Butanol production from wheat straw hydrolysate using <Emphasis Type="Italic">Clostridium beijerinckii</Emphasis>

In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation 48.9 g L⁻¹ glucose (initial sugar 62.0 g L⁻¹) was used to produce 20.1 g L⁻¹ ABE with a productivity and yield of 0.28 g L⁻¹ h⁻¹ and 0.41, respectively. In a similar experiment where WSH (60.2 g L⁻¹ total sugars obtained from hydrolysis of 86 g L⁻¹ wheat straw) was used, the culture produced 25.0 g L⁻¹ ABE with a productivity and yield of 0.60 g L⁻¹ h⁻¹ and 0.42, respectively. These results are superior to the control experiment and productivity was improved by 214%. When WSH was supplemented with 35 g L⁻¹ glucose, a reactor productivity was improved to 0.63 g L⁻¹ h⁻¹ with a yield of 0.42. In this case, ABE concentration in the broth was 28.2 g L⁻¹. When WSH was supplemented with 60 g L⁻¹ glucose, the resultant medium containing 128.3 g L⁻¹ sugars was successfully fermented (due to product removal) to produce 47.6 g L⁻¹ ABE, and the culture utilized all the sugars (glucose, xylose, arabinose, galactose, and mannose). These results demonstrate that C. beijerinckii P260 has excellent capacity to convert biomass derived sugars to solvents and can produce over 28 g L⁻¹ (in one case 41.7 g L⁻¹ from glucose) ABE from WSH. Medium containing 250 g L⁻¹ glucose resulted in no growth and no ABE production. Mixtures containing WSH + 140 g L⁻¹ glucose (total sugar approximately 200 g L⁻¹) showed poor growth and poor ABE production. Mention of trade names or commercial products in this article is solely for the purpose of providing scientific information and does not imply recommendation or endorsement by the United States Department of Agriculture.     

Production of a Metal-Binding Exopolysaccharide by Paenibacillus jamilae Using Two-Phase Olive-Mill Waste as Fermentation Substrate

The present study investigated the use of two-phase olive mill waste (TPOMW) as substrate for the production of exopolysaccharide (EPS) by the endospore-forming bacilli Paenibacillus jamilae. This microorganism was able to grow and produce EPS in aqueous extracts of TPOMW as a unique source of carbon. The effects of substrate concentration and the addition of inorganic nutrients were investigated. Maximal polymer yield in 100-ml batch-culture experiments (2 g l⁻¹) was obtained in cultures prepared with an aqueous extract of 20% TPOMW (w/v). An inhibitory effect was observed on growth and EPS production when TPOMW concentration was increased. Nutrient supplementation (nitrate, phosphate, and other inorganic nutrients) did not increase yield. Finally, an adsorption experiment of Pb (II), Cd (II), Cu (II), Zn (II), Co (II), and Ni (II) by EPS is reported. Lead was preferentially complexed by the polymer, with a maximal uptake of 230 mg/g EPS.     

Obtaining and selection of hexokinases-less strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for production of ethanol and fructose from sucrose

Saccharomyces cerevisiae hexokinase-less strains were produced to study the production of ethanol and fructose from sucrose. These strains do not have the hexokinases A and B. Twenty-three double-mutant strains were produced, and then, three were selected for presenting a smaller growth in yeast extract–peptone–fructose. In fermentations with a medium containing sucrose (180.3 g L⁻¹) and with cell recycles, simulating industrial conditions, the capacity of these mutant yeasts in inverting sucrose and fermenting only glucose was well characterized. Besides that, we could also see their great tolerance to the stresses of fermentative recycles, where fructose production (until 90 g L⁻¹) and ethanol production (until 42.3 g L⁻¹) occurred in cycles of 12 h, in which hexokinase-less yeasts performed high growth (51.2% of wet biomass) and viability rates (77% of viable cells) after nine consecutive cycles.     

Production and composition of extracellular polysaccharide synthesized by a Rhizobium isolate of Vigna mungo (L.) Hepper

An extracellular polysaccharide (EPS) was produced by a Rhizobium sp. isolated from the root nodules of Vigna mungo (L.) Hepper. Maximum EPS production (346 mg l⁻¹) was when the yeast extract basal medium was supplemented with mannitol (1%), biotin (1.5 mg l⁻¹) and asparagine (0.3%). Ribose (53%) and mannose (47%) were the principle monomers of the EPS. Chemical, chromatographic and spectroscopic analysis showed that this polymer, which has Man₄Rib₁ as an oligomeric subunit, has an apparent molecular mass of 750 kDa.     

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

A microorganism with the ability to catalyze the resolution of racemic phenyloxirane was isolated and identified as Aspergillus niger SQ-6. Chiral capillary electrophoresis was successfully applied to separate both phenyloxirane and phenylethanediol. The epoxide hydrolase (EH) involved in this resolution process was (R)-stereospecific and constitutively expressed. When whole cells were used during the biotransformation process, the optimum temperature and pH for stereospecific vicinal diol production were 35°C and 7.0, respectively. After a 24-h conversion, the enantiomer excess of (R)-phenylethanediol produced was found to be >99%, with a conversion rate of 56%. In fed-batch fermentations at 30°C for 44 h, glycerol (20 g L⁻¹) and corn steep liquor (CSL) (30 g L⁻¹) were chosen as the best initial carbon and nitrogen sources, and EH production was markedly improved by pulsed feeding of sucrose (2 g L⁻¹ h⁻¹) and continuous feeding of CSL (1 g L⁻¹ h⁻¹) at a fermentation time of 28 h. After optimization, the maximum dry cell weight achieved was 24.5±0.8 g L⁻¹; maximum EH production was 351.2±13.1 U L⁻¹ with a specific activity of 14.3±0.5 U g⁻¹. Partially purified EH exhibited a temperature optimum at 37°C and pH optimum at 7.5 in 0.1 M phosphate buffer. This study presents the first evidence for the existence of a predicted epoxide racemase, which might be important in the synthesis of epoxide intermediates.     

Prospects of using marine actinobacteria as probiotics in aquaculture

In the present study, optimum culture conditions for the production of extracellular polysaccharides (EPS) in submerged culture of an edible mushroom, Laetiporus sulphureus var. miniatus and their stimulatory effects on insulinoma cell (RINm5F) proliferation and insulin secretion were investigated. The maximum mycelial growth (4.1 g l⁻¹) and EPS production (0.6 g l⁻¹) in submerged flask culture were achieved in a medium containing 30 g l⁻¹ maltose, 2 g l⁻¹ soy peptone, and 2 mM MnSO₄·5H₂O at an initial pH 2.0 and temperature 25°C. In the stirred-tank fermenter under optimized medium, the concentrations of mycelial biomass and EPS reached a maximum level of 8.1 and 3.9 g l⁻¹, respectively. Interestingly, supplementation of deep sea water (DSW) into the culture medium significantly increased both mycelial biomass and EPS production by 4- and 6.7-fold at 70% (v/v) DSW medium, respectively. The EPS were proved to be glucose-rich polysaccharides and were able to increase proliferation and insulin secretary function of rat insulinoma RINm5F cells, in a dose-dependent manner. In addition, EPS also strikingly reduced the streptozotocin-induced apoptosis in RINm5F cells indicating the mode of the cytoprotective role of EPS on RINm5F cells.     

Enhanced fructooligosaccharides and inulinase production by a <Emphasis Type="Italic">Xanthomonas campestris</Emphasis> pv. <Emphasis Type="Italic">phaseoli</Emphasis> KM 24 mutant

Xanthomonas campestris pv phaseoli produced an extracellular endoinulinase (9.24 ± 0.03 U mL⁻¹) in an optimized medium comprising of 3% sucrose and 2.5% tryptone. X. campestris pv. phaseoli was further subjected to ethylmethanesulfonate mutagenesis and the resulting mutant, X. campestris pv. phaseoli KM 24 demonstrated inulinase production of 22.09 ± 0.03 U mL⁻¹ after 18 h, which was 2.4-fold higher than that of the wild type. Inulinase production by this mutant was scaled up using sucrose as a carbon source in a 5-L fermenter yielding maximum volumetric (21,865 U L⁻¹ h⁻¹) and specific (119,025 U g⁻¹ h⁻¹) productivities of inulinase after 18 h with an inulinase/invertase ratio of 2.6. A maximum FOS production of 11.9 g L⁻¹ h⁻¹ and specific productivity of 72 g g⁻¹ h⁻¹ FOS from inulin were observed in a fermenter, when the mutant was grown on medium containing 3% inulin and 2.5% tryptone. The detection of mono- and oligosaccharides in inulin hydrolysates by TLC analysis indicated the presence of an endoinulinase. This mutant has potential for large-scale production of inulinase and fructooligosaccharides.     

Comparison of exopolysaccharide production by strains of Lactobacillus rhamnosus and Lactobacillus paracasei grown in chemically defined medium and milk

Exopolysaccharide (EPS) production was compared among three strains of lactobacilli. Lactobacillus rhamnosus strain 9595M can be classified among the highest EPS-producing strains of lactic acid bacteria reported to date with a maximum EPS production of 1275 mg L⁻¹. Under controlled pH, no significant differences in the quantity of EPS produced could be detected between carbon source (glucose or lactose) or fermentation temperature (32 or 37°C). In milk, strains ATCC 9595M and R produced more than 280 mg L⁻¹ EPS whereas strain Type V produced less than 80 mg L⁻¹ EPS. Journal of Industrial Microbiology & Biotechnology (2000) 24, 251–255. Received 10 September 1999/ Accepted in revised form 22 December 1999     

A halotolerant and thermotolerant <Emphasis Type="Italic">Bacillus</Emphasis> sp. degrades hydrocarbons and produces tensio-active emulsifying agent

A Bacillus sp. strain DHT, isolated from oil-contaminated soil, grew and produced biosurfactant when cultured in variety of substrate at salinities of up to 100 g l⁻¹ and temperatures up to 45°C. It was capable of utilizing crude oil, fuels, various pure alkanes and PAHs as a sole carbon and energy source across a wide range of temperature and salinity. Over the range evaluated, the degradation of hydrocarbon and biosurfactant production was not influenced by salinity (0–10% wv⁻¹) and temperature (30–45°C). The biosurfactant produced by the organism emulsified a range of hydrocarbons with hexadecane as the best substrate and toluene as the poorest. From 16S rDNA analysis, strain DHT was related to Bacillus licheniformis.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid from sugarcane molasses,sugarcane juice and sugar beet juice by <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Lactobacillus delbrueckii was grown on sugarcane molasses, sugarcane juice and sugar beet juice in batch fermentation at pH 6 and at 40°C. After 72 h, the lactic acid from 13% (w/v) sugarcane molasses (119 g total sugar l⁻¹) and sugarcane juice (133 g total sugar l⁻¹) was 107 g l⁻¹ and 120 g l⁻¹, respectively. With 10% (w/v) sugar beet juice (105 g total sugar l⁻¹), 84 g lactic acid l⁻¹ was produced. The optical purities of d-lactic acid from the feedstocks ranged from 97.2 to 98.3%.     

Guggulsterone production in cell suspension cultures of the guggul tree, <Emphasis Type="Italic">Commiphora wightii</Emphasis>, grown in shake-flasks and bioreactors

Cell suspension cultures of Commiphora wightii, grown in modified MS medium containing 2,4-dichlorophenoxyacetic acid (0.5 mg l⁻¹) and kinetin (0.25 mg l⁻¹), produced ∼5 μg guggulsterone g⁻¹ dry wt. In a 2 l stirred tank bioreactor, the biomass was 5.5 g l⁻¹ and total guggulsterone was 36 μg l⁻¹.     

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 10⁸ cells ml⁻¹ and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Y _ps) and productivity (Q _p ) were 100 g l⁻¹, 0.42 g g⁻¹ and 1.67 g l⁻¹ h⁻¹ respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Y _ps and Q _p were 120 g l⁻¹, 0.48 g g⁻¹ and 1.11 g l⁻¹ h⁻¹ respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.     

Production and structural analysis of the polysaccharide secreted by <Emphasis Type="Italic">Trametes (Coriolus) versicolor</Emphasis> ATCC 200801

Trametes versicolor ATCC 200801 secretes 4.1 g L⁻¹ of exopolysaccharide (EPS) when synthetic minimal medium and low-shear bioreactor cultivation technique are used. Structural and compositional analyses by thin layer chromatography, gas chromatography–mass spectrometry, electrospray ionization tandem mass spectrometry, and nuclear magnetic resonance spectroscopy yielded predominantly glucose and small amounts of galactose, mannose, arabinose, and xylose. The main EPS is composed of β-1,3/β-1,6-linked d-glucose molecules which is identical with Schizophyllan but does not possess a triple helical arrangement as secondary structure. Two molar mass fractions were detected by size exclusion chromatography yielding weight-average molecular weights of 4,100 and 2.6 kDa. Protein content varies between 2–3.6% (w/w). The exopolysaccharide is different in the nature of the glycosidic linkage, composition of monosaccharides, protein content, and weight-average molecular weight compared to the well-known polysaccharopeptide (PSP) and polysaccharopeptide Krestin (PSK).     

Enhanced production of an alkaline pectinase from Streptomyces sp. RCK-SC by whole-cell immobilization and solid-state cultivation

An alkalophilic Streptomyces sp. RCK-SC, which produced a thermostable alkaline pectinase, was isolated from soil samples. Pectinase production at 45 °C in shaking conditions (200 rev min⁻¹) was optimal (76,000 IU l⁻¹) when a combination of glucose (0.25% w/v) and citrus pectin (0.25% w/v) was added along with urea (0.25% w/v) in the basal medium devoid of yeast extract and peptone. All the tested amino acids and vitamins greatly induced pectinase production and increased the specific productivity of pectinase up to 550%. In an immobilized cell system containing polyurethane foam (PUF), the pectinase production was enhanced by 32% (101,000 IU l⁻¹) compared to shake flask cultures. In solid-state cultivation (SSC) conditions, using wheat bran as solid substrate, pectinase yield of 4857 IU g⁻¹ dry substrate was obtained at substrate-to-moisture ratio of 1:5 after 72 h of incubation. The partially purified pectinase was optimally active at 60 °C and retained 80% of its activity at 50 °C after 2 h of incubation. The half life of pectinase was 3 h at 70 °C. Pectinase was stable at alkaline pH ranging from 6.0 to 9.0 for more than 8 h at room temperature retaining more than 50% of its activity. This revised version was published online in August 2006 with corrections to the Cover Date.     

Temperature profiles of cellular growth and exopolysaccharide synthesis by Botryococus braunii Kütz. UC 58

Temperature profiles (range 20–33 °C) were obtained for growth and exopolysaccharide (EPS) biosynthesis of the microalga Botryococcus braunii strain UC 58 under photoautotrophic conditions. The maximum temperature for growth was 32 °C and the temperature dependence of the specific growth rate was described by the Hinshelwood equation based on the Arrhenius relationship. The optimal range of temperatures for growth and extracellular EPS synthesis (25–30 °C) concurred and production of 4.5–5 g l⁻¹ of EPS was obtained routinely, leading to high broth viscosities. Below 23 °C EPS biosynthesis was negligible, although the specific growth rate maintained high values. At supraoptimal temperatures EPS biosynthesis decreased, accompanying the increase in doubling time. The polymers formed at temperatures within the optimal range for production, when dissolved in water, produced solutions (2 gl⁻¹) with the highest viscosity, suggesting that their molecular weight showed the highest values. The degree of polymerization of the EPS synthesized at suboptimal and supraoptimal temperatures was significantly below the values within the optimal range.     

Influence of feeding conditions on clavulanic acid production in fed-batch cultivation with medium containing glycerol

First, the effect of different levels of nitrogen source on clavulanic acid (CA) production was evaluated in batch cultivations utilizing complex culture medium containing glycerol and three different levels of soy protein isolate (SPI). Cellular growth, evaluated in terms of the rheological parameter K, was highest with a SPI concentration of 30 g.L⁻¹ (4.42 g.L⁻¹ N total). However, the highest production of CA (380 mg.L⁻¹) was obtained when an intermediate concentration of 20 g.L⁻¹ of SPI (2.95 g.L⁻¹ total N) was used. To address this, the influences of volumetric flow rate (F) and glycerol concentration in the complex feed medium (Cs_F) in fed-batch cultivations were investigated. The best experimental condition for CA production was F=0.01 L.h⁻¹ and Cs_F=120 g.L⁻¹, and under these conditions maximum CA production was practically twice that obtained in the batch cultivation. A single empirical equation was proposed to relate maximum CA production with F and Cs_F in fed-batch experiments.     

Evaluation of sugar cane hemicellulose hydrolyzate for cultivation of yeasts and filamentous fungi

Sugar cane bagasse hemicellulosic fraction submitted to hydrolytic treatment with 100 mg of sulfuric acid per gram of dry mass, at 140°C for 20 min, was employed as a substrate for microbial protein production. Among the 22 species of microorganisms evaluated, Candida tropicalis IZ 1824 showed TRS consumption rate of 89.8%, net cell mass of 11.8 g L⁻¹ and yield coefficient (Y_x/s) of 0.50 g g⁻¹. The hydrolyzate supplemented with rice bran (20.0 g L⁻¹), P₂O₅ (2.0 g L⁻¹) and urea (2.0 g L⁻¹) provided a TRS consumption rate of 86.3% and a cell mass of 8.4 g L⁻¹. At pH 4.0 cellular metabolism was inhibited, whereas at pH 6.0 the highest yield was obtained. The presence of furfural (2.0 g L⁻¹) hydroxymethylfurfural (0.08 g L⁻¹) and acetic acid (3.7 g L⁻¹) in the hydrolyzate did not interfere with cultivation at pH 6.0. Received 25 October 1996/ Accepted in revised form 10 March 1997     

Biotransformation of Isoeugenol to Vanillin by a Novel Strain of <Emphasis Type="Italic">Bacillus fusiformis</Emphasis>

A novel strain of Bacillus fusiformis, producing high amounts of vanillin from isoeugenol, was isolated from soil. Using 60% (v/v) isoeugenol as substrate and solvent and at pH 4.0, 37 °C and 180 rpm, vanillin was produced at 32.5 g l⁻¹ over 72 h. The unused isoeugenol was reusable.     

The Role of Sucrose in Regulation of Trunk Tissue Development in Betula pendula Roth

Comparative ultrastructural analysis of the conducting and non-conducting phloem cells in the common straight-grained silver birch (Betula pendula var. pendula) and the Karelian birch (B. pendula var. carelica) with abnormal patterned wood was carried out, leading to the conclusion that there is an elevated sucrose content in the conducting phloem of the Karelian birch. A connection between sucrose levels and formation of abnormalities in the development of conducting tissues in the Karelian birch trunk was surmised. Experiments in which exogenous sucrose was applied to the silver birch trunk tissues have demonstrated the effects of different sucrose concentrations (0 g L⁻¹, 10 g L⁻¹, 25 g L⁻¹, 50 g L⁻¹, 100 g L⁻¹) on the formation of xylem and phloem structural elements, and they yielded the types of tissue development that correspond to the abnormal tissue development in the Karelian birch trunk.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.