Mannitol production by heterofermentative <Emphasis Type="BoldItalic">Lactobacillus reuteri</Emphasis> CRL 1101 and <Emphasis Type="BoldItalic">Lactobacillus fermentum</Emphasis> CRL 573 in free and controlled pH batch fermentations

Certain lactic acid bacteria, especially heterofermentative strains, are capable to produce mannitol under adequate culture conditions. In this study, mannitol production by Lactobacillus reuteri CRL 1101 and Lactobacillus fermentum CRL 573 in modified MRS medium containing a mixture of fructose and glucose in a 6.5:1.0 ratio was investigated during batch fermentations with free pH and constant pH 6.0 and 5.0. Mannitol production and yields were higher under constant pH conditions compared with fermentations with free pH, the increase being more pronounced in the case of the L. fermentum strain. Maximum mannitol production and yields from fructose for L. reuteri CRL 1101 (122 mM and 75.7 mol%, respectively) and L. fermentum CRL 573 (312 mM and 93.5 mol%, respectively) were found at pH 5.0. Interestingly, depending on the pH conditions, fructose was used only as an alternative external electron acceptor or as both electron acceptor and energy source in the case of the L. reuteri strain. In contrast, L. fermentum CRL 573 used fructose both as electron acceptor and carbon source simultaneously, independently of the pH value, which strongly affected mannitol production by this strain. Studies on the metabolism of these relevant mannitol-producing lactobacilli provide important knowledge to either produce mannitol to be used as food additive or to produce it in situ during fermented food production.     

Effect of pH and lactic or acetic acid on ethanol productivity by Saccharomyces cerevisiae in corn mash

The effects of lactic and acetic acids on ethanol production by Saccharomyces cerevisiae in corn mash, as influenced by pH and dissolved solids concentration, were examined. The lactic and acetic acid concentrations utilized were 0, 0.5, 1.0, 2.0, 3.0 and 4.0% w/v, and 0, 0.1, 0.2, 0.4, 0.8 and 1.6% w/v, respectively. Corn mashes (20, 25 and 30% dry solids) were adjusted to the following pH levels after lactic or acetic acid addition: 4.0, 4.5, 5.0 or 5.5 prior to yeast inoculation. Lactic acid did not completely inhibit ethanol production by the yeast. However, lactic acid at 4% w/v decreased (P<0.05) final ethanol concentration in all mashes at all pH levels. In 30% solids mash set at pH ≤5, lactic acid at 3% w/v reduced (P<0.05) ethanol production. In contrast, inhibition by acetic acid increased as the concentration of solids in the mash increased and the pH of the medium declined. Ethanol production was completely inhibited in all mashes set at pH 4 in the presence of acetic acid at concentrations ≥0.8% w/v. In 30% solids mash set at pH 4, final ethanol levels decreased (P<0.01) with only 0.1% w/v acetic acid. These results suggest that the inhibitory effects of lactic acid and acetic acid on ethanol production in corn mash fermentation when set at a pH of 5.0–5.5 are not as great as that reported thus far using laboratory media.     

Optimal production of curdlan by Agrobacterium sp. with feedback inferential control of optimal pH profile

The pH control was important for curdlan production with Agrobacterium sp. ATCC31750. Specific cell growth rate was the highest at pH 7 and the specific curdlan production rate was at pH 5.5. The pH profiles maximizing curdlan production was changed from pH 7 optimal for cell growth to pH 5.5 optimal for curdlan production after ammonium consumption. The feedback inferential control methods, with easily measurable variables such as NaOH addition for pH control and dissolved oxygen (DO), were also applied. The pH was successfully controlled to follow optimal profiles and the maximal production of curdlan (60 g l^–1 in 120 h) was achieved with feedback optimal control.     

Temperature and pH affect the production of bacterial biofilm

The effect of different cultivation temperatures (30 and 37 °C) and pH of the media (5.5, 7.5, 8.5) on the biofilm production was compared in Pseudomonas aeruginosa, Klebsiella pneumoniae, and Vibrio cholerae non-O1 and O1 using the crystal-violet test for estimation of quantitative production of the biofilm. Decrease (46.4–98.4 %) in the biofilm production was observed at 37 °C in 8 of the tested strains (P. aeruginosa three strains, K pneumoniae two, V. cholerae non-O1 two, and V. cholerae O1 one strain) compared with the production at 30 °C. On the other hand, five strains (P. aeruginosa 1, K. pneumoniae 3, V. cholerae non-O1 1) exhibited under these conditions a higher biofilm production (103–143 %). However, this difference was not significant (p = 0.196). Increased pH lead to a higher biofilm production using all media tested. In P. aeruginosa the biofilm production at pH 8.5 was 139–244 %, at pH 7.5 136–164 % in comparison with pH 5.5. Similarly, in K. pneumoniae the biofilm production increased to 151–319 % at pH 8.5 while with the drop of pH to 7.5 the biofilm production was 113–177 % compared with pH 5.5. In V. cholerae non-O1 and O1 the biofilm production reached 204–329 % at pH 8.5, and 123–316 % at pH 7.5 (compared with the production at pH 5.5). An increase in biofilm production represented an average of 169 % (p = 0.001) at pH change from 5.5 to 7.5, with the rise of pH from 5.5 to 8.5 caused an average difference of 229 % (p = 0.001).     

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     

The effects of short-term pH decrease on the reproductive output of the copepod Acartia bifilosa – a laboratory study

This laboratory study reports some reproductive responses of the copepod Acartia bifilosa to rapid variations in pH. The imposed changes mimic those that copepods could experience due to coastal upwelling, changed mixing conditions or vertical migration. We measured effects of low pH on egg production, hatching and early nauplii development (H₀: no effects on response variables between low and ambient pH). On treatment with low pH, we found positive effects on egg production rate and nauplii development time. The positive response to low pH could be an initial stress response or show that A. bifilosa is tolerant to the experimental pH values. The result suggests that A. bifilosa is adapted to pH changes as it performs daily migrations between the depths with differing pH. It could also be advantageous for population development if eggs hatch at high speed and so reduce the possibility that they will sink into anoxic and low pH waters.     

Optimization of process parameters for the production of carbonyl reductase by <Emphasis Type="Italic">Candida viswanathii</Emphasis> in a laboratory-scale fermentor

The effect of pH, aeration and mixing on the growth and production of carbonyl reductase by Candida viswanathii was investigated in a 6.6-l fermentor. Controlling the pH at 8.0 had a very significant effect on the enzyme production. Aeration and agitation influenced the dissolved oxygen concentration which in turn affected growth as well as enzyme production. A maximum carbonyl reductase activity (53 Umg⁻¹) was attained in 24 h under the optimal cultivation conditions of controlled pH at 8.0, aeration rate 1 vvm and an agitation speed of 250 rpm at 25°C. The enzyme activity was twice as high (56 Umg⁻¹) in the fermentor as compared to a shake flask. Further, the duration of growth and enzyme production in the fermentor was shortened. Cells cultivated under the optimized conditions were used for the preparative scale reduction of N, N-dimethyl-(3-keto)-2-thienyl-propanamine to (S)-N, N-dimethyl-(3-hydroxy)-2-thienyl-propanamine, a key intermediate in the production of the important antidepressant drug (S)-duloxetine.     

Optimal pH control of batch processes for production of curdlan by Agrobacterium species

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L⁻¹) compared to that of constant pH operation (36 g L⁻¹). Received 24 November 1998/ Accepted in revised form 17 June 1999     

Effect of environmental factors and precursors on oxalic acid production,mycelial biomass and virulence of a potential bioherbicide isolate of Sclerotium rolfsii SC64 produced in liquid culture

The fungus Sclerotium rolfsii is presently under development as a bioherbicide for broadleaf weed species using fungus-infested substrates as application material in this laboratory. The effect of environmental factors and three precursors (citric acid, ascorbic acid, and sodium succinate) on mycelial growth, oxalic acid production, and virulence by SC64 in liquid culture were investigated. The results showed that for mycelia growth the optimum liquid medium was Modified Richard's solution (MRS) among the five tested media, but potato dextrose broth (PDB) produced the maximum oxalic acid production and virulence on detached Solidago canadensis leaves. When PDB was used as the basic medium, the oxalic acid/mycelial dry weight (mg g^–1) ratio reached the peak 4 days after inoculation. The optimum temperature for oxalic acid production was at 27°C, but increased mycelial dry weight and virulence were observed at 30°C. The optimum range of initial pH value for oxalic acid accumulation was 4.0–6.0, with the optimal pH 5.0; highest mycelial growth was with an initial pH 3.5–6.0 (optimum pH 5.0) and subsequently pH 3.5–5.5 (maximum at pH 3.5). Both mycelial dry weight and oxalic acid production showed a decreasing trend as a result of the precursor of oxalic acid being added to PDB. Among the three precursors, the greatest decrease in mycelial dry weight, and oxalic acid production was caused by sodium succinate. This clarification of optimal conditions for production of mycelial biomass while insuring high concentrations of oxalic acid and high virulence should be useful for further development of this fungus as biocontrol agent.     

Effect of phytate on aflatoxin formation by Aspergillus parasiticus and Aspergillus flavus in synthetic media

The effect of phytate on the production of aflatoxins by Aspergillus parasiticus and Aspergillus flavus grown on synthetic media was examined. In the absence of pH control (initial pH 4.5–6.5) for A. parasiticus, phytate (14.3 mM) caused a six-fold decrease in aflatoxins in the medium and a ten-fold decrease in those retained by the mycelia. When the initial pH of the medium was adjusted to 4.5 no effect on aflatoxin production was observed. With A. flavus or A. parasiticus grown on media with a higher initial pH value (6 to 7), the presence of phytate in the media caused an increase in aflatoxin production. These results are inconsistent with previous studies which indicated that phytate depresses aflatoxin production by rendering zinc, a necessary co-factor for aflatoxin biosynthesis, unavailable to the mold.     

Influence of pH regulation and nutrient content on cephalosporin C production in solid-state fermentation by Acremonium chrysogenum C10

Aims: To investigate the effect of pH regulation and nutrient concentration on cephalosporin C (CPC) production in solid‐state fermentation (SSF), using sugarcane bagasse as inert support, impregnated with liquid medium. Methods and Results: Solid‐state fermentation using different initial pH values, buffer and nutrient concentrations were performed. Results revealed pH as a key parameter in CPC SSF, as it hampered the antibiotic production not only above 7·8, but also under 6·4. Using initial pH lower than 6·8 and PB in the solid medium, it was possible to keep pH within the production range, increase the production period (from 1 to 3 days) and hence the CPC yield from 468 to 3200 μg gdm^?1 (g^?1 of dry matter). Conclusion: Parameters that help to keep pH in adequate values for CPC production in SSF, such as initial pH, buffering system and nutrient concentration, can greatly increase the production time and CPC yields in this fermentation technique. Significance and Impact of the Study: This is the first work on CPC production on impregnated support, and the only one revealing pH as a key parameter; it is also shown that high nutrient concentration can improve CPC yields in SSF as long as pH is kept under control.     

Generalized model of the effect of pH on lactate fermentation and citrate bioconversion in Lactococcus lactis ssp. Lactis biovar. diacetylactis

An aroma-imparting mesophilic lactic starter (Lactococcus lactis ssp. lactis biovar. diacetylactis) was studied in batch culture in medium with 50 g·l^–1 lactose and 2 g·l^–1 citrate. The effect of pH on the physiology of growth and the production of flavour compounds was investigated with a mathematical model. The specific rates of growth and of lactose fermentation obeyed a law of non-competitive inhibition by lactic acid produced, inhibition increasing as the pH of the medium decreased. The pH thus acted indirectly by increasing the proportion of non-dissociated lactic acid, identified as the inhibiting form of lactic acid. The generalized model, taking into account the effect of pH, was tested using fermentations at pH controlled at different values (4.5–6.5), as well as with a fermentation conducted at non-regulated pH. These simulations supported the working hypotheses. The effect of pH on the fermentation of citric acid resulted in an increase in the maximal specific rate of citrate utilization, in the bioconversion yield, and in the constant of diacetyl and acetoin reduction at acid pH. The production of flavour compounds is a complex phenomenon resulting from the interaction of pH, citric acid concentration, and the physiological state of the cells. These results are discussed with respect to the use of this strain in the preparation of manufactured dairy products.     

Influence of temperature and pH on production of two bacteriocins by Leuconostoc mesenteroides subsp. mesenteroides FR52 during batch fermentation

The influence of temperature and pH on growth of Leuconostoc mesenteroides subsp. mesenteroides FR52 and production of its two bacteriocins, mesenterocin 52A and mesenterocin 52B, was studied during batch fermentation. Temperature and pH had a strong influence on the production of the two bacteriocins which was stimulated by slow growth rates. The optimal temperature was 20 °C for production of mesenterocin 52A and 25 °C for mesenterocin 52B. Optimal pH values were 5.5 and 5.0 for production of mesenterocin 52A and mesenterocin 52B respectively. Thus, by changing the culture conditions, production of one bacteriocin can be favoured in relation to the other. The relationship between growth and specific production rates of the two bacteriocins, as a function of the culture conditions, showed different kinetics of production and the presence of several peaks in the specific production rates during growth. Received: 13 February 1998 / Received revision: 27 May 1998 / Accepted: 1 June 1998     

Effect of pH on the stability of Pleurotus eryngii versatile peroxidase during heterologous production in Emericella nidulans

Complementary DNA (cDNA) encoding the new versatile peroxidase from the ligninolytic basidiomycete Pleurotus eryngii has been expressed in the ascomycete Emericella nidulans. In recombinant E. nidulans cultures, the pH reached values as high as 8.3, correlating with a sharp decrease in peroxidase activity. Peroxidase was rapidly inactivated at alkaline pH, but was comparatively stable at acidic pH. The peroxidase inactivation in alkaline buffer could be reversed by adding Ca²⁺ and lowering the pH. However, reactivation did not result after incubating the enzyme in non-buffered E. nidulans cultures that reached pH 7.5. To optimize recombinant peroxidase production, the effect of controlling the pH in E. nidulans bioreactor cultures was studied. An extended growth period, and a significant increase in the recombinant peroxidase level (5.3-fold higher activity than in the bioreactor without pH control) was obtained when the pH was maintained at 6.8, showing that culture pH is an important parameter for recombinant peroxidase production.     

Influence of pH on growth and bacteriocin production byLactococcus lactis subsp.lactis 14ONWC during batch fermentation

The influence of pH on growth, and lactic acid and bacteriocin production byLactococcus lactis subsp.lactis 140 NWC was studied during batch fermentation in a lactose-based complex medium. Growth and lactic acid production were modelled using a simple logistic equation while substrate consumption was found to be a function growth and lactic acid production rate. The optimal pH for growth and lactic acid production was between 6.0 and 6.5. Bacteriocin production showed primary metabolite kinetics. pH had a dramatic effect on the production of the bacteriocin, lactococcin 140. A maximum activity of 15.4 × 10⁶ AU (arbitrary units) 1^–1 was obtained after 7 h at pH 5.5. Maximum bacteriocin activity was achieved before the end of growth and was followed by a decrease in activity, which was due to adsorption to the cells of the producing organism, possibly followed by degradation by specific proteases. Both bacteriocin production and degradation rates were higher at pH 5.0 and 5.5, resulting in sharper activity peaks than at pH 6.0 or 6.5. On the basis of the experimental results a qualitative model for bacteriocin production is proposed.     

Effect of pH on production of xylanase by Trichoderma reesei on xylan- and cellulose-based media

Trichoderma reesei VTT-D-86271 (Rut C-30) was cultivatedon media based on cellulose and xylan as the main carbon source in fermentors with different pH minimum controls. Production of xylanase was favoured by a rather high pH minimum control between 6.0 and 7.0 on both cellulose- and xylan-based media. Although xylanase was produced efficiently on cellulose as well as on xylan as the carbon source, significant production of cellulose was observed only on the cellulose-based medium and best production was at lower pH (4.0 minimum). Production of xylanase at pH 7.0 was shown to be dependent on the nature of the xylan in the cultivation medium but was independent of other organic components. Best production of xylanase was observed on insoluble, unsubstituted beech xylan at pH 7.0. Similar results were obtained in laboratory and pilot (200-l) fermentors. Downstream processing of the xylanase-rich, low-cellulose culture filtrate presented no technical problems despite apparent autolysis of the fungus at the high pH. Enzyme produced in the 200-l pilot fermentor was shown to be suitable for use in enzyme-aided bleaching of kraft pulp. Due to the high xylanase/cellulase ratio of enzyme activities in the culture filtrate, pretreatment for removal of cellulase activity prior to pulp bleaching was unnecessary. Correspondence to: M. J. Bailey     

Statistical optimization of culture media and conditions for production of mannan by Saccharomyces cerevisiae

In view of the increase in Saccharomyces cerevisiae mannan content, the culture medium and condition for S. cerevisiae were optimized in this study. The influence of culture medium ingredients such as carbon and nitrogen sources, inorganic ion, and enzyme activator on mannan production were evaluated using factional design. The mathematical model was established by the quadratic rotary combination design through response surface analysis. The optimized concentrations of culture medium were determined as follows: 4.98 g/100 mL, sucrose; 4.39 g/100 mL, soybean peptone; 3.10 g/100 mL, yeast extract; and 2.21 g/100 mL, glycerol. The optimized culture medium increased mannan production from 82.7 ± 3.4 mg/100 mL to 162.53 ± 3.47 mg/100 mL. The influence of original pH, inoculum size, temperature, and media volume on mannan production was evaluated and confirmed by orthogonale experimental design, with the order of effect as follows: media volume > temperature > initial pH > inoculation size. The optimized culture condition was pH, 5; inoculum size, 5 ml; temperature, 32°C; and media volume, 40 mL. The maximum mannan production increased to 258.5 ± 9.1 mg/100 mL at the optimum culture condition. It was evident that the mannan production was affected significantly by culture medium and condition optimization (p < 0.01).     

Production of xylanases byAspergillus fumigatus andAspergillus oryzae on xylan-based media

Aspergillus fumigatus andA. oryzae were cultivated in laboratory fermenters on media containing xylan as the main carbon source.A. fumigatus produced xylanase on unsubstituted, insoluble beech xylan but growth and enzyme production on soluble xylo-oligosaccharides from the steaming of hardwood were poor due to the presence of inhibitors. An essential prerequisite for good xylanase production byA. fumigatus was decrease in the pH of the cultivation below 3.0 At higher pH values, the production of proteolytic enzymes caused degradation of the xylanase activity already produced.A. oryzae produced rather less xylanase activity thanA. fumigatus on the beech xylan medium but, after adaptation, was capable of efficient enzyme production on the steamed substrate.M.J. Bailey and L. Viikari are with the VTT, Biotechnical Laboratory, PO Box 202, SF-02151 Espoo, Finland     

Optimization of culturing conditions of <Emphasis Type="Italic">Porphyridium cruentum</Emphasis> using uniform design

The red microalga Porphyridium contains many valuable compounds such as polysaccharides, polyunsaturated fatty acids, and phycoerythrin (PE). In this study, a uniform design method and regression analysis were used to investigate the effects of initial pH, light intensity, inoculation ratio, and liquid volume in flask on the optimal biomass, exopolysaccharides (EPS), and PE production of Porphyridium cruentum in a batch culture at laboratory scale. Using regression analysis, we obtained the models to clarify the effects of individual factors and their interactions on the biomass, EPS, and PE production of P. cruentum. The optimal condition for the biomass was the following: pH 5.0, light intensity 7098.0 lx, inoculation ratio 1:17.2, and liquid volume 100.0 ml; for EPS was pH 5.0, light intensity 4501.0 lx, inoculation ratio 1:20, and liquid volume 100.3 ml; while pH 8.0, light intensity 7100.0 lx, inoculation ratio 1:20, and liquid volume 100.3 ml was the best for PE production. The maximum biomass 3.27 g/l, EPS production 543.1 mg/l, and PE production 132.0 mg/l were demonstrated by confirmatory experiment to the optimum culture conditions in a reciprocal shaker. The statistical methods used in the present study are useful strategies for optimizing of culture conditions for other microalgae.