Application of statistically-based experimental designs in medium optimization for spore production of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> from distillery effluent

Spore production of Bacillus subtilis from distillery effluent was optimized using statistically-based experimental designs. The two-level Plackett–Burman design was applied to choose the nutrient supplements significantly influencing spore production. Among the seven variables we tested, the most significant variables influencing spore production were statistically elucidated for optimization, and included (NH₄)₂SO₄, corn flour and MgSO₄. The optimum concentration of each significant variable was then predicted using Box–Behnken design. A second-order polynomial was determined by the multiple regression analysis of this experimental data. The optimum values for the critical nutrient supplements for the maximum were obtained as followed: (NH₄)₂SO₄, 4.54%; corn flour, 1.2%; MgSO₄, 0.56% with the corresponding value of maximum spore production of 7.24 × 10⁸ spores/ml. A verification experiment performed under the optimum conditions resulted in 6.95 × 10⁸ spores/ml. The determination coefficient (R ²) was 0.98, which ensure an adequate credibility of the model.     

Production and properties of inulinase from Penicillium sp. NFCC 2768 grown on inulin-rich vegetal infusions

Inulinase production by Penicillium sp. NFCC 2768 isolated from the rhizosphere soil of dahlia was studied on media containing inulin-rich plant extracts. The maximum inulinase activity (64.54 nkat/ml) was observed with the tuber extract of dahlia (Dahlia pinnata). The fungus produced substantial inulinase activity on asparagus root powder (45.23 nkat/ml) and garlic extracts (41.32 nkat/ml). The apparent molecular weight of the purified inulinase was 68 kDa. The optimum pH and temperature for enzyme activity were 5.0 and 50°C, respectively. Mn²⁺ and Ca²⁺ were found to enhance the inulinase activity, while Hg²⁺ was found to be a strong inhibitor. Inulinase liberated fructose, glucose, sucrose, kestose (GF₂), nystose (GF₃), and inulooligosaccharides (IOS). This study suggested the use of dahlia tuber extract and asparagus root powder as suitable substrates for inulinase production by the newly isolated Penicillium sp. NFCC 2768, and its application in the generation of fructose and IOS.     

Extracellular lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NRRL3; production,partial purification and properties

Four strains of Aspergillus niger were screened for lipase production. Each was cultivated on four different media differing in their contents of mineral components and sources of carbon and nitrogen. Aspergillus niger NRRL3 produced maximal activity (325U/ml) when grown in 3% peptone, 0.05% MgSO₄.7H₂O, 0.05% KCl, 0.2% K₂HPO₄ and 1% olive oil:glucose (0.5:0.5). A. niger NRRL3 lipase was partially purified by ammonium sulphate precipitation. The majority of lipase activity (48%) was located in fraction IV precipitated at 50–60% of saturation with a 18-fold enzyme purification. The optimal pH of the partial purified lipase preparation for the hydrolysis of emulsified olive oil was 7.2 and the optimum temperature was 60°C. At 70°C, the enzyme retained more than 90% of its activity. Enzyme activity was inhibited by Hg²⁺ and K⁺, whereas Ca²⁺ and Mn²⁺ greatly stimulated its activity. Additionally, the formed lipase was stored for one month without any loss in the activity.     

Production of Indole Acetic Acid in Culture by a Rhizobium Species from the Root Nodules of a Monocotyledonous Tree,Roystonea regia

The study of the rhizobial root nodules of the monocotyledonous tree Roystonea regia revealed that the Rhizobium sp. isolated from the root nodules produced high amounts (45.6 μg/ml) of indole acetic acid (IAA) from L‐tryptophan supplemented basal medium. The IAA production reached its optimum using 3 mg/ml of L‐tryptophan. The preferred carbon and nitrogen sources were glucose and KNO₃ and the optimum concentrations 1% and 0.02%, respectively. FeSO₄ × 7 H₂O was found to be the only metal ion that increased IAA production. An optimum IAA production was also achieved when the basal medium was supplemented with glucose (1%), FeSO₄ × 7 H₂O (10 μg/ml), KNO₃ (0.02%) as well as EDTA (5 μg/ml) and L‐tryptophan (3 mg/ml). The possible role of IAA production in the monocotyledonous tree‐Rhizobium symbiosis is discussed. Hormone production is shown to be the beneficial aspect of this symbiosis as shown earlier in dicotyledonous plants.     

Production of Glucose Isomerase by Streptomyces flavogriseus

A microorganism that produces glucose isomerase was isolated from soil and identified as a strain of Streptomyces flavogriseus. The organism produced a large quantity of glucose isomerase when grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of ryegrass straw. The organism produced glucose isomerase both intra- and extra-cellularly. The highest level of intracellular glucose isomerase (3.5 U/ml) was obtained in about 36 h by a culture grown on straw hemicellulose; the extracellular enzyme (1.5 U/ml) appeared in cultures grown for about 72 h. About equal levels of enzyme were produced in cultures grown on straw hemicellulose, xylan, xylose, and H₂SO₄ hydrolysate of straw, but production of the enzyme was drastically reduced when the organism was grown on other carbon sources. As a nitrogen source, corn steep liquor produced the best results. Soy flour extract, yeast extract, and various peptones also were adequate substrates for glucose isomerase production. Addition of Mg²⁺, Mn²⁺, or Fe²⁺ to the growth medium significantly enhanced enzyme production. The organism, however, did not require Co²⁺, which is commonly required by microorganisms used in the production of glucose isomerase.     

Ethanol production from raw starch by simultaneous fermentation using Schizosaccharomyces pombe and a raw starch saccharifying enzyme from Corticium rolfsii

Alcoholic fermentation from raw corn starch using Schizosaccharomyces pombe AHU 3179 and a raw starch saccharifying enzyme (RSSE) from Corticium rolfsii AHU 9627 was investigated. The optimum ethanol production was achieved at pH 3.5, 27°C and under the yeast cell concentration of 2.7 × 10⁹ cells/ml. Addition of RSSE 5 units (as glucoamylase)/g raw corn starch was found sufficient. Under these optimum conditions, 18.5% (v/v, at 15°C) ethanol was obtained from 30% raw corn starch (30.8% as glucose) after incubation for 48 h.     

Characterization of Active Lentinula edodes Glucoamylase Expressed and Secreted by Saccharomyces cerevisiae

The gene encoding Lentinula edodes glucoamylase (GLA) was cloned into Saccharomyces cerevisiae, expressed constitutively and secreted in an active form. The enzyme was purified to homogeneity by (NH₄)₂SO₄ fractionation, anion exchange and affinity chromatography. The protein had a correct N-terminal sequence of WAQSSVIDAYVAS, indicating that the signal peptide was efficiently cleaved. The recombinant enzyme was glycosylated with a 2.4% carbohydrate content. It had a pH optimum of 4.6 and a pH 3.4–6.4 stability range. The temperature optimum was 50°C with stability ≤50°C. The enzyme showed considerable loss of activity when incubated with glucose (44%), glucosamine (68%), galactose (22%), and xylose (64%). The addition of Mn⁺⁺ activated the enzyme by 45%, while Li⁺, Zn⁺⁺, Mg⁺⁺, Cu⁺, Ca⁺⁺, and EDTA had no effect. The enzyme hydrolyzed amylopectin at rates 1.5 and 8.0 times that of soluble starch and amylose, respectively. Soluble starch was hydrolyzed 16 and 29 times faster than wheat and corn starch granules, respectively, with the hydrolysis of starch granules using 10× the amount of GLA. Apparent K_m and V_max for soluble starch were estimated to be 3.0 mg/ml and 0.13 mg/ml/min (40°C, pH 5.3), with an apparent k_cat of 2.9×10⁵ min⁻¹.     

Production of high-fructose syrup by a heat-fixed Lactobacillus sp.

A Lactobacillus sp. isolated from soil and capable of growing on xylose-containing medium exhibited high glucose isomerase activity. The enzyme was thermostable, stable toward dialysis, and activated by heat treatment. It did not show the presence of xylose or ribose isomerase activities; the K_m for glucose and xylose substrates were 0.48M and 0.513M, respectively. The heat treatment of ultrasonic crude extract gave insoluble fixed active glucose isomerase enzyme. The properties of free and immobilized enzyme in heat-fixed whole cells differed in many respects. The optimum temperature for enzyme activity changed from 70 to 85°C, the optimum substrate concentration changed from 1.0M to 2.4M, and the optimum pH from 7.4 to 6.0. Co²⁺ and Mg²⁺ ions activated the enzyme when used singly, but in combination they inhibited the enzyme and Mn²⁺ had no effect on the enzyme. Free and immobilized enzymes, when used in the used in the conversions of corn and bagasse hydrolysates to fructose, gave 58, 25.6%, and 50, 27.6% conversions, respectively. Immobilized enzyme retained a significant activity for more than 30 hr and was able to operate at higher glucose concentrations showing less products inhibition effect as compared to free enzyme. In the batch process it was able to operate for about eight cycles.     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

Optimization of Xylanase Production by Thermomyces Lanuginosus in Tomato Seed Meal Using Response Surface Methodology

Summary A 3² central composite experimental design was performed with the aim of optimizing xylanase production by Thermomyces lanuginosus grown on corn cobs in submerged cultures. Xylanase production was first tested on different nitrogen sources (tomato skin, tomato seed meal, corn steep liquor, meat peptone, bacto-tryptone and yeast extract). Tomato seed meal was the selected substrate to test the effect of two variables on xylanase production (corn cobs and tomato seed meal concentrations). A second-order quadratic model and a response surface method showed that the optimum condition for xylanase production was corn cobs 4.6% (w/v) and tomato seed meal 2.1% (w/v). The optimum conditions found were transferred to 7-l bioreactors, where activities as high as 1630 U/ml were obtained.     

Production and characterization of extracellular protease of mutant Aspergillus niger AB100 grown on fish scale

Fish scale, the chief waste material of fish processing industries was processed and tested for production of extracellular protease by mutant Aspergillus niger AB₁₀₀. Protease production by A. niger AB₁₀₀ was greatly enhanced in presence of processed fish scale powder. Where as among the three complex nutrients tested, soya bean meal shows maximum stimulatory effect over protease production (2,776 μmol/ml/min) when used in combination with glucose (5% w/v) and urea (2.5% w/v). The protease was optimally active at pH 7.0, retaining more than 60% of its activity in the pH range of 5–9. The enzyme was found to be most active at 50°C and stable at 30°C for 1 h. Purification of enzyme by CM-Cellulose and SDS-PAGE resulted in about 26-fold increase in the specific activity of the enzyme with a molecular weight of 30.9 kDa. HPLC study shows the purity of the enzyme as 75.92%. By the activating effect of divalent cations (Fe²⁺, Zn²⁺, Mn²⁺, Ca²⁺and Mg²⁺) and inhibiting effect of chelating agent (EDTA) and Hg²⁺, the enzyme was found to be a metalloprotease.     

Purification and characterization of a dextranase from Sporothrix schenckii

A dextranase (EC 3.2.1.11) was purified and characterized from the IP-29 strain of Sporothrix schenckii, a dimorphic pathogenic fungus. Growing cells secreted the enzyme into a standard culture medium (20 °C) that supports the mycelial phase. Soluble bacterial dextrans substituted for glucose as substrate with a small decrease in cellular yield but a tenfold increase in the production of dextranase. This enzyme is a monomeric protein with a molecular mass of 79 kDa, a pH optimum of 5.0, and an action pattern against a soluble 170-kDa bacterial dextran that leads to a final mixture of glucose (38%), isomaltose (38%), and branched oligosaccharides (24%). In the presence of 200 mM sodium acetate buffer (pH 5.0), the K _m for soluble dextran was 0.067 ± 0.003% (w/v). Salts of Hg²⁺, (UO₂)²⁺, Pb²⁺, Cu²⁺, and Zn²⁺ inhibited by affecting both V _max and K _m. The enzyme was most stable between pH values of 4.50 and 4.75, where the half-life at 55 °C was 18 min and the energy of activation for heat denaturation was 99 kcal/mol. S. schenckii dextranase catalyzed the degradation of cross-linked dextran chains in Sephadex G-50 to G-200, and the latter was a good substrate for cell growth at 20 °C. Highly cross-linked grades (i.e., G-10 and G-25) were refractory to hydrolysis. Most strains of S. schenckii from Europe and North America tested positive for dextranase when grown at 20 °C. All of these isolates grew on glucose at 35 °C, a condition that is typically associated with the yeast phase, but they did not express dextranase and were incapable of using dextran as a carbon source at the higher temperature. Received: 29 December 1997 / Accepted: 4 March 1998     

Thermostable α‐Amylase and Glucoamylase from Thermomyces lanuginosus F1

An α‐amylase and a glucoamylase produced by Thermomyces lanuginosus F₁ were separated by ion‐exchange chromatography on Q‐Sepharose fast flow. The enzymes were further purified to electrophoretic homogeneity by chromatography on Sephadex G‐100 and Phenyl‐Sepharose CL‐4B.The molecular weights and isoelectric points of the enzymes were 55,000 Da and pHi 4.0 for α‐amylase and 70,000 Da and pH_i 4.0 for glucoamylase, respectively. The optimum pH and temperatures for the enzymes were found to be 5.0 and 60 °C for α‐amylase, and 6.0 and 70 °C for glucoamylase,respectively. Both enzymes were maximally stable at pH 4.0 and retained over 80% of their activity between pH 5.0 and 6.0 for 24 h. After incubation at 90 °C (1 h), the α‐amylase and glucoamylase retained only 6% and 16% of their activity, respectively. The enzymes readily hydrolyzed soluble starch, amylose, amylopectin and glycogen but hydrolyzed pullulan very slowly. Glucoamylase and α‐amylase had highest affinity for soluble starch with K_M values of 0.80 mg/ml and 0.67 mg/ml, respectively. The α‐amylase hydrolyzed raw starch granules with a predominant production of glucose and maltose. The activities of α‐amylase and glucoamylase increased in the presence of Mn²⁺, Co²⁺, Ca²⁺, Zn²⁺ and Fe²⁺, but were inhibited by guanidine‐HCl, urea and disodium EDTA. Both enzymes possess pH and thermal stability characteristics that may be of technological significance.     

Optimization of conditions for the production of lignocellulolytic enzymes by Sphingobacterium sp. ksn-11 utilizing agro-wastes under submerged condition

Abstract

The present work was aimed at studying the production of lignocellulolytic enzymes, namely cellulase, xylanase, pectinase, mannanase, and laccase by a newly isolated bacterium Sphingobacterium sp. ksn-11, utilizing various agro-residues as a substrate under submerged conditions. The production of lignocellulolytic enzymes was found to be maximum at the loading of 10%(w/v) agro-residues. The enzyme secretion was enhanced by two-fold at 2?mM CaCO_3, optimum pH 7, and temperature 40°. The Field Emission Gun-Scanning Electron Microscope (FEG-SEM) results have shown the degradative effect of lignocellulases; cellulase, xylanase, mannanase, pectinase, and laccase on corn husk with 3.55?U/ml, 79.22?U/ml, 12.43?U/ml, 64.66?U/ml, and 21.12?U/ml of activity, respectively. The hydrolyzed corn husk found to be good adsorbent for polyphenols released during hydrolysis of corn husk providing suitable conditions for stability of lignocellulases. Sphingobacterium sp. ksn is proved to be a promising candidate for lignocellulolytic enzymes in view of demand for enzymes in the biofuel industry.     

Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH₄Cl, urea, and ammonium citrate); and metal ions (Zn²⁺, Fe³⁺, Mg²⁺, Mn²⁺, Ca²⁺, and K⁺). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg²⁺ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg²⁺. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.     

Conversion of biomass hydrolysates and other substrates to ethanol and other chemicals by Lactobacillus buchneri*

Aims: A Lactobacillus buchneri strain NRRL B‐30929 can convert xylose and glucose into ethanol and chemicals. The aims of the study were to survey three strains (NRRL B‐30929, NRRL 1837 and DSM 5987) for fermenting 17 single substrates and to exam NRRL B‐30929 for fermenting mixed substrates from biomass hydrolysates. Methods and Results: Mixed acid fermentation was observed for all three L. buchneri strains using various carbohydrates; the only exception was uridine which yielded lactate, acetate and uracil. Only B‐30929 is capable of utilizing cellobiose, a desired trait in a potential biocatalyst for biomass conversion. Flask fermentation indicated that the B‐30929 strain can use all the sugars released from pretreated hydrolysates, and producing 1·98–2·35 g l^?1 ethanol from corn stover hydrolysates and 2·92–3·01 g l^?1 ethanol from wheat straw hydrolysates when supplemented with either 0·25× MRS plus 1% corn steep liquor or 0·5× MRS. Conclusions: The L. buchneri NRRL B‐30929 can utilize mixed sugars in corn stover and wheat straw hydrolysates for ethanol and other chemical production. Significance and Impact of the Study: These results are valuable for future research in engineering L. buchneri NRRL B‐30929 for fermentative production of ethanol and chemicals from biomass.     

Production of uridine 5′-monophosphate by Corynebacterium ammoniagenes ATCC 6872 using a statistically improved biocatalytic process

Attempts were made with success to develop a two-step biocatalytic process for uridine 5′-monophosphate (UMP) production from orotic acid by Corynebacterium ammoniagenes ATCC 6872: the strain was first cultivated in a high salt mineral medium, and then cells were harvested and used as the catalyst in the UMP production reaction. Effects of cultivation and reaction conditions on UMP production were investigated. The cells exhibited the highest biocatalytic ability when cultivated in a medium containing corn steep liquor at pH 7.0 for 15 h in the exponential phase of growth. To optimize the reaction, both “one-factor-at-a-time” method and statistical method were performed. By “one-factor-at-a-time” optimization, orotic acid, glucose, phosphate ion (equimolar KH₂PO₄ and K₂HPO₄), MgCl₂, Triton X-100 were shown to be the optimum components for the biocatalytic reaction. Phosphate ion and C. ammoniagenes cell were furthermore demonstrated as the most important main effects on UMP production by Plackett–Burman design, indicating that 5-phosphoribosyl-1-pyrophosphate (PRPP) synthesis was the rate-limiting step for pyrimidine nucleotides production. Optimization by a central composition design (CCD) was then performed, and up to 32 mM (10.4 g l⁻¹) UMP was accumulated in 24 h from 38.5 mM (6 g l⁻¹) orotic acid. The yield was threefold higher than the original UMP yield before optimization.     

Characterization of a thermostable and alkaline xylanase from Bacillus sp. and its bleaching impact on wheat straw pulp

Delignification efficacy of xylanases to facilitate the consequent chemical bleaching of Kraft pulps has been studied widely. In this work, an alkaline and thermally stable cellulase-less xylanase, derived from a xylanolytic Bacillus subtilis, has been purified by a combination of gel filtration and Q-Sepharose chromatography to its homogeneity. Molecular weight of the purified xylanase was 61 kDa by SDS–PAGE. The purified enzyme revealed an optimum assay temperature and pH of 60°C and 8.0, respectively. Xylanase was active in the pH range of 6.0–9.0 and stable up to 70°C. Divalent ions like Ca²⁺, Mg²⁺ and Zn²⁺ enhanced xylanase activity, whereas Hg²⁺, Fe²⁺, and Cu²⁺ were inhibitory to xylanase at 2 mM concentration. It showed K _m and V _max values of 9.5 mg/ml and 53.6 μmol/ml/min, respectively, using birchwood xylan as a substrate. Xylanase exhibited higher values of turn over number (K _cat) and catalytic efficiency (K _cat/K _m) with birchwood xylan than oat spelt xylan. Bleach-boosting enzyme activity at 30 U/g dry pulp displayed the optimum bio-delignification of Kraft pulp resulting in 26.5% reduction in kappa number and 18.5% ISO induction in brightness at 55°C after 3 h treatment. The same treatment improved the pulp properties including tensile strength and burst index, demonstrating its potential application in pre-bleaching of Kraft pulp.     

Purification and characterization of maltooligosaccharide-forming amylase from Bacillus circulans GRS 313

A maltooligosaccharide-forming amylase that hydrolyzes starch into maltotriose and maltopentaose was found in the culture filtrate of a strain of Bacillus circulans GRS 313 isolated from local soil. The enzyme was purified by organic solvent fractionation, Sephadex G-100 gel filtration and CM-Sephadex column chromatography. Optimum pH and temperature of amylase were evaluated using response surface methodology (RSM) and were found to be 48°C and 4.9, respectively. The enzyme was stable up to 60°C and its pH stability was in the range of 5.0–8.0. The K _m and V _max of the amylase with starch were 11.66 mg/ml and 68.97 U, respectively, and the energy of activation, E _a, was 7.52 kcal/mol. Dextrin inhibited the enzyme competitively, with a K _i of 6.1 mg/ml, and glucose caused noncompetitive inhibition with a K _i of 9.5 mg/ml. The enzyme was inhibited by Hg²⁺, Mn²⁺, Fe³⁺ and Cu²⁺ and enhanced by Co²⁺ and Mg²⁺. EDTA reversed the inhibitory effect of the metals. Paper chromatographic and high-performance liquid chromatography analysis of the products of the amylolytic reaction showed the presence of maltotriose, maltotetraose, maltopentaose, maltose and glucose in the starch hydrolysate. Journal of Industrial Microbiology & Biotechnology (2002) 28, 193–200 DOI: 10.1038/sj/jim/7000220 Received 11 December 2000/ Accepted in revised form 22 October 2001     

Interactions between substrate,fermentation end-products,buffering systems and gas production upon fermentation of different carbohydrates by mixed rumen microorganisms in vitro

Summary In animal nutrition, incubation of feed samples with CO₂/HCO₃-buffered rumen fluid is used to predict the nutritional values of the feed. During fermentation, volatile fatty acids (VFAs) are produced, which release CO₂ from the buffer through their H⁺ ions. This indirect gas production amounted to 20.8 ml gas per mmol VFA. By incubating glucose, rice starch and cellulose, the relationship between direct and indirect gas production in relation to fermentation kinetics was studied. The total amount of gas formed was found to be dependent on the composition of the fermentation end-products formed. This could be described by: ml gas = M_v·mmol HAc + 2M_v·mmol HB + 0.87M_v·mmol Tot. VFA where HAc = acetic acid; HB = butyric acid; and M_v = molar gas volume. No clear relationship was found between the rate of fermentation and total gas production. From rice starch more total gas was produced than from glucose and cellulose, which were fermented faster and slower, respectively. Correspondence to: S. F. Spoelstra