SO(3), SO(4), S(2)O(3), MoO(4), and Wo(4) showed an excitant effect. p-Chloromercuribenzoic acid and sodium iodoacetate were inhibitory, whereas cysteine, reduced glutathione, thiourea, beta-mercaptoethanol, and sodium glycerophosphate afforded protection to enzyme activity. alpha-Amylase was fairly resistant to EDTA treatment at 30 degrees C, but heating at 90 degrees C in presence of EDTA resulted in the complete loss of enzyme activity, which could be recovered partially by the addition of Cu and Fe but not by the addition of Ca or any other divalent ions. |
Thiosulfate-oxidizing enzyme has been demonstrated in cell-free extracts of the marine, thiosulfate-oxidizing pseudomonad strain 16B. The enzyme, partially purified by ion-exchange chromatography and calcium phosphate gel treatment, catalyzed the oxidation of thiosulfate to tetrathionate with the concomitant reduction of ferricyanide. Native but not mammalian cytochrome c was also reduced by the enzyme in the presence of thiosulfate. The enzyme was located exclusively in the supernatant of ultracentrifuged cell extracts. The most purified enzyme preparation, like intact cells, exhibited a temperature optimum of 30 to 31 degrees C. However, it exhibited no definite pH optimum. At pH 6.1 to 6.3 and 30 degrees C, the K(m) for thiosulfate was 1.57 mM. At lower temperatures, the apparent K(m) for thiosulfate increased, but the apparent maximum velocity remained virtually unchanged. Thiosulfate oxidation in intact cells exhibited an increase in the pH optimum at lower temperatures. The thiosulfate-oxidizing enzyme of marine heterotroph 16B is compared with thiosulfate-oxidizing enzymes from other bacteria, and the effect of temperature on the relationship between pH and thiosulfate oxidation is discussed with reference to the natural habitat of the bacterium. |
Biotransformation of 7-ethoxycoumarin by Streptomyces griseus resulted in the accumulation of two metabolites which were isolated and identified as 7-hydroxycoumarin and 7-hydroxy-6-methoxycoumarin. A novel series of biotransformation reactions is implicated in the conversion of the ethoxycoumarin substrate to these products, including O-deethylation, 6-hydroxylation to form a 6,7-dihydroxycoumarin catechol, and subsequent O-methylation. Either 7-hydroxycoumarin or 6,7-dihydroxycoumarin was biotransformed to 7-hydroxy-6-methoxycoumarin by S. griseus. Trace amounts of the isomeric 6-hydroxy-7-methoxycoumarin were detected when 6,7-dihydroxycoumarin was used as the substrate. Efforts to obtain a cell-free catechol-O-methyltransferase enzyme system from S. griseus were unsuccessful. However, [methyl-C]methionine was used with cultures of S. griseus to form 7-hydroxy-6-[C]methoxycoumarin. |
A new mesophilic methanogenic bacterial species isolated from marine sediments collected in the Cayman Islands is described. Cells are small rods occuring singly without filaments, are not motile, and do not possess flagella. Colonies are semitransparent and off-white in color. After 2 weeks of incubation at 37 degrees C colonies are 1 to 2 mm in size, circular, and have entire edges. Only hydrogen-carbon dioxide is a substrate for growth and methane formation. Cells can tolerate a variety of organic secondary buffers (bicarbonate-CO(2) being the primary buffer). Cells do not require yeast extract or Trypticase, but do require acetate, for growth. The optimum growth temperature is 40 degrees C. The optimum sodium concentration is 0.15 M. The optimum pH for growth is 7.0. The minimum generation time is 4.8 h. The DNA base composition is 44.9 mol% guanine plus cytosine. The name Methanomicrobium paynteri is proposed in honor of M. J. B. Paynter. The type strain is G-2000 (=ATCC 33997, =DSM 2545). |
Partial characterization of an extracellular xylanase isolated by chromatography from Bacillus subtilis gave a molecular weight of 32,000 and optimum pH and temperature of 5.0 and 50 degrees C, respectively. K(m) and V(max) values, determined with a soluble larchwood xylan, were 0.16% and 7.0 x 10 mumol min mg of enzyme respectively. The amino acid composition showed more basic amino acid residues than in a previously characterized xylanase from a white-rot fungus. |
Nitrate reductase activity was evaluated by four approaches, using four strains of Rhizobium japonicum and 11 chlorate-resistant mutants of the four strains. It was concluded that in vitro assays with bacteria or bacteroids provide the most simple and reliable assessment of the presence or absence of nitrate reductase. Nitrite reductase activity with methyl viologen and dithionite was found, but the enzyme activity does not confound the assay of nitrate reductase. |
Tannase isolated from Penicillium chrysogenum was purified 24-fold with 18.5% recovery after ammonium sulfate precipitation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration. Optimum enzyme activity was recorded at pH 5.0 to 6.0 and at 30 to 40 degrees C. The enzyme was stable up to 30 degrees C and within the pH range of 4.0 to 6.5. The K(m) value was found to be 0.48 x 10 M when tannic acid was used as the substrate. Metal salts at 20 mM inhibited the enzyme to different levels. |
Eight strains of obligately anaerobic, mesophilic, cellulolytic bacteria were isolated from mud of freshwater environments. The isolates (C strains) were rod-shaped, gram negative, and formed terminal spherical to oval spores that swelled the sporangium. The guanine plus cytosine content of the DNA of the C strains ranged from 30.7 to 33.2 mol% (midpoint of thermal denaturation). The C strains fermented cellulose with formation primarily of acetate, ethanol, CO(2), and H(2). Reducing sugars accumulated in the supernatant fluid of cultures which initially contained >/=0.4% (wt/vol) cellulose. The C strains resembled Clostridium cellobioparum in some phenotypic characteristics and Clostridium papyrosolvens in others, but they were not identical to either of these species. The C strains differed from thermophilic cellulolytic clostridia (e.g., Clostridium thermocellum) not only in growth temperature range but also because they fermented xylan and five-carbon products of plant polysaccharide hydrolysis such as d-xylose and l-arabinose. At 40 degrees C, cellulose was degraded by cellulolytic mesophilic cells (strain C7) at a rate comparable to that at which C. thermocellum degrades cellulose at 60 degrees C. Substrate utilization and growth temperature data indicated that the C strains contribute to the anaerobic breakdown of plant polymers in the environments they inhabit. |
Nostoc sp. colonies from field collections were cultured and propagated on silica sand with aqueous N-free BG-11 medium. Laboratory experiments were conducted to characterize the in vivo freeze-recovery physiology of nitrogenase activity. Nitrogenase activity was monitored by the acetylene reduction technique. Frozen Nostoc sp. colonies were thawed and warmed to 10, 15, 20, 25, or 30 degrees C. At 25 and 30 degrees C, nitrogenase activity was detected within 6 h after thawing. At 20 degrees C or lower, nitrogenase activity was not detected until 12 h after thawing. Optimum thawing temperature with respect to the recovery of nitrogenase activity was 25 degrees C. In subsequent experiments, laboratory-grown Nostoc colonies were used along with the following conditions: prefreezing treatment of 3 days of exposure to light or darkness, freezing, and then thawing to 25 degrees C in light or darkness with or without metabolic inhibitors [3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU), monofluoroacetate, or chloramphenicol]. Approximately 30% of the energy in the initial recovery of nitrogenase activity (to 12 h after thawing) appeared to be supplied via the utilization of carbon compounds stored before freezing. Photosynthetic conditions (i.e., light and without DCMU) were necessary for maximum recovery of nitrogenase activity. In the presence of the protein synthesis inhibitor chloramphenicol, nitrogenase activity was still detected at 12 to 48 h after thawing. Although damage may occur to nitrogenase, some of the enzyme was capable of surviving the freeze-thaw period in vivo. However, complete recovery of nitrogenase activity (equal to prefreezing activity) may entail some de novo synthesis of nitrogenase. |
Five species of the genus Dunaliella (D. tertiolecta, D. primolecta, D. parva, D. bardawil, and D. salina) were examined for glycerol accumulation, growth rate, cell density, and protein and chlorophyll content. The suitability of each algal species for use as a fermentation substrate was judged according to glycerol accumulation and quantities of neutral solvents produced after sequential bacterial fermentations. When grown in 2 M NaCl, with 24 mM NaHCO(3) or 3% CO(2) at 28 degrees C and with 10,000 to 15,000 lx of incident light on two sides of a glass aquarium, four of the five species tested produced ca. 10 to 20 mg of glycerol per liter of culture. Clostridium pasteurianum was found to convert an algal biomass mixture supplemented with 4% glycerol to ca. 16 g of mixed solvents (n-butanol, 1,3-propanediol, and ethanol) per liter. Acetone was not detected. Additionally, it has been demonstrated that Dunaliella concentrates of up to 300-fold can be directly fermented to an identical pattern of mixed solvents. Overall solvent yields were reduced by >50% when fermentations were performed in the presence of 2% NaCl. These results are discussed in terms of practical application in tropical coastal zones. |
The structural gene for a thermostable alpha-amylase from Bacillus stearothermophilus was cloned in plasmids pTB90 and pTB53. It was expressed in both B. stearothermophilus and Bacillus subtilis. B. stearothermophilus carrying the recombinant plasmid produced about fivefold more alpha-amylase (20.9 U/mg of dry cells) than did the wild-type strain of B. stearothermophilus. Some properties of the alpha-amylases that were purified from the transformants of B. stearothermophilus and B. subtilis were examined. No significant differences were observed among the enzyme properties despite the difference in host cells. It was found that the alpha-amylase, with a molecular weight of 53,000, retained about 60% of its activity even after treatment at 80 degrees C for 60 min. |
The occurrence of ferrichrome-type hydroxamate siderophores in soil was confirmed. In the presence of the iron-scavenging chelator ethylenediamine[di(o-hydroxyphenylacetic)acid], soil extract stimulated the growth of an Escherichia coli strain possessing the ferrichrome transport protein (TonA) but did not stimulate growth of a strain lacking this protein (TonA). The siderophore concentration in a 1:1 (soil-water) extract was estimated to be approximately 78 nM. Specificity of the assay was supported by the absence of significant differential strain responses to ferric citrate, ferric 2,3-dihydroxybenzoate, enterochelin, ferrioxamine B, coprogen, and triacetylfusigen. |
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