Production of isomeric 9,10,13 (9,12,13)-trihydroxy-11E (10E)-octadecenoic acid from linoleic acid by Pseudomonas aeruginosa PR3

Trihydroxy unsaturated fatty acids with 18 carbons have been reported as plant self-defense substances. Their production in nature is rare and is found mainly in plant systems. Previously, we reported that a new bacterial isolate, Pseudomonas aeruginosa PR3, converted oleic acid and ricinoleic acid to 7,10-dihydroxy-8(E)-octadecenoic acid and 7,10,12-trihydroxy-8(E)-octadecenoic acid, respectively. Here we report that strain PR3 converted linoleic acid to two compounds: 9,10,13-trihydroxy-11(E)-octadecenoic acid (9,10,13-THOD) and 9,12,13-trihydroxy-10(E)-octadecenoic acid (9,12,13-THOD). Stereochemical analyses showed the presence of 16 different diastereomers — the maximum number possible. The optimum reaction temperature and pH for THOD production were 30°C and 7.0, respectively. The optimum linoleic acid concentration was 10 mg/ml. The most effective single carbon and nitrogen sources were glucose and sodium glutamate, respectively. However, when a mixture of yeast extract (0.05%), (NH₄)₂HPO₄ (0.2%), and NH₄NO₃ (0.1%) was used as the nitrogen source, THOD production was higher by 8.3% than when sodium glutamate was the nitrogen source. Maximum production of total THOD with 44% conversion of substrate was achieved at 72 h of incubation, after which THOD production plateaued up to 240 h. THOD production and cell growth increased in parallel with glucose concentration up to 0.3%, after which cell growth reached its maximum and THOD production did not increase. These results suggested that THODs were not metabolized by strain PR3. This is the first report of microbial production of 9,10,13- and 9,12,13-THOD from linoleic acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 109–115. Received 18 March 2000/ Accepted in revised form 09 June 2000     

Effect of ammonium sulphate concentration and agitation speed on the kinetics of alginate production by Azotobacter vinelandii DSM576 in batch fermentation

Growth and alginate production by Azotobacter vinelandii DSM576 as a function of initial ammonium sulphate concentration (0.45–1.05 g l⁻¹) and agitation speed (300–700 rpm) were studied in batch fermentations at controlled pH. The time course of growth, alginate production and substrate consumption and the effect of nitrogen concentration and agitation speed on kinetic parameters and on maximum alginate molecular weight (MW) was modelled using empirical equations. The kinetics of growth, alginate production and polymerization were deeply affected by agitation speed and, to a lesser extent, by inorganic nitrogen concentration. Average and maximum specific growth rate and maximum alginate MW all increased with agitation speed, and were higher at intermediate ammonium sulphate concentration. Maximum alginate MW (>250,000) was obtained at high agitation speed (600–700 rpm) and alginate depolymerization was limited or did not occur at all when the agitation speed was higher than 500 rpm, while at 400 rpm depolymerization significantly reduced the alginate. However, alginate yield was negatively affected by increasing agitation speed. A good compromise between alginate yield (>2 g l⁻¹) and quality (MW>250,000) was obtained with agitation speed of 500–600 rpm and 0.75–0.90 g l⁻¹ of ammonium sulphate. Journal of Industrial Microbiology & Biotechnology (2000) 25, 242–248. Received 23 February 2000/ Accepted in revised form 04 August 2000     

Effect of culture conditions on the production of an extracellular proteinase byThermus sp. Rt41A

Thermus sp. Rt41A produced a single extracellular proteinase, as determined by fast protein liquid chromatography and isoelectric focusing. Proteinase activity was expressed from very early in the log phase, and halted when the growth substrate was exhausted. There was no continued proteinase production in the stationary phase. Proteinase production was not stimulated by O₂ limitation, not repressed by amino acid growth substrates, and its production could not be correlated to the type or oxidation state of the carbon and energy source or the growth rate on different carbon and energy sources. Growth on certain substrates, e.g. glutamate and glucose, resulted in production of high levels of proteinase, whereas others, such as acetate, resulted in low proteinase levels. Acetate repressed proteinase production in cultures growing on L-glutamate. In continuous culture on L-glutamate, acetate or pyruvate, proteinase production was highest at higher growth (dilution) rates. The kinetics of proteinase production in continuous culture on L-glutamate can be interpreted as evidence for the constitutive nature of proteinase expression byThermus sp. Rt41A. The data obtained show that the control of proteinase production is different to that postulated forThermus sp. Ok6.A1.     

Efficient production of menaquinone (vitamin K2) by a menadione-resistant mutant of Bacillus subtilis

Efficient production of menaquinone (MK) by Bacillus subtilis was achieved. An edible strain of B. subtilis, isolated from the traditional Japanese food natto, was mutated to improve MK productivity. A menadione-resistant mutant producing 30% more MK than its parent strain was obtained. Soybean extract and glycerol were the best nitrogen and carbon sources, respectively, among the sources tested. Addition of yeast extract also increased MK productivity. The maximum concentration of MK reached about 35.0 mg/l after 4 days of culture in a jar fermenter. The pH of the medium decreased to 5.5 after the start of cultivation, then spontaneously increased to 7.7–8.0. This pH change might be important in the production of MK because only small amounts of MK were obtained when pH was controlled at 5.7, 6.0, 7.0, 7.5 or 8.0. Journal of Industrial Microbiology & Biotechnology (2001) 26, 115–120. Received 24 April 2000/ Accepted in revised form 14 August 2000     

Kinetics of kojic acid fermentation by Aspergillus flavus using different types and concentrations of carbon and nitrogen sources

Kinetics of kojic acid fermentation by Aspergillus flavus Link 44-1 using various sources of carbon [glucose, xylose, sucrose, starch, maltose, lactose or fructose] and nitrogen [NH₄Cl, (NH₄)₂S₂O₈, (NH₄)₂NO₃, yeast extract or peptone] were analyzed using models based on logistic and Luedeking–Piret equations. The highest kojic acid production (39.90 g l⁻¹) in submerged batch fermentation was obtained when 100 g l⁻¹ glucose was used as a carbon source. Organic nitrogen sources such as peptone and yeast extract were favorable for kojic acid production as compared to inorganic nitrogen sources. Yeast extract at 5 g l⁻¹ was optimal. The optimal carbon to nitrogen (C/N) ratio for kojic acid fermentation was 93.3. In a resuspended cell system, the rate of glucose conversion to kojic acid by cell-bound enzymes increased with increasing glucose concentration up to 70 g l⁻¹, suggesting that the reaction followed the Michaelis–Menten enzyme kinetic model. The value of K _m and V _max for the reaction was 18.47 g l⁻¹ glucose and 0.154 g l⁻¹ h⁻¹, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 20–24. Received 13 October 1999/ Accepted in revised form 02 April 2000     

丙酮酸和乙酰-CoA协同促进L-亮氨酸的合成

【目的】通过理性改造柠檬酸合酶(citrate synthase,CS)、丙酮酸脱氢酶系E1p (pyruvate dehydrogenase complex,PDHC,编码基因aceE)和ATP-柠檬酸裂解酶(ATP-Citrate lyase,ACL),有效供应胞内丙酮酸和乙酰-CoA,以提高L-亮氨酸产量。【方法】以谷氨酸棒杆菌(Corynebacterium glutamicum)为底盘细胞,分析不同CS和PDHC酶活水平对L-亮氨酸合成的影响。随后,考查协同改造CS和PDHC或引入绿硫菌(Chlorobium tepidum)中ACL对L-亮氨酸合成的影响。【结果】低强度的CS酶活(即重组菌XL-3 P_(dapA-R2)gltA)有利于L-亮氨酸的合成,L-亮氨酸产量达到17.5±0.6 g/L。而改变PDHC酶活水平不利于L-亮氨酸的合成。此外,以启动子P_(dapA-R2)控制CS表达,而以启动子P_(gapA)控制PDHC表达时(即重组菌XL-4),可实现胞内丙酮酸和乙酰-CoA的有效供给,L-亮氨酸产量达到20.2±1.7 g/L,且显著降低副产物产量。若在重组菌XL-4中引入C.tepidum,ACL会显著抑制菌体生长而不利于L-亮氨酸合成,而引入到出发菌XL-3中因胞内丙酮酸和乙酰-CoA得到有效供给,目标重组菌XL-5L-亮氨酸产量达到18.5±1.2 g/L,比出发菌株XL-3增加了14.2%。【结论】重组菌XL-4中因协同控制CS和PDHC酶活,从而实现胞内丙酮酸和乙酰-CoA有效供给,促进L-亮氨酸的合成。该研究结果对后续利用代谢工程技术强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。     

茎瘤固氮根瘤菌ORS571中motA基因的功能分析

[目的] MotA是细菌的鞭毛马达蛋白,是跨膜质子通道的重要组成结构之一,在调控鞭毛运动中具有至关重要的作用。本研究探究了Azorhizobium caulinodans ORS571中鞭毛马达基因motA对菌株表型和植物互作的影响。[方法] 通过同源重组原理和三亲接合转移方法构建突变菌株∆motA,测定野生型与突变体在菌体生长、运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力的差异。[结果] 与野生型相比,突变体菌体生长没有明显差异,但其运动能力完全丧失,固氮、胞外多糖合成、生物膜形成及根系定殖能力减弱。[结论] MotA鞭毛马达蛋白对A.caulinodans ORS571的运动、固氮、胞外多糖合成、生物膜形成及根系定殖能力均有调控作用。     

Regulation of proteinase synthesis in Lactococcus lactis

The synthesis of extracellular serine proteinase of Lactococcus lactis was studied during the growth in a batch and a continuous culture on chemically defined media. In a batch culture the proteinase synthesis started during the exponential phase of growth and the highest proteinase concentrations were found at the end of the exponential and beginning of the stationary phase of growth. During the growth in a lactose-limited chemostat with amino acids as the sole source of nitrogen, the specific rate of proteinase synthesis was maximal at a μof 0.23 h^?1. At higher growth rates the proteinase productin declined. The proteinase synthesis was dependent on the amino acid sources in the medium. In batch cultures of L. lactis grown on a chemically defined medium with amino acids, the proteinase production was increased four-fold compared to media containing casein or a tryptic digest of casein as the sole source of nitrogen. The inhibition of the rate of proteinase synthesis by casein and peptides was also observed during the growth in a chemostat. The addition of the dipeptide leucylproline (final concentration of 100 μM) to a lactose-limited continuous culture during the steady state (D = 0.23 h^?1) resulted in a transient inhibition of the rate of proteinase synthesis. This suggested that exogenously supplied peptides control the regulation of proteinase synthesis of L. lactis.     

Kinetic analysis on formation of poly(3-hydroxybutyrate) from acetic acid by Ralstonia eutropha under chemically defined conditions

Batch cultures of Ralstonia eutropha in chemically defined media with acetic acid (HAc) as the sole carbon source were conducted to investigate acetate utilization, formation of poly(3-hydroxybutyrate) (PHB) and growth of active biomass (ABM) under different carbon to nitrogen (C/N) weight ratios. The specific acetate utilization rate based on ABM approached 0.16 g/g ABM h⁻¹, which was not affected very much by the extracellular HAc concentration from 1 to 5 g/l, but was affected by the C/N weight ratio. A low C/N ratio or high nitrogen supply sped up the specific acetate utilization rate to produce more ABM and less PHB. A high HAc concentration (>6 g/l), however, depressed acetate utilization as well as the ABM growth and PHB formation. A high cell mass concentration enhanced the tolerance of R. eutropha to the toxicity of HAc at pH 7 to 8.5. The viscosity-average molecular size of PHB generally increased first and then declined in batch cultures. Larger PHB molecules and less PHB per ABM were produced at a low C/N ratio with enough nutrient nitrogen than those under a high C/N ratio with less nutrient nitrogen available. Journal of Industrial Microbiology & Biotechnology (2001) 26, 121–126. Received 06 June 2000/ Accepted in revised form 21 October 2000     

Influence of nutritional and environmental factors on polysaccharide production by Azotobacter vinelandii cultured on 4-hydroxybenzoic acid

The capacity of 4-hydroxybenzoic acid to support exopolysaccharide (EPS) biosynthesis was investigated. Carbon source concentration, nitrogen supplementation, and other nutritional and environmental factors were optimized to obtain maximal EPS recovery. Higher EPS yields were obtained in nitrogen-free media amended with 20–30 mM 4-hydroxybenzoic acid. In general, modifications in inorganic salt concentration did not alter EPS production, except in the case of magnesium ions. Increased levels of this cation were correlated to greater EPS yields. Production was strongly influenced by certain environmental factors. Optimal values of 34°C, 80 rpm and neutral or slightly basic conditions were selected. Under these conditions, more than 25% of the carbon source supplied was converted to EPS and the production was improved about 42% in comparison to that observed in the initial media. Journal of Industrial Microbiology & Biotechnology (2001) 27, 5–10. Received 05 November 2000/ Accepted in revised form 30 April 2001     

Effect of nitrogen source and pH on siderophore production by Rhodotorula strains and their application to biocontrol of phytopathogenic moulds

The production of rhodotorulic acid, a siderophore synthesized by Rhodotorula strains, was improved with the objective of achieving the biocontrol of phytopathogenic moulds. Rhodotorulic acid increased up to 60% in the presence of urea as a nitrogen source, pH near to 8 and a C:N ratio of 8:1. The siderophore-containing spent medium showed in vitro antifungal activity against important plant pathogens including Botrytis cinerea, which causes grey mould on a wide variety of host plants including numerous commercial crops. The antifungal activity was related to siderophore concentration. Journal of Industrial Microbiology & Biotechnology (2001) 26, 226–229. Received 06 June 2000/ Accepted in revised form 28 January 2001     

Conversion of gamma-butyrobetaine to L-carnitine by Achromobacter cycloclast

L -Carnitine is an ubiquitous substance that plays a major role in the transportation of long-chain fatty acids. We investigated crucial factors that influence microbial conversion of γ-butyrobetaine to L-carnitine using an Achromobacter cycloclast strain. Two-stage culture results showed that γ-butyrobetaine induced enzymes essential for the conversion, which suggests that the precursor should be present in the initial cell growth stage. The addition of yeast extract enhanced L-carnitine production whereas inorganic nitrogen sources inhibited it. Under nitrogen-limiting conditions, the cells accumulated poly-β-hydroxybutyrate instead of L-carnitine. Among the trace elements tested, nickel addition enhanced L-carnitine production by almost twice that of the control and copper strongly inhibited the conversion. L-Carnitine production was reduced when the medium contained inorganic salts of sodium, potassium, and calcium at a concentration greater than 2 g l⁻¹. A higher L-carnitine yield was achieved when cells were incubated in a lower culture volume. The optimal pH for L-carnitine production was 5 to 5.5, whereas that of growth was 7.0, indicating that a pH shift was required. Under optimal conditions, L-carnitine concentrations as high as 15 g l⁻¹ were obtained in 62 h with a 45% molar conversion yield. Journal of Industrial Microbiology & Biotechnology (2001) 26, 309–315. Received 26 November 2000/ Accepted in revised form 27 February 2001     

The jasmonate pathway is involved differentially in the regulation of different defence responses in tobacco cells

Jasmonic acid, a product of the lipoxygenase (LOX) pathway, has been proposed to be a signal transducer of defence reactions in plants. We have reported previously that methyl jasmonate (MJ) induced accumulation of proteinase inhibitors in tobacco cell suspensions (Rickauer et al., 1992, Plant Physiol Biochem 30: 579–584). The role of this compound in the induction of this and of other defence reactions is further studied in this paper. Treatment of tobacco cell suspensions with an elicitor from Phytophthora parasitica var. nicotianae induced a rapid and transient increase in jasmonic acid levels, which was abolished when cells were preincubated with eicosatetraynoic acid (ETYA), an inhibitor of LOX. Pretreatment with ETYA also inhibited the induction of proteinase inhibitors by fungal elicitor, but not by MJ. Linolenic acid, a precursor of jasmonate biosynthesis, induced this defence response, whereas linoleic acid had no effect. Expression of defence-related genes encoding proteinase inhibitor II, hydroxyproline-rich or glycine-rich glycoproteins, glucanase and chitinase, was induced in a basically similar manner by fungal elicitor or MJ. However, ETYA did not inhibit, or only partially inhibited, the elicitation of these defence genes. Expression of the sesquiterpene cyclase (5-epi-aristolochene synthase) gene was not induced by MJ, but only by fungal elicitor, and ETYA pretreatment had no effect on this induction. The obtained results indicate that synthesis of jasmonate via the LOX pathway seems to be only part of a complex regulatory mechanism for the onset of many, but not all, defence reactions. Received: 4 July 1996 / Accepted: 23 November 1996     

Effect of plant root symbionts on performance of native woody species in competition with an invasive grass in multispecies microcosms

The majority of terrestrial plants form mutualistic associations with arbuscular mycorrhizal fungi (AMF) and rhizobia (i.e., nitrogen‐fixing bacteria). Understanding these associations has important implications for ecological theory and for restoration practice. Here, we tested whether the presence of AMF and rhizobia influences the performance of native woody plants invaded by a non‐native grass in experimental microcosms. We planted eight plant species (i.e., Acacia acuminata, A. microbotrya, Eucalyptus loxophleba subsp. loxophleba, E. astringens, Calothamnus quadrifidus, Callistemon phoeniceus, Hakea lissocarpha and H. prostrata) in microcosms of field‐conditioned soil with and without addition of AMF and rhizobia in a fully factorial experimental design. After seedling establishment, we seeded half the microcosms with an invasive grass Bromus diandrus. We measured shoot and root biomass of native plants and Bromus, and on roots, the percentage colonization by AMF, number of rhizobia‐forming nodules and number of proteaceous root clusters. We found no effect of plant root symbionts or Bromus addition on performance of myrtaceous, and as predicted, proteaceous species as they rely little or not at all on AMF and rhizobia. Soil treatments with AMF and rhizobia had a strong positive effect (i.e., larger biomass) on native legumes (A. microbotrya and A. acuminata). However, the beneficial effect of root symbionts on legumes became negative (i.e., lower biomass and less nodules) if Bromus was present, especially for one legume, i.e., A. acuminata, suggesting a disruptive effect of the invader on the mutualism. We also found a stimulating effect of Bromus on root nodule production in A. microbotrya and AMF colonization in A. acuminata which could be indicative of legumes’ increased resource acquisition requirement, i.e., for nitrogen and phosphorus, respectively, in response to the Bromus addition. We have demonstrated the importance of measuring belowground effects because the aboveground effects gave limited indication of the effects occurring belowground.     

Effect of the regulation system of metabolic nitrogen exchange on biosynthesis of serine proteinases from <Emphasis Type="Italic">Bacillus intermedius</Emphasis>

The regulatory link between biosynthesis of Bacillus intermedius subtilisin-like serine proteinase and nitrogen metabolism in B. intermedius cells was determined. The level of the enzyme biosynthesis by the recombinant strain of Bacillus subtilis in the medium containing ammonium ions was three- to fivefold less than that in the medium with poorly utilized sodium nitrate. Accumulation of glutamyl endopeptidase in a culture liquid of this microorganism did not depend on the source of nitrogen present in the medium. During cultivation in the rich medium, the productivity of subtilisin-like proteinase in the recombinant B. subtilis strain carrying a mutation in the NrgB sensor protein was demonstrated to increase threefold compared to that of the control strain. In the minimal culture medium, mutation in the nrgB gene abolished the effect of a nitrogen source on the level of the subtilisin-like proteinase gene expression. At the same time, this mutation did not affect glutamyl endopeptidase biosynthesis. Thus, expression of the gene coding for subtilisin-like proteinase from B.intermedius is suggested to be positively regulated by the regulatory system of nitrogen metabolism.     

Effects of acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a minimal medium

Specific growth rates (μ) of two strains of Saccharomyces cerevisiae decreased exponentially (R ²>0.9) as the concentrations of acetic acid or lactic acid were increased in minimal media at 30°C. Moreover, the length of the lag phase of each growth curve (h) increased exponentially as increasing concentrations of acetic or lactic acid were added to the media. The minimum inhibitory concentration (MIC) of acetic acid for yeast growth was 0.6% w/v (100 mM) and that of lactic acid was 2.5% w/v (278 mM) for both strains of yeast. However, acetic acid at concentrations as low as 0.05–0.1% w/v and lactic acid at concentrations of 0.2–0.8% w/v begin to stress the yeasts as seen by reduced growth rates and decreased rates of glucose consumption and ethanol production as the concentration of acetic or lactic acid in the media was raised. In the presence of increasing acetic acid, all the glucose in the medium was eventually consumed even though the rates of consumption differed. However, this was not observed in the presence of increasing lactic acid where glucose consumption was extremely protracted even at a concentration of 0.6% w/v (66 mM). A response surface central composite design was used to evaluate the interaction between acetic and lactic acids on the specific growth rate of both yeast strains at 30C. The data were analysed using the General Linear Models (GLM) procedure. From the analysis, the interaction between acetic acid and lactic acid was statistically significant (P≤0.001), i.e., the inhibitory effect of the two acids present together in a medium is highly synergistic. Journal of Industrial Microbiology & Biotechnology (2001) 26, 171–177. Received 06 June 2000/ Accepted in revised form 21 September 2000     

Role of Chrysonilia sitophila in the quality of cork stoppers for sealing wine bottles

The contribution of Chrysonilia sitophila in cork stopper manufacture was studied and a simulation of the industrial processing of cork stoppers was performed. Stoppers cut from slabs where mold development was inhibited were compared with others cut from slabs colonized by C. sitophila alone or with several molds, in terms of physical properties and chemical taints. C. sitophila does not produce 2,4,6-trichloroanisole, guaiacol, or 1-octene-3-ol on cork slabs incubated for 66 days. Since some chlorophenol-related compounds contaminate cork slabs during the production processes, metabolic tests were performed to investigate the capability of molds to produce 2,4,6-trichloroanisole by methylation of 2,4,6-trichlorophenol. Degradation of 2,4,6-trichlorophenol by C. sitophila resulted in a very high level of degradation without production of 2,4,6-trichloroanisole. C. sitophila restricted growth of other molds on maturing slabs for at least 30 days. These results show that C. sitophila can be exploited by industrial producers of cork stoppers since it is able to inhibit the development of other molds and it does not produce the compounds responsible for ‘cork-taint’, even in the presence of chlorophenols. Journal of Industrial Microbiology & Biotechnology (2000) 24, 256–261. Received 28 July 1999/ Accepted in revised form 05 January 2000     

Measurement of potential activity of fixed nitrifying bacteria in biological filters used in drinking water production

Nitrification during biological filtration is being used more and more in drinking water production to remove ammonia, which can be the source of several water quality problems during distribution. In this process, ammonia is converted into nitrite and then into nitrate by fixed autotrophic nitrifying bacteria. The purpose of this work was to develop a technique to estimate fixed nitrifying biomass (sum of ammonia- and nitrite-oxidizing populations). The quantification of autotrophic nitrifying biomass was determined by potential nitrifying activity measurement. The production of oxidized forms of inorganic nitrogen (nitrates and nitrites) was measured after an incubation of 2 cm³ of colonized solid support in the presence of a 5-ml nitrifier medium containing 10 mg N-NH₄ L⁻¹ for 30 min at 32°C. The production rate of oxidized nitrogen in optimal conditions was measured and converted into nitrifying biomass by using the maximum specific oxidizing activity. This technique was shown to be appropriate for conditions encountered in the biological filters used in drinking water production and sufficiently simple to be used for routine measurements. Journal of Industrial Microbiology & Biotechnology (2000) 24, 161–166. Received 28 July 1999/ Accepted in revised form 11 November 1999     

Extracellular proteinase fromEnterococcus faecalis subsp.liquefaciens

Growth and extracellular proteinase production byEnterococcus faecalis subsp.liquefaciens was studied on several culture media and under different incubation conditions. The organisms grew well and developed extracellular proteinase activity on proteinaceous media, but when it grew on Collins basal medium (lacking of protein), growth was poor and proteinase activity was not detected. The activation energy for growth was estimated to be 116 kJ/mol, the optimum being at 37°C. Proteinase production was not affected by temperature in the range studied (7–45°C). Growth rate was not affected by aeration although a higher amount of microorganisms was observed on shaking the culture during incubation. Likewise, extracellular proteolytic activity was about twice higher in cultures shaken at 2.3 or 3.3 Hz than in those shaken at 0 or 1.3 Hz.     

Optimization of erythritol production by Candida magnoliae in fed-batch culture

A two-stage fed-batch process was designed to enhance erythritol productivity by the mutant strain of Candida magnoliae. The first stage (or growth stage) was performed in the fed-batch mode where the growth medium was fed when the pH of the culture broth dropped below 4.5. The second stage (or production stage) was started with addition of glucose powder into the culture broth when the cell mass reached about 75 g dry cell weight l⁻¹. When the initial glucose concentration was adjusted to 400 g l⁻¹ in the production stage, 2.8 g l⁻¹ h⁻¹ of overall erythritol productivity and 41% of erythritol conversion yield were achieved, which represented a fivefold increase in erythritol productivity compared with the simple batch fermentation process. A high glucose concentration in the production phase resulted in formation of organic acids including citrate and butyrate. An increase in dissolved oxygen level caused formation of gluconic acid instead of citric acid. Journal of Industrial Microbiology & Biotechnology (2000) 25, 100–103. Received 25 February 2000/ Accepted in revised form 08 June 2000