The influence of the oxidation-reduction potential of the medium upon the growth ofAzotobacter chroococcum

Summary 1. Under aerobic conditions the development ofAzotobacter is suppressed by the presence of reducing substances in concentrations lowering the aerobic potential more than ±120 mV.2. The development ofAzotobacter is not only retarded by the presence of these reducing substances, but the cells are killed.3. Under anaerobic conditions no development ofAzotobacter could be observed even when the solution was poised at a high redox potential. The cells were not killed by the anaerobic conditions; as soon as aerobic conditions were restored, a rapid development ofAzotobacter was observed.4. When the anaerobic conditions coincide with a low redox potentiale.g. in culture solutions containing glucose, the cells ofAzotobacter were killed.5. The limits of potential endured byAzotobacter appeared to be dependent on the relations between the ions in the medium.6. The cells ofAzotobacter secrete certain substances enabling them to develop at low potentials.7. The conclusion is drawn that the development ofAzotobacter is in general influenced by the redox potential of the medium. However, this influence may be very complex, as the potential of the medium is only important in so far as the redox potential(s) in the cell are changed. The factors influencing the relation between the redox potential of the medium and the redox potentials of the cell are discussed.8. ForAzotobacter, oxygen cannot be replaced by other redox systems with a suitable redox potential, most probably because these redox systems have to react with the respiratory enzymes of the cell.     

Effect of redox potential regulation on succinic acid production by Actinobacillus succinogenes

The effects of redox potential used as a control parameter on the process of succinic acid production in batch cultures of Actinobacillus succinogenes NJ113 have been investigated. In batch fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of ?100 to ?450 mV. From the results, the ORP level of ?350 mV was preferable, which resulted in high succinic acid yield (1.28 mol mol^?1), high succinic acid productivity (1.18 g L^?1 h^?1) and high mole ratio of succinic acid to acetic acid (2.02). The mechanism of redox potential regulation was discussed by metabolic flux analysis and the ratio of NADH/NAD⁺. We expected that redox potential can be used as a valuable parameter to monitor and control much more anaerobic fermentation production.     

The effect of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae

Summary The effects of acetic acid and specific growth rate on acetic acid tolerance and trehalose content of Saccharomyces cerevisiae CBS 2806 were studied using anaerobic chemostat cultures. Cells grown in the presence of acetic acid at a defined specific growth rate showed a higher acetic acid tolerance and a slightly lower trehalose content. Cells grown at a low specific growth rate showed a lower energy demand, a higher acetic acid tolerance, and a higher trehalose content. These results indicate that trehalose plays a growth rate dependent role in the tolerance of S. cerevisiae to acetic acid.     

Acetic acid production by Acetobacter aceti in a silicone tube bioreactor

Summary Continuous acetic acid fermentation was carried out using a column reactor, in which 20 to 200 thin silicone tubes (0.33 mm in outer diameter) were packed to supply oxygen by permeation. The highest value of volumetric oxygen transfer coefficient determined by the sulfite oxidation method was 2,860 h^–1, which was comparable to that of a well agitated and aerated fermentor. The maximum production rate of acetic acid by the bacterial films of Acetobacter aceti M7 grown on the shell-side surface of the tubes was 38.0 g/lh at an acetic acid concentration of 44.5 g/l. This was 29 times that of a continuous culture using a jar fermentor.     

Kinetic study on succinic acid and acetic acid formation during continuous cultures of Anaerobiospirillum succiniciproducens grown on glycerol

Succinic acid-producing Anaerobiospirillum succiniciproducens was anaerobically grown in a glycerol-fed continuous bioreactor in order to investigate the physiological responses of the cell to different pH values (5.9, 6.2, or 6.5) and various dilution rates, D. In these experiments, A. succiniciproducens showed a pH-dependent glycerol consumption behavior. When pH was maintained at 5.9 or 6.5, glycerol started to accumulate even at a very low D of 0.027 h⁻¹. Succinic acid yield was not significantly affected by the pH of the culture or the Ds. However, more acetic acid formation was observed when the growth rate of A. succiniciproducens was fast on glycerol at pH 6.2 (at D ≥ 0.15 h⁻¹). The highest obtainable succinic acid/acetic acid ratio was 40:1, which was 10 times higher than that obtained by batch cultures grown on glucose. The maximum obtainable productivity of succinic acid was 2.1 g L⁻¹ h⁻¹), which was 14 times higher than that obtained by batch culture.     

Growth promotion of groundnut by IAA producing rhizobacteria Bacillus licheniformis MML2501

B. licheniformis MML2501 which was isolated from groundnut rhizosphere soil showed increased populations on spermozphere colonisation and significantly increased the seed germination and other growth parameters in groundnut under in vitro conditions. B. licheniformis MML2501 did not solubilise phosphate but produced indole acetic acid (IAA), with a maximum of 23 μg/ml under optimised conditions such as pH 7.0, temperature 35°C, tryphtophan at a concentration of 16 mM and at 200 rpm shaken conditions. The production of IAA by B. licheniformis MML2501 was further confirmed by TLC and HPLC analyses, in which the R_f value and retention time of 0.66 and 4.1 min respectively, match with that of the authentic IAA. Seed treatment of B. licheniformis MML2501 in groundnut showed a significant increase in seed germination, other growth parameters and yield parameters under potted plant experiments.     

Structural analysis of the HiPIP from the acidophilic bacteria: Acidithiobacillus ferrooxidans

Hip is a high-potential iron–sulfur protein (HiPIP) isolated from the acidophilic bacterium, Acidithiobacillus ferrooxidans. In the present work, a structural model of Hip suggests that the role of proline residues is essential to stabilize the protein folding at very low pH. The presence of an unusual disulfide bridge in Hip is demonstrated using mass spectrometry and nuclear magnetic resonance. This disulfide bridge is necessary to anchor the N-terminal extremity of the protein, but is not involved in the acid stability of Hip. The structural parameters correlated with the pH dependence of Hip redox potential are also analysed on the basis of this model. Given that the same structural features can enhance acidic stability and lead to elevated redox potentials, modulation of the redox potentials of electron carriers may be necessary to achieve electron transfer at very low pH.     

Influence of submerged macrophytes,temperature, and nutrient loading on the development of redox potential around the sediment–water interface in lakes

Redox potential is a significant factor in aquatic systems to regulate the availability of nutrients and some metals. To assess the driving variables regulating redox potential, background parameters (dissolved oxygen, pH, temperature, chlorophyll-a, soluble reactive and total phosphorus content of water, coverage and height of submerged macrophytes) and redox potential profiles around the sediment–water interface (SWI) were measured in simulated shallow lake ecosystems. There were two nutrient regimes (enriched and non-enriched) and three temperature scenarios (unheated; +3.5°C; +5°C) installed in the experimental setups, which were constructed to study the effects of global climate change. Temperature did not have any detectable effect on redox potentials, and we presume that nutrient addition had only indirect positive effects through triggering phytoplankton dominance which causes macrophyte absence. When submerged macrophytes were present in high density (80–100% coverage), redox potentials at the SWI varied between 60–215 mV and the mean redox potential was 133 ± 34 mV (mean ± 1 SD). In contrast to this, when phytoplankton dominance was coupled to low macrophyte density (0–20% coverage), the range of redox potentials at the SWI was 160–290 mV and the mean redox potential was 218 ± 34 mV. The results revealed the primary importance of submersed macrophytes; macrophyte coverage determined alone the redox potential of the sediment–water interface by 81%. This study suggests that possible positive effects of macrophytes on redox potential can be suppressed by their negative effects in case of 80–100% coverage and total inhabitation of the water column.     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

Aeration-controlled formation of acetic acid in heterolactic fermentations

Summary Controlled aeration ofLeuconostoc mesenteroides was studied as a possible mechanism for control of the formation of acetic acid a metabolite of major influence on the taste of lactic fermented foods. Fermentations were carried out in small scale in a medium in which growth was limited by the buffer capacity only. Ethanol and acetic acid formed during the fermentation were analyzed by rapid head space gas chromatography, and the ratio of the molar concentrations of these two volatiles quantitatively predicted the balance between the formation of acetic acid and lactic acid. The oxygen concentration during the fermentations decreased rapidly to zero, meaning that oxygen transfer was limited by the volumetric oxygen transfer rate,k ₁ aC ^*. A linear correlation between k₁aC^* and the quantity of acetic acid produced was established, and it is suggested that such oxygenated heterolactic fermentation processes should be analyzed as fed-batch fermentations with oxygen as the limiting substrate. Addition of fructose in limited amounts leads to the formation of one half mole of acetic acid for each mole fructose, thus offering an alternative mechanism for controlling acetic acid formation.     

Modeling of microbial substrate conversion,growth and product formation in a recycling fermentor

Paracoccus denitrificans and Bacillus licheniformis were grown in a carbon- and energy source-limited recycling fermentor with 100% biomass feedback. Experimental data for biomass accumulation and product formation as well as rates of carbon dioxide evolution and oxygen consumption were used in a parameter optimization procedure. This procedure was applied on a model which describes biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of the biomass growth rate. The fitting procedure yielded two growth domains for P. denitrificans. In the first domain the values for the maximal growth yield and the maintenance coefficient were identical to those found in a series of chemostat experiments. The second domain could be described best with linear biomass increase, which is equal to a constant growth yield. Experimental data of a protease producing B. licheniformis also yielded two growth domains via the fitting procedure. Again, in the first domain, maximal growth yield and maintenance requirements were not significantly different from those derived from a series of chemostat experiments. Domain 2 behaviour was different from that observed with P. denitrificans. Product formation halts and more glucose becomes available for biomass formation, and consequently the specific growth rate increases in the shift from domain 1 to 2. It is concluded that for many industrial production processes, it is important to select organisms on the basis of a low maintenance coefficient and a high basic production of the desired product. It seems less important that the maximal production becomes optimized, which is the basis of most selection procedures.     

Physico-chemical properties of aqueous solutions, prepared in a membrane electrolyzer

The physicochemical properties of aqueous solutions resulting from membrane electrolysis were studied. It was shown that the catholyte contains hydrogen peroxide at a concentration of 10(-7), which is formed during the reduction of soluble oxygen. It was found that the relaxation of the catholyte redox potential is caused by the transition of the reducing agent to the gaseous phase. The relaxation characteristics of the redox potentials of the catholyte and molecular hydrogen solution were compared. The similarity of the relaxation characteristics of the catholyte and the hydrogen solution as well as the fact that the catholyte, despite its low redox potential, does not reduce either potassium ferricyanide or 5-5'-dithiobis(2-nitrobenzoic acid) support the suggestion that the redox potential of the catholyte is due to molecular hydrogen. However, based on this suggestion, it is impossible to explain the increase in the relaxation time of the catholyte with increasing ionic strength and the fact that, as the redox potential of the catholyte decreases, the concentration of other gases dissolved in the catholyte remains unchanged. Thus, the question regarding the nature of the reducing agent remains open.     

Basic Aspects of Electrode Potential Change in Submerged Fermentation

The platinum electrode potentials relative to the standard half cell depended on a pH value, dissolved oxygen concentration, equilibrium constant and oxidation reduction potentials of the liquid The overall potential change in submerged fermentation gave no independent information on these individual factors A thermostatic and pH-static apparatus excluded influences of temperatures and pH values on the electrode pontentials If the determination was completed for short time duration, potentials were governed by the dissolved oxygen tension. While the oxygen concentration was maintained at a same level, redox potential changes became a dominant. This measurement of redox potential, which gave the concentration of extremely low dissolved oxygen that could not be detected by the membrane-coated oxygen electrode, was practically useful for the control of aerobic fermentation     

Bacteria isolated from roots and rhizosphere of Vitis vinifera retard water losses,induce abscisic acid accumulation and synthesis of defense‐related terpenes in in vitro cultured grapevine

Eleven bacterial strains were isolated at different soil depths from roots and rhizosphere of grapevines from a commercial vineyard. By 16S rRNA gene sequencing 10 different genera and 8 possible at species level were identified. From them, Bacillus licheniformis Rt4M10 and Pseudomonas fluorescens Rt6M10 were selected according to their characteristics as plant growth promoting rhizobacteria (PGPR). Both produced abscisic acid (ABA), indole‐3‐acetic acid (IAA) and the gibberellins A₁ and A₃ in chemically‐defined medium. They also colonized roots of in vitro grown Vitis vinifera cv. Malbec plants. As result of bacterization ABA levels in 45 days‐old in vitro plants were increased 76‐fold by B. licheniformis and 40‐fold by P. fluorescens as compared to controls. Both bacteria diminished plant water loss rate in correlation with increments of ABA. Twenty and 30 days post bacterization the plants incremented terpenes. The monoterpenes α‐pinene, terpinolene, 4‐carene, limonene, eucalyptol and lilac aldehyde A, and the sesquiterpenes α‐bergamotene, α‐farnesene, nerolidol and farnesol were assessed by gas chromatography‐electron impact mass spectrometry analysis. α‐Pinene and nerolidol were the most abundant (µg per g of tissue in plants bacterized with P. fluorescens). Only α‐pinene, eucalyptol and farnesol were identified at low concentration in non‐bacterized plants treated with ABA, while no terpenes were detected in controls. The results obtained along with others from literature suggest that B. licheniformis and P. fluorescens act as stress alleviators by inducing ABA synthesis so diminishing water losses. These bacteria also elicit synthesis of compounds of plant defense via an ABA independent mechanism.     

Respiration of the Roots of Flood-Tolerant and Flood-Intolerant Senecio Species: Affinity for Oxygen and Resistance to Cyanide

The affinity of respiration for oxygen in the roots of six Senecio species studied was low compared with the affinity of cytochrome oxidase for oxygen. Half saturation values of approximately 22 μM oxygen were measured. Root respiration was to a large extent insensitive to cyanide in flood-tolerant as well as in flood-sensitive species. The evidence presented suggests that high activity of salicylhydroxamic acid (SHAM)-sensitive oxidase in Senecio roots was the basis for the low oxygen affinity and for the high cyanide-insensitivity of root respiration in the Senecio species. Methods are described to determine the in vivo activity of the SHAM-sensitive oxidase. It was estimated that it contributed 70% to the total root respiration. The presence of SHAM-sensitive oxidase activity could explain a higher efficiency of root growth respiration under a low oxygen tension if this alternate oxidase was inhibited at a low oxygen concentration in the root medium. However, the SHAM-sensitive oxidase was not specifically involved in either growth respiration or maintenance respiration. Its significance in regulation of the redox state of the cells is discussed.     

Inhibition of fermentation and growth in batch cultures of the yeast Brettanomyces intermedius upon a shift from aerobic to anaerobic conditions (Custers effect)

Aerobic growth of the yeast Brettanomyces intermedius CBS 1943 in batch culture on a medium containing glucose and yeast extract proceeded via a characteristic pattern. In the first phase of growth glucose was fermented to nearly equal amounts of ethanol and acetic acid. After glucose depletion, growth continued while the ethanol produced in the first phase was almost quantitatively converted to acetic acid. Finally, after a long lag phase, growth resumed with concomitant consumption of acetic acid.When the culture was made anaerobic during the first phase, growth, glucose consumption and metabolite production stopped immediately. This Custers effect (inhibition of alcoholic fermentation as a result of anaerobic conditions) was transient. After 7–8 h the culture was adapted to anaerobiosis, and growth and ethanol production resumed. The lag phase could be shortened at will by the introduction of hydrogen acceptors, such as oxygen or acetoin, into the culture. Glycerol production was not observed during any phase of growth. These results support the hypothesis that the Custers effect in this yeast is due to a disturbance of the redox balance, resulting from the tendency of the organism to produce acetic acid, and its inability to restore the balance by production of glycerol.     

Fine-tuning ethanol oxidation pathway enzymes and cofactor PQQ coordinates the conflict between fitness and acetic acid production by Acetobacter pasteurianus

The very high concentrations required for industrial production of free acetic acid create toxicity and low pH values, which usually conflict with the host cell growth, leading to a poor productivity. Achieving a balance between cell fitness and product synthesis is the key challenge to improving acetic acid production efficiency in metabolic engineering. Here, we show that the synergistic regulation of alcohol/aldehyde dehydrogenase expression and cofactor PQQ level could not only efficiently relieve conflict between increased acetic acid production and compromised cell fitness, but also greatly enhance acetic acid tolerance of Acetobacter pasteurianus to a high initial concentration (3% v/v) of acetic acid. Combinatorial expression of adhA and pqqABCDE greatly shortens the duration of starting-up process from 116 to 99 h, leading to a yield of 69 g l^-1 acetic acid in semi-continuous fermentation. As a final result, average acetic acid productivity has been raised to 0.99 g l^-1 h^-1, which was 32% higher than the parental A. pasteurianus. This study is of great significance for decreasing cost of semi-continuous fermentation for producing high-strength acetic acid industrially. We envisioned that this strategy will be useful for production of many other desired organic acids, especially those involving cofactor reactions.     

Comparative Toxicity of Three Organic Acids to Freshwater Organisms and Their Impact on Aquatic Ecosystems

The comparative toxicity of lactic acid, acetic acid, and benzoic acid to tilapia (Oreochromis mossambicus), cladoceran crustacea (Moina micrura), and oligochaete worm (Branchiura sowerbyi) were determined using static bioassay tests. Worms were found most sensitive to all the acids whereas the cladoceran was found most resistant to lactic acid and the fish most resistant to acetic acid and benzoic acid. The 96h LC₅₀ values of lactic acid, acetic acid, and benzoic acid, were, respectively, 257.73, 272.87, and 276.74 mg L^?1 for O. mossambicus; 329.12, 163.72, and 71.65 mg L^?1 for M. micrura and 50.82, 14.90, and 39.47 mg L^?1 for B. sowerbyi. Tilapia lost appetite at sub-lethal concentrations as low as 2.18 mg L^?1 lactic acid, 1.26 mg L^?1 acetic acid, and 13.84 mg L^{? 1} of benzoic acid. Growth and reproduction of the fish were affected following 90-day chronic exposure to sub-lethal concentrations of the acids. Minimum effective concentration of the acids that significantly reduced food conversion efficiency (FCE), percent increase of weight, specific growth rate, yield and fecundity of the fish were 2.18, 1.47, and 3.95 mg · L^?1 of lactic acid, acetic acid, and benzoic acid, respectively. Effects of acetic acid and benzoic acid on FCE, weight increase, and yield were not significantly different from each other whereas lactic acid produced different effects from acetic acid as well as benzoic acid. Mean values of dissolved oxygen, primary productivity, and plankton populations of the test medium significantly reduced from control at 16.94 mg L^?1 lactic acid, 16.79 mg L^?1 acetic acid, and 13.84 mg L^?1 benzoic acid.     

Substrate-energy relationships in Acetomonas oxydans

Summary Acetomonas oxydans is not able to grow on ethanol because of the lack of enzymes of the tricarboxylic acid cycle. Ethanol is merely oxidized to acetic acid.However, it was shown that Am. oxydans can utilize the energy from the oxidation of ethanol to acetic acid for growth. In this respect alcohol can be replaced by lactate.P/O ratios were measured with cell-free extracts and the following substrates: ethanol, lactate, pyruvate, acetaldehyde, NADH₂ and NADPH₂. The P/O values were identical when the cells were grown on the same medium. Glucose grown cells gave a P/O ratio for ethanol or lactate of 0.08. But with glucose-ethanol grown cells P/O ratios of 0.28 were obtained. Ethanol can be replaced by lactate for cell cultivation and as a substrate for the oxidative phosphorylation.In each oxidation step, i.e. ethanolacetaldehyde, lactatepyruvate, and acetaldehydeacetate, the same amount of ATP is produced per mole oxygen consumed when the cells were grown under comparable conditions.     

Effect of cation binding agents on sludge solubilization potential of bacteria

The aim of the present study is to increase sludge solubilization potential of bacteria by the addition of cation binding agents. During the study, three strains of bacteria B1, B2 and B3 were isolated from waste activated sludge acclimatized to a thermophilic condition (55°C). Using these strains the mixed liquor suspended solids degradation was 67, 59, and 33% and the chemical oxygen demand solubilization enhancement was 71, 62, and 36% compared with the control. Cation binding agents such as citric acid, ethylenediaminetetraacetate and sodium tripolyphosphate were added to enhance the sludge solubilization further. Among these, citric acid along with B1 was more effective in solubilization with the mixed liquor suspended solids degradation of 110% and the chemical oxygen demand solubilization enhancement of 115%. 16s rRNA technique was used to identify the bacterial species B1 and it was found to be Bacillus licheniformis. It was also observed that mixed liquor suspended solids reduced rapidly when more soluble chemical oxygen demand was released, thereby increasing sludge solubilization.