Trehalose accumulation from corn starch by Saccharomycopsis fibuligera A11 during 2-l fermentation and trehalose purification

In this study, corn starch was used as the substrate for cell growth and trehalose accumulation by Saccharomycopsis fibuligera A11. Effect of different aeration rates, agitation speeds, and concentrations of corn starch on direct conversion of corn starch to trehalose by S. fibuligera A11 were examined using a Biostat B2 2-l fermentor. We found that the optimal conditions for direct conversion of corn starch to trehalose by this yeast strain were that agitation speed was 200 rpm, aeration rate was 4.0 l/min, concentration of corn starch was 2.0% (w/v), initial pH was 5.5, fermentation temperature was 30°C. Under these conditions, over 22.9 g of trehalose per 100 g of cell dry weight was accumulated in the yeast cells, cell mass was 15.2 g/l of the fermentation medium, 0.12% (w/v) of reducing sugar, and 0.21% (w/v) of total sugar were left in the fermented medium within 48 h of the fermentation. It was found that trehalose in the yeast cells could be efficiently extracted by the hot distilled water (80°C). After isolation and purification, the crystal trehalose was obtained from the extract of the cells.     

Simultaneous degradation of phytic acid and starch by an industrial strain of Saccharomyces cerevisiae producing phytase and α-amylase

Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis α-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the δ-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and α-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.     

Protein enrichment of grain sorghum by submerged culture of the amylolytic yeastsSchwanniomyces occidentalis andLipomyces kononenkoae

Cultivation of aSchwanniomyces occidentalis derepressed mutant in a 10% (w/v) gelatinized grain sorghum slurry increased the crude protein content of the biomass from an initial value of 12% to 41% (dry) within 20 h, with no detectable residual starch. Co-cultivation ofCandida utilis with theS. occidentalis mutant improved the final crude protein content to 47% within 18 h, whereas a co-culture ofC. utilis with aLipomyces kononenkoae mutant resulted in a cultivation time of 50 h with a significantly lower protein content and a low final -amylase activity. In a 15% (w/v) grain sorghum slurry aC. utilis/S. occidentalis co-culture increased the protein content to about 44% within 30 h. Yeast cultivation increased the lysine and threonine content of the final biomass considerably.     

Production of Short Side Chain-Poly[Hydroxyalkanoate] by a Newly Isolated Ralstonia Pickettii Strain

In order to obtain better bacterial species or strains for production of short side chain-poly[hydroxyalkanoate](ssc-PHA) from cheap carbon sources, a bioprospecting programme was performed in a subtropical rainforest soil. From 398 bacterial isolates, one produced high amounts of ssc-PHA when grown on sugarcane molasses or sucrose as detected by spectrophotometric scanning and gas chromatography coupled to mass spectrometry. Also, the GC—MS analysis indicated that the polymer was composed basically of poly[3-hydroxybutyrate](PHB). Phylogenetic studies using 16S rDNA analysis showed that the isolated bacterium belonged to the Ralstonia pickettii species and had a high identity/similarity with 16S rDNA obtained from total DNA of uncultured strains of soils and with unidentified bacteria at species level. The new strain was named R. pickettii 61A6. Spectrofluorometric analysis showed that the best rates of ssc-PHA accumulation within the cells occurred in 10%(w/v) sucrose and in 5%(w/v) sugarcane molasses at the stationary phase, with a yield of 231 and 357 mg/l of ssc-PHA per g dry cell weight, respectively.     

Effects of inoculum concentration,temperature, and carbon sources on tannase production during solid state fermentation of cashew apple bagasse

In this study, the optimization of tannase production by solid state fermentation was investigated using cashew apple bagasse (CAB), an inexpensive residue produced by the cashew apple agroindustry, as a substrate. To accomplish this, CAB was enriched with 2.5% (w/w) tannic acid and 2.5% (w/w) ammonium sulphate and then moistened with water (60 mL/100 g of dry CAB). The influence of inoculum concentration (10⁴ to 10⁷ spores/g), temperature (20, 25, 30, and 35°C) and several additional carbon sources (glucose, starch, sucrose, maltose, analytical grade glycerol, and glycerol produced during biodiesel production) on enzyme production by Aspergillus oryzae was then evaluated. Supplementation with maltose and glycerol inhibited tannase synthesis, which resulted in lower enzyme activity. Starch and sucrose supplementation increased enzyme production, but decreased the enzyme productivity. The maximum tannase activity (4.63 units/g of dry substrate) was obtained at 30°C, using 10⁷ spores/g and 1.0% (w/v) sucrose as an additional carbon source.     

Glycerol as substrate for biomass and β-carotene production byRhodotorula lactosa

Glycerol was used as the sole carbon and energy source for growingRhodotorula lactosa. The maximum biomass yield (0.53 g/g substrate) was obtained after 20 h with 21.5 g glycerol/l; growth was inhibited with 28.0 g glycerol/l and cell morphology was changed. At this time, the cells were not pigmented. After 48 h of cultivation, -carotene was at 1.8 mg/g dry cells, yielding 22.0 mg/l. When cells were grown for 20 h, washed, suspended in distilled water and aerated for 24 hours, more -carotene (2.66 mg/g dry cells or 28.0 mg/l of the original culture) was produced. Cell protein content after 48 h was 36 to 38% (w/w) before extraction and 45 to 47% (w/w) for acetone-extracted cells.     

Growth and butyric acid production by Clostridium populeti

The growth of Clostridium populeti in 2% (w/v) glucose medium containing 0.2% (w/v) yeast extract was optimal with 10 mM NH₄Cl as the nitrogen source. Although the maximum specific growth rate (=0.32 h^-1) with 5 mM NH₄Cl was similar, the biomass yield was about 30% lower than that at the optimum. Either sodium sulphide or cysteine-HCl at an optimum concentration of 0.33 mM and 5.0 mM respectively, could serve as the sole sulphur source for growth. The growth rate was unaffected by initial glucose concentrations of up to 10% (w/v), but in the presence of 15% glucose it declined by about 35%. The molar yield of butyric acid (mol/mol glucose) declined from 0.70 in 1% (w/v) initial glucose medium to 0.39 in 10% glucose medium. In 5.7% initial glucose medium, butyric acid levels of 6.3 g/l were obtained (0.56 mol butyrate/mol glucose) after 72 h of incubation in 2.5 l batch cultures. A decrease of about 50% in the maximum specific growth rate of C. populeti was observed in the presence of an initial concentration of either 1.2 g/l of butyric acid or 18.9 g/l of acetic acid.This paper is issued as NRCC No. 29032     

Single-cell protein of Rhodotorula sp. Y-38 from ethanol, acetic acid and acetaldehyde

Summary Continuous cultivation of Rhodotorula sp. Y-38 was carried out on ethanol, acetic acid or acetaldehyde. At a feed concentration of 1.0 % (w/v) ethanol, the cell yield of 64 g/100 g ethanol and crude protein of 52 g/100 g biomass were obtained at D=0.5 h^-1. The respective value of the content of amino acids and nucleic acids was 42.6 and 9.4 g/100 g biomass. At 2.0 % (w/v) acetic acid, cell yield was found to be 50 g/100 g acetic acid at D=0.4 h^-1. The optimum dilution rate ranged between 0.3 and 0.4 h^-1. At 0.05 % (w/v) acetaldehyde, the maximum cell yield was obtained at D=0.14 h^-1.     

Direct alcohol fermentation of starch by a derepressed mutant ofSchwanniomyces castellii

Summary Production of ethanol by direct fermentation of soluble starch was carried out using a derepressed mutant M-6 ofSchwanniomyces castellii NRRL Y-2477. Under optimal fermentation conditions, 58.4g/l ethanol was produced utilizing 86% of 150g/l soluble starch. With a mixed culture of mutant M-6 andSaccharomyces cerevisiae STV 89, 64.3g/l of ethanol was produced utilizing 94% of 150g/l soluble starch.     

Optimization for growth of Rhodocyclus gelatinosus in seafood processing effluents

Tuna condensate was a better substrate than shrimp-blanching water or effluent from a frozen-seafood plant for growing Rhodocyclus gelatinosus under anaerobic conditions in the light. One strain out of four examined, R7, gave the highest biomass (4.0 g/l), cell yield (0.32 g cell/g COD), and COD removal (78%) in 1:10 (v/v) diluted tuna condensate. Shrimp-blanching water added to the tuna condensate further increased growth rate, biomass and COD removal. Optimal growth was at pH 7.0 and 3000 Lux light intensity. Acetate, pyruvate, glucose, glutamate, propionate or malate added to the tuna condensate did not increase cell yield, carotenoid or bacteriochlorophyll content or biomass protein. A maximum cell mass of 5.6 g/l (containing 50% protein) and 86% COD removal were obtained after 5 days' incubation under optimal conditions.     

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.     

Isolation and growth of the phototrophic bacterium Rhodopseudomonas palustris strain B1 in sago-starch-processing wastewater

An indigenous strain of the purple non-sulphur phototrophic bacterium, Rhodopseudomonas palustris strain B1, was selected for the utilization and treatment of wastewater from a sago-starch-processing decanter. Growth of Strain B1 under anaerobic–light conditions in the carbohydrate-rich effluent was optimized by using 50% (v/v) effluent diluted in a basal minimal mineral medium with the addition to 0.1% (w/v) yeast extract. The optimum level of nitrogen source supplement, ammonium sulphate, was 1.0g/l. Highest cell mass concentration was achieved by using tungsten lamps as the light source with a light intensity of 4 klux. Under these optimal conditions, a maximum biomass of about 2.5g dry cell/l with a pigment content of about 1.1mg carotenoid/g dry weight cell was achieved after 96h of anaerobic cultivation. There was a 77% reduc n the chemical oxygen demand (COD) of the effluent. A cell yield of about 0.59g dry weight cell/g COD was obtained.     

Effect of C/N ratio and starch concentration on ethanol production from sago starch using recombinant yeast

Recombinant Saccharomyces cerevisiae YKU 131 (capable of expressing glucoamylase) was used to produce ethanol from sago starch. The optimum C/N ratio for ethanol production by the recombinant yeast was 7.9, where 4.7 and 10.1 g/l ethanol was produced from 20 and 40 g/l sago starch, respectively. At sago starch concentration higher than 40 g/l and C/N ratio higher than 10.4, glucoamylase production and rate of starch hydrolysis were reduced, which in turn, reduced ethanol production significantly. The theoretical yield of ethanol based on sago starch consumed in fermentation using 40 g/l was 72.6%. This yield was slightly lower than those obtained in fermentation using soluble starch such as potato and corn starch, which ranged from 80–90% as reported in the literature. However, S. cerevisiae YKU 131 could only utilize 62% of the total amount of starch added to a medium.     

Heterologous protein secretion by Aspergillus niger growing in submerged culture as dispersed or aggregated mycelia

The secreted production of a heterologous enzyme, hen egg-white lysozyme, by Aspergillus niger was studied in shake flasks containing media of different initial viscosities. Raising the viscosity of the medium by addition of polyvinylpyrrolidone (PVP) brought about a transition in the form of growth from aggregated mycelia (pellets) to dispersed mycelia. The specific yield of lysozyme in cultures containing an initial concentration of 5% (w/v) starch was 8 mg lysozyme/g dry weight. Addition of 2% (w/v) PVP to the medium resulted in a specific yield of 14 mg lysozyme/g dry weight.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

The effect of oxygen tension on the production of Bacillus thuringiensis subsp. israelensis toxin active against Aedes aegypti larvae

An optimized batch production of Bacillus thuringiensis subsp. israelensis was made in a stirred Bioflo III reactor using a previously selected medium, and operating conditions in the range of 100–500 rev/min stirrer speeds and 0.2–1 air flow/culture medium volume/minute (v/v/m) aeration rates, including five combinations; at the end of fermentation, dry powders were recovered and evaluated against Aedes aegypti larvae at 0.05 mg/l. Later, the lethal concentration inducing 50% mortality (LC₅₀) was determined for the two most toxic powders. The bioinsecticide yields varied from 9.1 to 15.7 g/l and the total fermentation times ranged between 18 and 30.3 h. The toxicity varied for two powders much more than for the others. These were for combinations with 300 rev/min:1 v/v/m and 500 rev/min:0.6 v/v/m, giving mortality percentages of 47.2 and 59.7, with LC₅₀ values of 0.2675 and 0.0685 mg/l, respectively. A t test showed no significant difference. However, the larvicidal powder produced with 300 rev/min:1 v/v/m gave more variable toxicity than the powder obtained with 500 rev/min:0.6 v/v/m.     

Cell Growth and Shikonin Production of Arnebia euchroma in a Periodically Submerged Airlift Bioreactor

Arnebia euchroma was grown in a 2-l periodically submerged, airlift bioreactor (PSAB) in which the non-submerged (immobilization culture) and submerged (suspension culture) operations were controlled automatically. PSAB had advantages in improving cell growth, shikonin content, shikonin production and cell aggregation compared with suspension culture. Under the optimal submerged/non-submerged period of 10 min/15 h, the shikonin content (4.6%, w/w) and, cell dry mass (16.8 g/l) were 229 and 26% higher than those in suspension culture. Revisions requested 31 October 2005 and 6 December 2005; Revisions received 2 December 2005 and 13 January 2006     

Nitrogen fixation by the hydrogen-oxidizing bacterium Alcaligenes latus

The aerobic hydrogen-oxidizing bacterium Alcaligenes latus represented by three strains was found to be able to grow with dinitrogen as the sole nitrogen source: The doubling time of total (Kjeldahl) nitrogen during growth on glucose at 30°C under an atmosphere containing 2% (v/v) oxygen in dinitrogen amounted to 39 h, while that in the presence of ammonium was 3 h. Nitrogen fixation did apparently not occur under air. During diazotrophic growth the cells accumulated up to 75% (w/dry weight) poly--hydroxybutyric acid. The efficiency of nitrogen fixation varied between 10 and 15 mg N per g glucose utilized. The specific nitrogenase activity measured in the acetylene reduction assay amounted to 5–17 nmol C₂H₄ formed per min and mg protein.     

Rhamnolipid production in batch and fed-batch fermentation using<Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> BYK-2 KCTC 18012P

The optimization of culture conditions for the bacteriumPseudomonas aeruginosa BYK-2 KCTC 18012P, was performed to increase its rhamnolipid production. The optimum level for carbon, nitrogen sources, temperature and pH, for rhamnolipid production in a flask, were identified as 25 g/L fish oil, 0.01% (w/v) urea, 25 and pH 7.0, respectively. Optimum conditions for batch culture, using a 7-L jar fermentor, were 200 rpm of agitation speed and a 2.0 L/min aeration rate. Under the optimum conditions, on fish oil for 216 h, the final cell and rhamnolipid concentrations were 5.3 g/L and 17.0 g/L respectively. Fed-batch fermentation, with different feeding conditions, was carried out in order to increase, cell growth and rhamnolipid production by thePseudomonas aeruginosa, BYK-2 KCTC 18012P. When 2.5 g of fish oil and 100 mL basal salts medium, containing 0.01% (w/v) urea, were fed intermittently during the fermentation, the final cell and rhamnolipid concentrations at 264 h, were 6.1 and 22.7 g/L respectively. The fed-batch culture resulted in a 1.2-fold increase in the dry cell mass and a 1.3-fold increase in rhamnolipid production, compared to the production of the batch culture. The rhamnolipid production-substrate conversion factor (0.75 g/g) was higher than that of the batch culture (0.68 g/g).     

Continuous single cell protein production fromCellulomonas sp. andCandida utilis grown in mixture on barley straw

Summary Continuous fermentations with mixed cultures of the cellulolytic bacteriaCellulomonas sp. and the yeastCandida utilis were examined. Fermentations were carried out in an aerated 5-l fermenter with different preparations of wet disintegrated barley straw as the cellulose source (3.6–4.2%). The straw was pretreated with NaOH (3.2–8.5 kg NaOH/100 kg dry straw) under high pressure and temperature in a feedstuff pellet press. The quantity of dry cell mass produced and the breakdown of the straw were measured. Crude protein and ash content in cell dry matter and residual fiber were determined. The experiments showed thatCellulomonas sp. andCandida utilis may be grown together in a continuous culture (dilution rate D=0.12–0.14 h^–1) for at least 3 days without washing out one of the organisms. Highest productivity was 1.39 g cell dry matter/l/h when using straw pretreated with 5.7% NaOH. The dry cell product contained 58–66% crude protein and up to 51% of the organic fiber dry matter was solubilized. The yield constants were 0.32–0.61 g cell dry matter per g solubilized organic fibers.