Degradation of aniline by newly isolated,extremely aniline-tolerant <Emphasis Type="Italic">Delftia </Emphasis>sp. AN3

A bacterial strain, AN3, which was able to use aniline or acetanilide as sole carbon, nitrogen and energy sources was isolated from activated sludge and identified as Delftiasp. AN3. This strain was capable of growing on concentrations of aniline up to 53.8 mM (5000 mg/l). Substituted anilines such as N-methylaniline, N, N-dimethylaniline, 2-methylaniline, 4-methylaniline, 2-chloroaniline, 3-chloroaniline, o-aminoaniline, m-aminoaniline, p-aminoaniline, and sulfanilic acid did not support the growth of strain AN3. The optimal temperature and pH for growth and degradation of aniline were 30 degrees C and 7.0, respectively. The activities of aniline dioxygenase, catechol 2,3-dioxygenase and other enzymes involved in aniline degradation were determined, and results indicated that all of them were inducible. The K (m) and V (max) of aniline dioxygenase were 0.29 mM and 0.043 mmol/mg protein/min, respectively. The K (m) and V (max) of catechol 2, 3-dioxygenase for catechol were 0.016 mM and 0.015 mmol/mg protein/min, respectively. Based on the results obtained, a pathway for the degradation of aniline by Delftiasp. AN3 was proposed. The importance of the strain to the operation of municipal wastewater treatment plants is discussed.     

Effects of carbon and nitrogen sources,carbon-to-nitrogen ratio,and initial pH on the growth of nematophagous fungus <Emphasis Type="Italic">Pochonia chlamydosporia</Emphasis> in liquid culture

The effects of carbon and nitrogen sources, carbon-to-nitrogen ratio (C:N) and initial pH value on the growth and sporulation of the nematophagous fungus Pochonia chlamydosporia in liquid culture were examined. Among the 21 carbon sources and 15 nitrogen compounds tested, the optimal carbon and nitrogen sources for mycelial growth were sweet potato and L-tyrosine, and for sporulation were sweet potato and casein peptone. A C:N ratio of 10:1 at pH 3.7 gave the maximum yield of conidia and a C:N ratio of 40:1 at pH 6.8 gave the maximum biomass. The initial pH value had a significant effect on mycelial growth and conidial production, with the optimal ranges being 3.5–4.5 for sporulation and 5–6 for growth. Maximum conidial production was obtained at an initial pH of 4.0 and the maximum biomass at pH 6.0. The results also showed that the final pH after 7 days cultivation was always higher than the initial value. The variability in growth and sporulation of seven strains of P. chlamydosporia in liquid culture was also compared and discussed.     

Influence of aromatic hydrocarbons on growth,plant growth promoting activities and survival in soil of Azotobacter chroococcum strain JL104

This study was undertaken to determine the effect of aromatic hydrocarbons on growth and plant growth promoting activities of Azotobacter chroococcum strain JL104. The organism was grown on Jensen’s media without sucrose, supplemented with different concentrations of aromatic hydrocarbons. Azotobacter chroococcum strain JL104 was able to grow in the presence of benzene, toluene, aniline and benzoic acid and was able to utilize these as sole carbon source as well. The culture showed the highest growth in presence of 0.5% concentrations of aniline and benzoic acid and 0.01% concentrations of benzene and toluene. Maximum indole acetic acid (IAA) production and acetylene reduction activity (ARA) were recorded with benzene and benzoic acid, respectively. Among other substituted benzene derivatives such as xylene, p-hydroxybenzoic acid, di-nitrophenol and di-chlorophenol, xylene was observed to be the least toxic and di-nitrophenol the most toxic hydrocarbon. The highest soil survival was found in soil amended with 1% sucrose however, the population of A. chroococccum strain JL104 declined continuously in unamended soil. Amongst various hydrocarbons, 0.1% toluene amended soil supported the maximum survival, indicating it to be least toxic aromatic hydrocarbon carbon in soil.     

A novel acid-producing fungus isolated from bauxite residue: the potential to reduce the alkalinity

Abstract

Globally, bauxite residue is creating extensive environmental problems. Nevertheless, there are numerous functional microorganisms that exist in this extreme environment, and fungus has an essential role in the pedogenesis of bauxite residue. This study describes a microbial method to reduce the alkalinity of bauxite residue through the actions of a functional fungus to produce acidic substances. Bauxite residue samples were screened for fungal activity, and fungal tolerance was assessed using saline and alkaline media of varying concentrations, using aniline blue-PDA medium to evaluate acid production. A tolerant fungus with the ability to produce acidic substances was selected and named EEEL01 (Environmental Ecological Engineering Laboratory No. 01). Further morphological and molecular characterization identified this fungus as Penicillium oxalicum. Factors including pH, NaCl concentration, carbon and nitrogen sources were used to test the efficiency of acid production by the fungus. Based on optimal growth conditions, bauxite residue inoculated with EEEL01 reduced pH from 10.26 to 6.48 over 11?days. These results suggest that EEEL01 could effectively grow and release organic acids under extreme alkaline and saline conditions, which provide the potential for remediation.     

Effect of pH and nitrogen source on pigment production by Monascus purpureus

The effect of pH and nitrogen source on pigment production by Monascus purpureus 192F using glucose as the carbon and energy source, was studied in pH-controlled, batch fermentor cultures using HPLC analysis to determine individual pigment concentrations. A maximum of four pigments were detected in fungal extracts. These were the yellow pigments monascin and ankaflavin, the orange rubropunctatin and the red pigment monascorubramine. Monascorubramine was present as the major product in all instances. Fungal growth and ankaflavin synthesis were favoured at low pH (pH 4.0), whereas production of the other pigments was relatively independent of pH. The nature of the nitrogen source affected fungal growth and pigment production, independent of pH. Ammonium and peptone as nitrogen sources gave superior growth and pigment concentrations compared to nitrate. Ankaflavin was not detected in nitrate cultures. The highest red pigment production was obtained using a glucose-peptone medium at pH 6.5, due to the secretion of red pigments into the medium under these conditions. Correspondence to: M. R. Johns     

Effect of Different Carbon Sources on Endochitinase Production by Colletotrichum gloeosporioides

The present work analyzes the production of endochitinase by Colletotrichum gloeosporioides, a phytopathogenic fungus, using six different carbon sources and two pH values. For quantitative assay of endochitinase activity in solution, the synthetic substrate 4-methylumbelliferyl-β-D-N,N’,N”-triacetylchitotrioside was used. The major productions were obtained at pH 7.0 and 9.0, when colloidal chitin and glucose were used, whereas xylose and lactose were not good carbon sources. When testing different concentrations of colloidal chitin, glucose and glucosamine, colloidal chitin 0.5% was the best substrate, giving values of 2.4 U at the fifth day. When using glucose, best production occurred at 0.3% concentration, after 5 days growth, with values of 1.31 U. Endochitinase production was markedly decreased in high levels of glucose and in all glucosamine concentrations tested. SDS-PAGE co-polymerized with glycol-chitin analysis showed three major activity bands of 200, 100, and 95 kDa, when incubated at 50°C.     

Utilization of methanol by rhodospirillaceae

Enrichment culture of organisms growing anaerobically in the light in methanol-bicarbonate medium resulted in isolation of strains of Rhodopseudomonas gelatinosa and Rhodopseudomonas acidophila. The pH optimum for growth on methanol for all strains tested was approximately one unit higher than for growth on carbon sources containing more than one carbon atom. At the appropriate pH, 17 strains of Rhodospirillaceae out of 39 in a culture collection grew anaerobically in the light on methanol-bicarbonate. Rhodopseudomonas acidophila strain 10050 showed the most abundant growth and was studied in more detail. Its growth on methanol was stimulated by yeast extract or vitamin-free casamino acids. The organism grew on methanol-bicarbonate, methanol-formate or formate alone as the sole carbon sources. No growth was observed on methylamine or formaldehyde. In the presence of excess bicarbonate a maximum yield of 98 g cell material from 100 g methanol was obtained. Ribulose diphosphate carboxylase was present in the methanol-bicarbonate-grown organism at six times the specific activity of that in the succinate-grown organism.     

CULTURE AND HETEROTROPHY OF THE FRESHWATER DINOFLAGELLATE,PERIDINIUM CINCTUM FA. OVOPLANUM LINDEMAN1

A defined minimal medium was developed for an axenic strain of Peridinium (Indiana Culture No. LB 1336). Thiamine, biotin, and vitamin B₁₂ did not stimulate growth. Of 15 organic carbon sources tried in light, fructose, galactose, glucose, malate, malonate, and pyruvate enhanced growth but propionate retarded growth. In dark-grown cultures only media with succinate permitted growth above the survival level. Stimulation of growth by organic carbon sources was markedly pH dependent.     

Heterotrophic growth of Thiobacillus acidophilus in batch and chemostat cultures

Heterotrophic growth of the facultatively chemolithoautotrophic acidophile Thiobacillus acidophilus was studied in batch cultures and in carbon-limited chemostat cultures. The spectrum of carbon sources supporting heterotrophic growth in batch cultures was limited to a number of sugars and some other simple organic compounds. In addition to ammonium salts and urea, a number of amino acids could be used as nitrogen sources. Pyruvate served as a sole source of carbon and energy in chemostat cultures, but not in batch cultures. Apparently the low residual concentrations in the steady-state chemostat cultures prevented substrate inhibition that already was observed at 150 M pyruvate. Molar growth yields of T. acidophilus in heterotrophic chemostat cultures were low. The Y _max and maintenance coefficient of T. acidophilus grown under glucose limitation were 69 g biomass · mol^–1 and 0.10 mmol · g^–1 · h^–1, respectively. Neither the Y _max nor the maintenance coefficient of glucose-limited chemostat cultures changed when the culture pH was increased from 3.0 to 4.3. This indicates that in T. acidophilus the maintenance of a large pH gradient is not a major energy-requiring process. Significant activities of ribulose-1,5-bisphosphate carboxylase were retained during heterotrophic growth on a variety of carbon sources, even under conditions of substrate excess. Also thiosulphate- and tetrathionate-oxidising activities were expressed under heterotrophic growth conditions.     

Nutritional studies on Colletotrichum gloeosporioides Penz

Summary Colletotrichum gloeosporioides isolated from the diseased leaves ofPolyscias balfuriana could grow and sporulate on a wide range of pH (viz. from 3.0 to 9.0). Maximum growth was recorded at pH 5.5. Mannitol was the best carbon source for growth. Good growth as well as good or excellent sporulation was also recorded on glucose, fructose, maltose and starch. Organic acids (malic and tartaric) supported poor growth.Present organism could utilize a number of nitrogen sources. Nitrates in general were comparatively better sources than ammonium compounds. Aspartic acid was found to be the best nitrogen source for growth. Nitrites were toxic at lower pH values though they supported growth at alkaline medium. Best growth of the organism was obtained on MgSO₄, 7H₂O. The urea supported poor growth. ZnSO₄ inhibited the growth completely. The present organism was incapable of growing in media lacking carbon, nitrogen or sulphur.     

Carbon Catabolite Repression-Independent and pH-Dependent Production of Indoles by Rubrivivax benzoatilyticus JA2

Rubrivivax benzoatilyticus JA2 produces indole derivatives (indoles) from aniline, anthranilate or l-tryptophan. Glucose repressed indole production in R. benzoatilyticus JA2, while malate had no effect. Growth of R. benzoatilyticus JA2 on glucose resulted in decrease in culture pH (6.4) compared with malate (8.4). Growth of R. benzoatilyticus JA2 on sugar carbon sources decreased culture pH (6.4–6.6) and indole production. Further, culture pH of 6.4 repressed the indole production, and pH 8.4 promoted the production irrespective of carbon sources used for growth. Moreover, correlation between indole production and culture pH was observed, where acidic pH inhibited indole production, while alkaline pH promoted the production, suggesting the role of pH in indole production. Tryptophan-catabolizing enzyme activities are significantly high in malate-grown cultures (pH 8.4) compared with that of the glucose (pH 6.4)-grown cultures and corroborated well with indole production, indicating their role in indole production. These results confirm that indole production in R. benzoatilyticus JA2 is pH dependent rather than carbon catabolite repression.     

A physiological study of Saksenaea vasiformis Saksena

Summary The physiological conditions governing growth and sporulation ofSaksenaea vasiformis Saksena, a fungus with outstanding morphological features quite peculiar for Mucorales, were determined. Earlier studies made byTiwari (1955) on a strain of the same species had shown that this fungus is incapable of sporulating on any synthetic medium normally employed for growing fungi.The fungus had been found to have a high tolerance for very low and high pH values. It showed maximum growth at two pH values, one near neutral point, at pH 6, and another at high alkaline pH value, i.e., pH 11. Reason for this behaviour of this fungus has already been discussed. The most suitable temperature for the growth of the fungus was found to be between 30–35° C.Nearly all the carbon, nitrogen and sulphur sources which generally favour growth of fungi were found to support vegetative growth of this fungus as well. However, sporulation in this fungus had peculiar nutritional needs. Only some of the carbon sources, viz., arabinose, rhamnose, sorbose, galactose, lactose and citric acid which supported poor growth, were found to support good or excellent sporulation. But it may be stated that not all carbon sources supporting poor growth could induce sporulation of the organism. Also none of the nitrogen or sulphur sources could induce the fungus to sporulate in presence of glucose as carbon source.     

Effects of Carbon Source, Carbon Concentration, and Chlorination on Growth Related Parameters of Heterotrophic Biofilm Bacteria

Abstract To investigate growth of heterotrophic biofilm bacteria, a model biofilm reactor was developed to simulate a drinking water distribution system. Controlled addition of three different carbon sources (amino acids, carbohydrates, and humics) at three different concentrations (500, 1,000, and 2,000 ppb carbon) in the presence and absence of chlorine were used in separate experiments. An additional experiment was run with a 1:1:2 mixture of the above carbon sources. Biofilm and effluent total and culturable cells in addition to total and dissolved organic carbon were measured in order to estimate specific growth rates (SGRs), observed yields, population densities, and bacterial carbon production rates. Bacterial carbon production rates (μg C/L day) were extremely high in the control biofilm communities (range = 295–1,738). Both growth rate and yield decreased with increasing carbon concentrations. Therefore, biofilm growth rates were zero-order with respect to the carbon concentrations used in these experiments. There was no correlation between growth rate and carbon concentration, but there was a significant negative correlation between growth rate and biofilm cell density (r=−0.637, p= 0.001 control and r=−0.57, p= 0.021 chlorinated biofilms). Growth efficiency was highest at the lowest carbon concentration (range = 12–4.5%, amino acids and humics respectively). Doubling times ranged from 2.3–15.4 days in the control biofilms and 1–12.3 days in the chlorinated biofilms. Growth rates were significantly higher in the presence of chlorine for the carbohydrates, humics, and mixed carbon sources (p= 0.004, < 0.0005, 0.013, respectively). The concept of r/K selection theory was used to explain the results with respect to specific growth rates and yields. Humic removal by the biofilm bacteria (78% and 56% for the control and chlorinated biofilms, respectively) was higher than previously reported literature values for planktonic bacteria. A number of control experiments indicated that filtration of drinking water was as effective as chlorination in controlling bacterial biofilm growth. Received: 26 March 1999; Accepted: 3 August 1999; Online Publication: 15 February 2000     

Medium optimization of antifungal lipopeptide,iturin A,production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> in solid-state fermentation by response surface methodology

Iturin A, a lipopeptide antibiotic produced by Bacillus subtilis RB14-CS, suppresses the growth of various plant pathogens. Here, enhancement of iturin A production in solid-state fermentation (SSF) on okara, a soybean curd residue produced during tofu manufacturing, was accomplished using statistical experimental design. Primary experiments showed that the concentrations of carbon and nitrogen sources were the main factors capable of enhancing iturin A production, whereas initial pH, initial water content, temperature, relative humidity, and volume of inoculum were only minor factors. Glucose and soybean meal were the most effective among tested carbon and nitrogen sources, respectively. Based on these preliminary findings, response surface methodology was applied to predict the optimum amounts of the carbon and nitrogen sources in the medium. The maximum iturin A concentration was 5,591 μg/g initial wet okara under optimized condition. Subsequent experiments confirmed that iturin A production was significantly improved under the predicted optimal medium conditions. The SSF product generated under the optimized conditions exhibited significantly higher suppressive effect on the damping-off of tomato caused by Rhizoctonia solani K-1 compared with the product generated under the non-optimized conditions.     

Factors affecting pH-dependent photo-inhibition of division in Euglena gracilis

Visible light of moderate intensity (1200 ft-cd) can severely inhibit cell division of a non-photosynthetic mutant of Euglena gracilis when growth is supported by butanol, ethanol, or fumarate as sole carbon source. The degree of inhibition is pH dependent, being greatest at pH 4 to 5. A wide variety of other carbon sources permitted growth in the light without inhibition.     

RESPONSES OF THE ACIDOPHILIC ALGA EUGLENA MUTABILIS (EUGLENOPHYCEAE) TO CARBON ENRICHMENT AT pH 31

A defined medium (MAM) simulating acid mine drainage waters was developed which supported reproducible growth rates of three axenic strains of Euglena mutabilis Schmitz. Growth responses to various pHs and carbon sources were examined under defined culture conditions. A lab strain and two 5eld isolates, tested over pH range 1.5-9.0, grew best under acidic conditions (pH < 5.5) with highest growth rates at pH 3-4. Photoauxotrophic growth rates of all strains at pH 3 were improved significantly over unstirred batch controls by bubbling with air and even more by enrichment with 5% CO₂ in air. These results confirmed inorganic carbon limitation in batch culture. Organic carbon substrates were tested as possible carbon supplements in batch culture at pH 3. None of the strains survived in the dark on any of the twenty organic sources added. In the light, the lab strain exhibited some photoheterotrophic growth potential on glucose, sucrose, ethanol, and amino acids but growth was inhibited by acetate. Field strains showed little or no growth improvement with any organic substrate addition. Under simultaneous enrichment with acetate and 5% CO₂ acetate continued to be inhibitory. Simultaneous enrichment with glucose and 5% CO₂ gave higher yields of the lab strain than with CO₂ alone but did not enhance growth of the field strain. We conclude that E. mutabilis is an acidophilic photoauxotroph which appears unable to use organic carbon supplements for growth even under conditions of carbon limitation.     

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

Comparative studies on the metabolism of aniline and chloroanilines by Pseudomonas multivorans strain An 1

Summary Pseudomonas multivorans strain An 1 used aniline but not chloroanilines as the sole source of carbon and energy for growth. The aniline-adapted cells, however, were able to oxygenate chloroanilines. Relative oxygenation rates for aniline, 2-chloroaniline, 3-chloroaniline, 4-chloroaniline, and 3,4-dichloroaniline were 100, 46, 66, 20, and 3%, respectively.The first intermediates in the metabolism of chloroanilines were chlorocatechols. 3-Chlorocatechol accumulated during growth of the organism in the presence of 2-chloroaniline, whereas 4-chlorocatechol was an intermediate metabolite of 3-chloroaniline and 4-chloroaniline.Chloroanilines were able to induce synthesis of the aniline oxygenating enzyme system of Pseudomonas multivorans strain An 1. In continuous culture experiments, induction of this enzyme system appeared to depend on cell density, concentration, toxicity, and pK-values of aniline or chloroanilines.Studies with ¹⁴C-labelled 3-chloroaniline and 4-chloroaniline showed that the turnover of chloroanilines did not cease with the formation of chlorocatechols, because radioactivity was detected in the CO₂ released and in bacterial cell components. The results suggest that the turnover of chloroanilines is due to metabolism rather than to cometabolism.     

Isolation and Culture Conditions of Thermophilic Hydrogen Bacteria

Isolation of thermophilic hydrogen bacteria was performed at 50°C using enrichment culture method. One of the four strains isolated, strain TH-1 grew most rapidly. Culture conditions of strain TH-1 were investigated. Optimum temperature and pH for growth proved to be 52°C and 7.0, respectively. There existed a positive correlation between the specific growth rate and the partial pressure of carbon dioxide in the gas phase. Ammonium and nitrate are the good nitrogen sources in that order. Effect of concentrations of nitrogen source, magnesium, ferrous and phosphate ions on the cell growth was also investigated. The maximum specific growth rate (μ_max) of strain TH-1 was determined as 0.68 hr^?1 by the cultivation at 52°C in a jar fermentor containing the optimal medium at pH 7.0.