Pilot-scale production of carboxymethylcellulase from rice hull by Bacillus amyloliquefaciens DL-3

Optimal conditions for pilot-scale production of the carboxymethylcellulase (CMCase) by Bacillus amyloliquefaciens DL-3 were investigated. The best carbon and nitrogen sources for the production of CMCase by B. amyloliquefaciens DL-3 were found to be rice hull and peptone and their optimal concentrations were 5.0 and 0.20% (w/v), respectively. Optimal temperature and initial pH for the production of CMCase were 37°C and 6.8. Optimal agitation speed and aeration rate for the production of CMCase were 300 rpm and 1.0 vvm in a 7 L bioreactor, which were different from those for the cell growth of B. amyloliquefaciens DL-3. The highest productions of CMCase by B. amyloliquefaciens DL-3 from 5.0% (w/v) rice hull as a carbon source under optimal conditions in a 7 or 100 L bioreactor were 220 and 367 U/mL, respectively.     

Plant cell growth under different levels of oxygen and carbon dioxide

Summary An experimental system, in which gases of known composition were passed through flasks, was used to systematically study the effects of oxygen and carbon dioxide on plant cell growth. As expected, oxygen limiting conditions resulted in suppressed growth of Catharanthus roseus cultures. Oxygen limitations did not alter the amount of cell mass produced per gram of sugar consumed which suggests that the production of fermentative metabolites was limited. Varying levels of carbon dioxide were observed to have no effect on the growth rates of either C. roseus or Daucus carota cultures. The amount of C. roseus cell mass generated per gram of sugar consumed appeared to be slightly increased at higher carbon dioxide levels.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

Rapid identification of target genes for 3-methyl-1-butanol production in Saccharomyces cerevisiae

Extracellular conditions determine the taste of fermented foods by affecting metabolite formation by the micro-organisms involved. To identify targets for improvement of metabolite formation in food fermentation processes, automated high-throughput screening and cDNA microarray approaches were applied. Saccharomyces cerevisiae was cultivated in 96-well microtiter plates, and the effects of salt concentration and pH on the growth and synthesis of the fusel alcohol-flavoured substance, 3-methyl-1-butanol, was evaluated. Optimal fermentation conditions for 3-methyl-1-butanol concentration were found at pH 3.0 and 0% NaCl. To identify genes encoding enzymes with major influence on product formation, a genome-wide gene expression analysis was carried out with S. cerevisiae cells grown at pH 3.0 (optimal for 3-methyl-1-butanol formation) and pH 5.0 (yeast cultivated under standard conditions). A subset of 747 genes was significantly induced or repressed when the pH was changed from pH 5.0 to 3.0. Expression of seven genes related to the 3-methyl-1-butanol pathway, i.e. LAT1, PDX1, THI3, ALD4, ILV3, ILV5 and LEU4, strongly changed in response to this switch in pH of the growth medium. In addition, genes involved in NAD metabolism, i.e. BNA2, BNA3, BNA4 and BNA6, or those involved in the TCA cycle and glutamate metabolism, i.e. MEU1, CIT1, CIT2, KDG1 and KDG2, displayed significant changes in expression. The results indicate that this is a rapid and valuable approach for identification of interesting target genes for improvement of yeast strains used in industrial processes.     

Growth yields and glucose metabolism of N<Subscript>2</Subscript>-fixing <Emphasis Type="Italic">Gluconacetobacter diazotrophicus</Emphasis> at different culture pH values

Gluconacetobacter diazotrophicus was grown in chemostat under N₂-fixing conditions at different culture pH values (from 2.5 to 7.5) with glucose as the C-source. Maximum glucose and oxygen utilization yields were observed at pH values between 5.0 and 6.5. Yields, although lower, were not severely affected at acidic (2.5–4.5) and moderate alkaline (7.5) pH values. But, at pH values just over 7.5, cultures became unstable and washed out. Maximum biomass yields coincided with optimal activity (and minimal synthesis) of pyrroloquinoline quinone (PQQ)-linked glucose dehydrogenase (PQQ-GDH). At external pH values of 7.0 and above, whereas PQQ-GDH was actively synthesized, a very low in situ activity could be detected. The lack of PQQ-GDH activity at moderate alkaline pH values seems to be the cause of lack of growth of this organism under these conditions.     

The adaptive acid tolerance response in root nodule bacteria and Escherichia coli

Root nodule bacteria and Escherichia coli show an adaptive acid tolerance response when grown under mildly acidic conditions. This is defined in terms of the rate of cell death upon exposure to acid shock at pH 3.0 and expressed in terms of a decimal reduction time, D. The D values varied with the strain and the pH of the culture medium. Early exponential phase cells of three strains of Rhizobium leguminosarum (WU95, 3001 and WSM710) had D values of 1, 6 and 5 min respectively when grown at pH 7.0; and D values of 5, 20 and 12 min respectively when grown at pH 5.0. Exponential phase cells of Rhizobium tropici UMR1899, Bradyrhizobium japonicum USDA110 and peanut Bradyrhizobium sp. NC92 were more tolerant with D values of 31, 35 and 42 min when grown at pH 7.0; and 56, 86 and 68 min when grown at pH 5.0. Cells of E. coli UB1301 in early exponential phase at pH 7.0 had a D value of 16 min, whereas at pH 5.0 it was 76 min. Stationary phase cells of R. leguminosarum and E. coli were more tolerant (D values usually 2 to 5-fold higher) than those in exponential phase. Cells of R. leguminosarum bv. trifolii 3001 or E. coli UB1301 transferred from cultures at pH. 7.0 to medium at pH 5.0 grew immediately and induced the acid tolerance response within one generation. This was prevented by the addition of chloramphenicol. Acidadapted cells of Rhizobium leguminosarum bv. trifolii WU95 and 3001; or E. coli UB1301, M3503 and M3504 were as sensitive to UV light as those grown at neutral pH.     

Effect of pH on organic acid production byClostridium propionicum in test tube and fermentor cultures

Summary Clostridum propionicum is a chemical autotroph that metabolizes alanine to propionic acid (reduction product) and acetic acid (oxidation product). The ratio of propionate/acetate predicted by the electron balance is 2:1. This study reports the effect of pH on growth and organic acid production by this organism when grown in both test tube cultures initially buffered from pH 7.0 to 5.0, and in fermentors maintained at pH 7.0 and 6.5. Highest growth and organic acid production was found at pH 7.0 in both cases. HPLC analysis showed that at pH 7.0, the ratios of propionate to acetate were 0.45:1 (stationary tube, 24 h). The highest ratio observed was 1.8:1 (stationary tube, pH 6.0, 24h). This tube produced 8.5% of the acids produced in the pH 7.0 culture tube. The identify of the major portion of the reduction products of the organism remains unknown.     

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.     

Factors affecting tyramine production in <Emphasis Type="Italic">Enterococcus durans</Emphasis> IPLA 655

The decarboxylation of tyrosine by certain lactic acid bacteria leads to the undesirable presence of tyramine in fermented foods. Tyramine is the most frequent biogenic amine found in cheese and is also commonly found in other fermented foods and beverages. The tyramine-producing strain Enterococcus durans IPLA 655 was grown in a bioreactor under different conditions to determine the influence of carbon source, tyrosine and tyramine concentrations, and pH on tyramine production. The carbon source appeared to have no significant effect on the production of tyramine. In contrast, tyrosine was necessary for tyramine production, while the presence of tyramine itself in the growth medium inhibited such production. pH showed by far the greatest influence on tyramine synthesis; tyramine was produced in the greatest quantities at pH 5.0, although this was accompanied by a reduced growth rate.     

Production of a raw starch saccharifying amylase byBacillus alvei grown on different agricultural substrates

Maximum activity of the amylase ofBacillus alvei was attained after growth of the organism on sorghum starch. Rice, corn, yam, cassava and potato starch gave high enzyme activities as did soluble starch. Glucose, maltose and glycerol were less effective. Optimum conditions for both growth and enzyme production were pH 6.8 at 40°C.     

Mutants of Alcaligenes eutrophus defective in autotrophic metabolism

Forty-four mutants of Alcaligenes eutrophus H 16 were isolated which grew poorly or not at all under autotrophic conditions. Four types were characterized with respect to their defects and their physiological properties. One mutant lacked both enzymes specific for autotrophic CO₂ fixation, another one lacked both hydrogenases, and two mutants lacked either the membrane-bound or the soluble hydrogenase. Comparing the results of studies on these mutant types, the following conclusions were drawn: the lack of each hydrogenase enzyme could be partially compensated by the other one; the lack of membrane-bound hydrogenase did not affect autotrophic growth, whereas the lack of the soluble hydrogenase resulted in a decreased autotrophic growth rate. When pyruvate as well as hydrogen were supplied to the wild-type, the cell yield was higher than in the presence of pyruvate alone. Mutant experiments under these conditions indicated that either of both hydrogenases was able to add to the energy supply of the cell. Only the soluble hydrogenase was involved in the control of the rate of hydrogen oxidation by carbon dioxide; the mutant lacking this enzyme did not respond to the presence or absence of CO₂. The suppression of growth on fructose by hydrogen could be mediated by either of both hydrogenases alone.     

Phosphate starvation responses are mediated by sugar signaling in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Phosphate (Pi) is one of the least available plant nutrients in soils. It is associated with dynamic changes in carbon fluxes and several crucial processes that regulate plant growth and development. Pi levels regulate the expression of large number of genes including those involved in photosynthesis and carbon metabolism. Herein we show that sugar is required for Pi starvation responses including changes in root architecture and expression of phosphate starvation induced (PSI) genes in Arabidopsis. Active photosynthesis or the supplementation of sugar in the medium was essential for the expression of PSI genes under Pi limiting conditions. Expression of these genes was not only induced by sucrose but also detected, albeit at reduced levels, with other metabolizable sugars. Non-metabolizable sugar analogs did not induce the expression of PSI genes. Although sugar input appears to be downstream of initial Pi sensing, it is absolutely required for the completion of the PSI signaling pathway. Altered expression of PSI genes in the hexokinase signaling mutant gin2 indicates that hexokinase-dependent signaling is involved in this process. The study provides evidence for requirement of sugars in PSI signaling and evokes a role for hexokinase in some components of Pi response mechanism.     

Identification of Escherichia coli genes whose expression increases as a function of external pH

Summary Using transposon TnphoA and a plate screening method, we have isolated a set of Escherichia coli strains carrying phoA fusions with genes whose expression is modulated as a function of external pH. Besides fusions with the ompF gene and the malB locus, thirteen independent fusions were analysed whose expression is maximal during growth at pHs ranging from 7.0 to 8.5 and minimal during growth at pH 5.0. Six different genetic loci, called phmA, phmB, phmC, phmD, phmE and phmF (for pH modulated) were characterized and localized on the E. coli chromosome at approx. 12, 18, 41, 45, 75 and 84 min, respectively. Expression of phmA: :phoA fusions is also influenced when internal pH or environmental conditions such as osmolarity or anaerobiosis are modified. EnvZ protein is not involved in the regulation of phm : :phoA fusions.     

Utilization of phosphorus by pasture plants supplied with myo-inositol hexaphosphate is enhanced by the presence of soil micro-organisms

A range of pasture grass (Danthonia richardsonii and Phalaris aquatica) and legume (Medicago polymorpha, M. sativa, Trifolium repens and T. subterraneum) species showed limited capacity to obtain phosphorus (P) from inositol hexaphosphate (IHP), when grown in either sterile agar (pH 5.0 or 5.5) or sand-vermiculite media (pH 5.0). The total P content of shoots from IHP-supplied plants grown in agar was between 20% and 34% of that for seedlings supplied with an equivalent amount of P as inorganic phosphate (P_i), while in sand-vermiculite, the total P content of IHP-grown plants was between 5 and 10% of control plants. The poor ability of plants to utilize P from IHP resulted in significantly lower tissue P concentrations and, in general, reduced plant dry weight accumulation. In contrast, the P nutrition of plants supplied with IHP was significantly improved by inoculating media with either a cultured population of total soil micro-organisms or with a specific isolate of Pseudomonas sp., selected for its ability to release phosphate from IHP (strain CCAR59; Richardson and Hadobas, 1997 Can. J. Micro. 43, 509-516). In agar and sand-vermiculite media, respectively, the P content of IHP-grown plants increased with inoculation by up to 3.9- and 6.8-fold, such that the dry weight and P content of the plant material were equivalent to those observed for control plants supplied with P_i. However, the response to inoculation was dependent on the growth medium and the source of micro-organisms used. In sand-vermiculite, the cultured population of soil micro-organisms was effective when IHP was supplied at an equivalent level of P_i required for maximum plant growth. By comparison, inoculation of plants with the Pseudomonas strain was only effective at very high levels of IHP supply (×36), whereas in agar a response to inoculation occurred at all levels of IHP. The ability of pasture plants to acquire P from phytate was, therefore, influenced by the availability of IHP substrate, which was further affected by the presence of soil micro-organisms. Our results show that in addition to having an effect on the sorption characteristics of the growth media, soil micro-organisms also provided a source of phytase for the dephosphorylation of phytate for subsequent utilization of P_i by plants.     

The pH profile for acid-induced elongation of coleoptile and epicotyl sections is consistent with the acid-growth theory

The acid-growth theory predicts that a solution with a pH identical to that of the apoplast of auxintreated tissues (4.5–5.0) should induce elongation at a rate comparable to that of auxin. Different pH profiles for elongation have been obtained, however, depending on the type of pretreatment between harvest of the sections and the start of the pH-incubations. To determine the acid sensitivity under in vivo conditions, oat (Avena sativa L.) coleoptile, maize (Zea mays L.) coleoptile and pea (Pisum sativum L.) epicotyl sections were abraded so that exogenous buffers could penetrate the free space, and placed in buffered solutions of pH 3.5–6.5 without any preincubation. The extension, without auxin, was measured over the first 3 h. Experiments conducted in three laboratories produced similar results. For all three species, sections placed in buffer without pretreatment elongated at least threefold faster at pH 5.0 than at 6.0 or 6.5, and the rate elongation at pH 5.0 was comparable to that induced by auxin. Pretreatment of abraded sections with pH-6.5 buffer or distilled water adjusted to pH 6.5 or above gave similar results. We conclude that the pH present in the apoplast of auxin-treated coleoptile and stems is sufficiently low to account for the initial growth response to auxin.Abbreviations FS free space - IAA indole-3-acetic acid This research was supported by a grant from the National Adonautics and space Administration (NASA), NAGW 1394 to R.E.C., NASA grant NAGW-297 to M.L.E., and NASA grant NAG 1849 to D.L.R.