Evaluation of xylitol production from corn cob hemicellulose hydrolysate by Candida parapsilosis

Candida parapsilosis was grown for 59 h in a medium containing corn cob hydrolysate consisting of 50 g xylose l^–1, 3.0 g glucose l^–1, 2.0 g arabinose l^–1, and 0.9 g acetic acid l^–1. A biomass of 9.1 g l^–1 was produced with 36 g xylitol l^–1 and 2.5 g ethanol l^–1. In a medium containing 50 g xylose l^–1 instead of corn cob hydrolysate, the concentrations of cells, xylitol, and ethanol were 8.6 g l^–1, 33 g l^–1, and 0.2 g l^–1, respectively. The differences between two cultures were due to the glucose and arabinose in the corn cob hydrolysate stimulating growth and the low concentration of acetic acid stimulating xylitol production.     

Mixed inhibitions by methanol, furfural and acetic acid on xylitol production by Candida guilliermondii

Xylose production by Candida guilliermondii FTI 20037 was carried out in a synthetic medium in the presence of 0–100 g methanol l^–1, 0–0.7 g furfural l^–1 or 0–1.3 g acetic acid l^–1. Kinetic results show a mixed inhibition mechanism in all three cases. Maximum specific productivity and saturation constant for product formation were, in the absence of inhibition, 3.6 g_P g_X ^–1 h^–1 and 232 g_S l^–1, respectively, while the inhibition constants, K _i and K _i, were 17 and 50 g methanol l^–1, 0.62 and 7.0 g furfural l^–1, 0.69 and 3.5 g acetic acid l^–1, which suggests the following order of inhibition: furfural > acetic acid > methanol.     

Glucose repression of xylitol production in Candida tropicalis mixed-sugar fermentations

Glucose repressed xylose utilization inCandida tropicalis pre-grown on xylose until glucose reached approximately 0–5 g l^–1. In fermentations consisting of xylose (93 g l^–1) and glucose (47 g l^–1), xylitol was produced with a yield of 0.65 g g^–1 and a specific rate of 0.09 g g^–1 h^–1, and high concentrations of ethanol were also produced (25 g l^–1). If the initial glucose was decreased to 8 g l^–1, the xylitol yield (0.79 g g^–1) and specific rate (0.24 g g^–1 h^–1) increased with little ethanol formation (<5 g l^–1). To minimize glucose repression, batch fermentations were performed using an aerobic, glucose growth phase followed by xylitol production. Xylitol was produced under O₂ limited and anaerobic conditions, but the specific production rate was higher under O₂ limited conditions (0.1–0.4 vs. 0.03 g g^–1 h^–1). On-line analysis of the respiratory quotient defined the time of xylose reductase induction.     

Production of gluconic acid by immobilized in pumice stones mycelium of Aspergillus niger using unconventional oxygenation of culture

Gluconic acid was produced in repeated batch processes with Aspergillus niger AM-11, immobilized in pumice stone particles using an unconventional oxygenation of culture media based on the addition of H₂O₂, decomposed by catalase to O₂ and water. The highest gluconic acid productivity of 8.2 g l^–1 h^–1 was reached with 30 g immobilized mycelium per 150 ml, 10% (w/v) glucose, at 24 °C and pH 6.5, with O₂ at 100% saturation. The immobilized mycelium was successfully reused up to 8 times in 1-h batches with only a slight loss (11%) of gluconic acid productivity.     

Multiple growth inhibition of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> in 1,3-propanediol fermentation

The inhibition of substrate and product on the growth of Klebsiella pneumoniae in anaerobic and aerobic batch fermentation for the production of 1,3-propanediol was studied. The cells under anaerobic conditions had a higher maximum specific growth rate of 0.19 h^–1 and lower tolerance to 110 g glycerol l^–1, compared to the maximum specific growth rate of 0.17 h^–1 and tolerance to 133 g glycerol l^–1 under aerobic conditions. Acetate was the main inhibitory metabolite during the fermentation under anaerobic conditions, with lactate and ethanol the next most inhibitory. The critical concentrations of acetate, lactate and ethanol were assessed to be 15, 19, 26 g l^–1, respectively. However, cells grown under aerobic conditions were more resistant to acetate and lactate but less resistant to ethanol. The critical concentrations of acetate, lactate and ethanol were assessed to be 24, 26, and 17 g l^–1, respectivelyRevisions requested 8 september; Revisions received 2 November 2004     

Culture conditions of tannase production by Lactobacillus plantarum

Lactobacillus plantarum produced an extracellular tannase after 24 h growth on minimal medium of amino acids containing 2 g tannic acid l^–1. Enzyme production (6 U ml^–1) was optimal at 37 °C and pH 6 with 2 g glucose l^–1 and 7 g tannic acid l^–1 in absence of O₂.     

Growth characteristics and oxidative capacity of<Emphasis Type="Italic"> Acetobacter aceti</Emphasis> IFO 3281: implications for <Emphasis Type="SmallCaps">l</Emphasis>-ribulose production

We studied the growth characteristics and oxidative capacities of Acetobacter aceti IFO 3281 in batch and chemostat cultures. In batch culture, glycerol was the best growth substrate and growth on ethanol occurred only after 6 days delay, although ethanol was rapidly oxidized to acetic acid. In continuous culture, both glycerol and ethanol were good growth substrates with similar characteristics. Resting cells in a bioreactor oxidized ribitol to l-ribulose with a maximal specific rate of 1.2 g g^–1 h^–1). The oxidation of ribitol was inhibited by ethanol but not by glycerol. Biomass yield (Y_SX; C-mmol/C-mmol) on ethanol and glycerol was low (0.21 and 0.17, respectively). In the presence of ribitol the yield was somewhat higher (0.25) with ethanol but lower (0.13) with glycerol, with respectively lower and higher CO₂ production. In chemostat cultures the oxidation rate of ribitol was unaffected by ethanol or glycerol. Cell-free extract oxidized ethanol very slowly but not ribitol; the oxidative activity was located in the cell membrane fraction. Enzymatic activities of some key metabolic enzymes were determined from steady-state chemostat with ethanol, glycerol, or ethanol/glycerol mixture as a growth limiting substrate. Based on the measured enzyme activities, metabolic pathways are proposed for ethanol and glycerol metabolism.     

Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping

Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H₂ and CO₂ as the carrier gases. This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l^–1 and 60 g l^–1, respectively. In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities. In a control batch reactor, the culture used 45.4 g glucose l^–1 and produced 17.6 g total solvents l^–1 (yield 0.39 g g^–1, productivity 0.29 g l^–1 h^–1). Using the integrated fermentation-gas stripping product-recovery system with CO₂ and H₂ as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity. The fed-batch reactor was operated for 201 h. At the end of the fermentation, an unusually high concentration of total acids (8.5 g l^–1) was observed. A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth. The average solvent yield and productivity were 0.47 g g^–1 and 1.16 g l^–1 h^–1, respectively.     

Angiostatin production in cultivation of recombinant Pichia pastoris fed with mixed carbon sources

A recombinant strain of Pichia pastoris with a phenotype of Mut^S was used to produce angiostatin. Due to the low methanol consumption rate of this strain, both methanol and glycerol feedings, that produced oscillation in dissolved O₂ concentration, were used during the expression phase to improve cell growth and angiostatin expression. However, enhanced cell growth led to nitrogen limitation that suppressed further production of angiostatin, but addition of ammonia allowed angiostatin concentration to reach 108 mg l^–1 after an expression period of 96 h. The ratio of consumed glycerol to methanol of 1.5:1 (w/w) in the expression phase suggested that methanol played an important role in the metabolism of carbon sources.     

Enhancement of xylitol productivity and yield using a xylitol dehydrogenase gene-disrupted mutant of Candida tropicalis under fully aerobic conditions

The effects of glycerol and the oxygen transfer rate on the xylitol production rate by a xylitol dehydrogenase gene (XYL2)-disrupted mutant of Candida tropicalis were investigated. The mutant produced xylitol near the almost yield of 100% from d-xylose using glycerol as a co-substrate for cell growth and NADPH regeneration: 50 g d-xylose l⁻¹ was completely converted into xylitol when at least 20 g glycerol l⁻¹ was used as a co-substrate. The xylitol production rate increased with the O₂ transfer rate until saturation and it was not necessary to control the dissolved O₂ tension precisely. Under the optimum conditions, the volumetric productivity and xylitol yield were 3.2 g l⁻¹ h⁻¹ and 97% (w/w), respectively.     

Production of highly concentrated vinegar in fed-batch culture

Vinegars of 170 g acetic acid l^–1 were obtained by fed-batch fermentation. Acetobacter grew up to a limit of 120 g acetic acid l^–1. Beyond this value, the total number of cells, approx. 10⁸ m^–1, decreased systematically irrespective of fermentation parameters.     

Lignin Deposition and Effect of Postharvest Treatment on Lignification of Green Asparagus (Asparagus officinalis L.)

To understand how lignin synthesis is regulated after harvest, detached green asparagus stalks (Asparagus officinalis L.) were treated with 1 μl l⁻¹ of 1-methylcyclopropene (1-MCP), 50 μg l⁻¹ gibberellic acid (GA₃), 2% (v:v) ethanol or 1 μl l⁻¹ ethylene. The results showed that lignin concentration in asparagus stalks stored at room temperature rapidly increased. Three conventional precursors of lignin, 4-hydroxycinnamic acid (coumaric acid), 3,4-dihydroxycinnamic acid (caffeic acid) and 4-hydroxy-3-mythoxycinnamic acid (ferulic acid), were found to be the major phenolics in the asparagus stalks. Furthermore, the concentrations of O₂⁻ in asparagus stalks steadily increased during the storage. Deposition of lignin in harvested asparagus was significantly reduced by treating the stalks with GA₃, 1-MCP or ethanol. The concentration of lignin in stalks treated with GA₃, 1-MCP or ethanol was 32, 20 or 27% lower, respectively, than in controls 3 days after treatment. Treating stalks with ethylene enhanced lignin synthesis (p<0.05). The concentration of total phenol in stalks was also significantly reduced by GA₃, 1-MCP and ethanol, but was enhanced by ethylene treatment. However, the concentration of active oxygen (O₂^−⋅) in stalks was significantly reduced by treatment with GA₃, 1-MCP and ethanol, but was enhanced by treatment with ethylene. Our study show that postharvest treatment with 1-MCP, GA₃ or ethanol may be applied to improve the quality of green asparagus.     

Optimization of glycerol feeding for clavulanic acid production by Streptomyces clavuligerus with glycerol feeding

Glycerol at 10–20 g l^–1 increased clavulanic acid production by Streptomyces clavuligerus in shake-flask culture. The biosynthesis of clavulanic acid continued for longer by feeding glycerol and production increased to 250 mg l^–1 compared with 115 mg l^–1 without feeding. In fermenter batch culture, degradation of clavulanic acid began after 72 h. With glycerol feeding in fed-batch culture, clavulanic acid production was not only increased further to about 280 mg l^–1 but also remained stable up to 130 h.     

Reducing by-product formation in L-lactic acid fermentation by Rhizopus oryzae

During L-lactic acid fermentation by Rhizopus oryzae, increasing the phosphate level in the fermentation medium from 0.1 g l^–1 to 0.6 g l^–1 KH₂PO₄ reduced the maximal concentration of L-lactic acid and fumaric acid from 85 g l^–1 to 71 g l^–1 and from 1.36 g l^–1 to 0.18 g l^–1, respectively; and it decreased the fermentation time from 72 h to 52 h. Phosphate at 0.40 g l^–1 KH₂PO₄ was suitable for both minimizing fumaric acid accumulation and benefiting L-lactic acid production.     

Effects of oxygen supply on yeast growth and metabolism in continuous fermentation

The yield changes in cell mass and metabolites with changes in the oxygen supply rate were investigated in continuous ethanol fermentation. With increases in oxygen concentration in the purging gas from 5.3 to 39.3 %, the specific oxygen uptake rate (qO₂) increased from 0.158 to 1.24 mmol/g/h. With this change, cell mass increased from 13.2 to 14.9 g/l and glycerol decreased from 4.8 to 0.99 g/l, although little change in ethanol yield was observed. At a higher oxygen concentration and/or at a lower respiratory quotient (RQ), glycerol disappeared, acetaldehyde, acetoin and 2,3-butanediol increased, and ethanol started to decrease. The yields of iso-butylalcohol and iso-amylalcohol also increased with increases in the oxygen supply rate when RQ was lower than approximately 10. Reduction in the redox balance (NADH/NAD) in the cells by qO₂, appeared to reduce initially the rate of glycerol-3-phosphate formation and next the rate of ethanol formation, resulting in the accumulation of acetaldehyde and formation of 2,3-butanediol through acetoin. Fatty acid composition changed with changes in the oxygen supply rate. The value for unsaturation, Δ mol⁻¹, increased from 0.745 to 0.836 with the increase in qO₂ from 0.158 to 1.79 mmol/g/h. Increases in oleic acid (C18:1) and decreases in palmitic acid (C16:0) were the major changes with the increases in Δ mol⁻¹.     

Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> using plastic composite support bioreactors

Continuous and repeat-batch biofilm fermentations using Actinobacillus succinogenes were performed with immobilized and suspended-cell systems. For the immobilized continuous system, plastic composite supports (PCS) containing 50% (w/w) polypropylene (PP), 35% (w/w) ground soybean hulls, 5% (w/w) dried bovine albumin, 2.5% (w/w) soybean flour, 2.5% (w/w) yeast extract, 2.5% (w/w) dried red blood cells, and 2.5% (w/w) peptone, or PP tubes (8.5 cm in length) were arranged around the agitator shaft in a grid formation. Agitation was controlled at 125 rpm and 150 rpm. Samples were taken at dilution rates of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 h^–1 and analyzed for succinic acid production and glucose consumption (g l^–1). For PCS bioreactors, the highest final succinic acid concentrations (10.1 g ^–1, 10.4 g l^–1) and percentage yields (62.6%, 71.6%) occurred at the dilution rate of 0.2 h^–1. PCS disks were evaluated in a repeat-batch biofilm reactor. Suspended-cell batch fermentations were performed in flasks and a repeat-batch bioreactor. The maximum concentration of succinic acid produced was 40 g l^–1. Peak succinic acid percentage yields in continuous and repeat-batch fermentations of A. succinogenes were observed in suspended-cell continuous fermentations at a dilution rate of 1.0 h^–1 (76.2%) and in PCS repeat-batch fermentations with an initial glucose concentration of 40 g l^–1 (86.7%).     

Plant regeneration from thin cell layers in Spinacia oleracea

Caulogenesis and somatic embryogenesis were induced from transverse thin cell layers (tTCLs) of two European (Spinacia oleracea L.) spinach genotypes. Regeneration occurred mostly when tTCLs had been excised from seedlings grown on a preconditioning medium consisting of White's macroelements, Nitsch's microelements, Murashige and Skoog's (MS) vitamins, 6 g l^–1 agar and 20 g l^–1 glucose. The explants were cultured on MS medium supplemented with sucrose (10, 30, 50 or 80 g l^–1) or fructose (5, 10 or 30 g l^–1) and several combinations of indole-3-acetic acid (IAA), -naphtalene acetic acid (NAA), 6-benzylaminopurine (BAP) and gibberellic acid (GA₃). Most of the regeneration events were obtained from root explants of the cultivar Carpo. The best result was observed on MS medium supplemented with 50 g l^–1 sucrose, 100 M NAA, 1 M BAP and 10 M GA₃. After an 8-week culture, the calluses were transferred onto MS medium where shoots and somatic embryos appeared 1 week later. The best root development was obtained on MS medium supplemented with 4.9 M indole-3-butyric acid (IBA) and 8 g l^–1 Phytagel. The plantlets were, then, transferred to soil and developed into well-conformed, fertile plants.     

Optimization of cell growth and poly(glutamic acid) production in batch fermentation by Bacillus subtilis

Poly(glutamic acid) was produced maximally by Bacillus subtilis in batch fermentations at pH 7 and using glycerol at 20 g l^–1 in a glutamic acid/citric acid medium. Poly(glutamic acid) reached 23 g l^–1 after 30 h.     

Photoautotrophic micropropagation of Russet Burbank Potato

The photoautotrophic micropropagation of potato cv. Russet Burbank was investigated. Single node microcuttings were grown for four weeks on Murashige and Skoog (MS) medium with or without sucrose (30 g l^–1) in the growth room at 21/19 °C day/night temperature, with 16-h photoperiod at 150 mol m^–2 s^–1, with or without supplemental CO₂ at 1500 l l^–1. A 20% increase in the number of nodes per stem (from 7.5 to 9.4) and a 50% increase in stem dry weight were observed in cultures grown on media with sucrose and in CO₂ enriched atmosphere comparing to the conventionally micropropagated cultures or the cultures grown photoautotrophically on media without sucrose but in air supplemented with 1500 l l^–1CO₂. Stems of these cultures (from media with sucrose in CO₂ enriched air) almost doubled in length the stems of cultures from the other two treatments. No significant differences were observed between Control (MS medium supplemented with sucrose, 30 g l^–1) and photoautotrophic cultures coming from MS medium with no sucrose grown under 1500 l l^–1 of CO₂. Photoautotrophic cultures produced stems averaging 43.3 mm, with 7 nodes and weighing 9.2 mg (dry weight), similar to conventionally grown in vitro cultures (47.9 mm with 7.5 nodes, 9.7 mg dry weight). Growers may consider photoautotrophic culturing of potato in areas where the high sterility levels are difficult to maintain. Supplementing air in the growth room with 1500 l l^–1 of CO₂ could be beneficial for potato plantlet production even on media containing sucrose since it significantly improved quality, size and biomass of produced plantlets, speeding up the multiplication.     

Optimized production of L-β-hydroxybutyric acid by a mutant of Yarrowia lipolytica

A mutant strain of Yarrowia lipolytica was developed which produced 8.0 g l--hydroxybutyric acid l^–1 from butyric acid in a batch culture. The optimum culture conditions in the fermenter for maintenance of a high cell activity, determined by chemostat analyses, were a specific growth rate of 0.06 h^–1, a glucose concentration of 2.0 g l^–1, and a butyric acid concentration of 8.1 g l^–1. A fed-batch fermentation was performed under these conditions resulting in an l--hydroxybutyric acid yield of 31 g l^–1.