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We investigated an effective method for the pretreatment of Japanese cedar for efficient enzymatic saccharification and ethanol production. A 45-atm steam explosion provided a comparatively large amount of glucose and reducing sugars. Addition of polyethylene glycol (PEG) influenced the digestibility of holocellulose in a 35-atm steam-exploded sample. However, we observed a negative effect on enzymatic saccharification when sodium hydroxide was used in the pretreatment. The maximum values of glucose and reducing sugars produced using consecutive pretreatments with a 25-atm steam explosion and an ionic liquid were 408 and 462 mg/(g initial dry sample), respectively. The most positive effects on the enzymatic saccharification kinetics were observed when the above consecutive pretreatment methods were used. However, using the organosolv treatment of wood chips without the steam explosion is a more cost-effective pretreatment method for the enzymatic saccharification of Japanese cedar, and this results in 386 and 426 mg/(g initial dry sample) of glucose and reducing sugars, respectively.

Bamboo is an alternative feedstock for the production of fine chemicals, such as fuel ethanol and lactic acid, because bamboo has a large amount of sugars. An effective pretreatment method for enzyme saccharification is required for the efficient production of these materials. Enzyme saccharification (48 h) using a 35 atm and 5 min steam exploded bamboo produced 426 and 488 mg/(g initial dry sample) of glucose and reducing sugar, respectively. In addition, pretreatments using 20 atm and 5 min steam explosion or mechanical milling for 5 min followed by 10 wt.% sodium hydroxide treatment at 121 °C for 60 min were attempted in order to enhance the digestibility of the holocellulose component. Both of these pretreatment methods had a large positive effect on the production of sugars by subsequent enzymatic hydrolysis. In particular, the maximum value of glucose production was obtained by the 20 atm steam explosion and 10 wt.% sodium hydroxide treatment. This produced 456 mg/(g initial dry sample) of glucose and 460 mg/(g initial dry sample) of reducing sugar. In comparison, the mechanical milling and 10 wt.% sodium hydroxide treatment produced 383 and 485 mg/(g initial dry sample) of glucose and reducing sugar, respectively. From these results, it was concluded that the pretreatment with 20 atm steam explosion and 10 wt.% sodium hydroxide treatment was the most effective pretreatment method for the production of glucose from bamboo by enzyme saccharification. However, since this pretreatment method requires the severe conditions of both high pressure and temperature steam explosion and high concentration sodium hydroxide, an alkaline peroxide pretreatment without a steam explosion and high concentration sodium hydroxide was also attempted. A comparatively large amounts of glucose and reducing sugar production, i.e. 399 and 568 mg/(g initial dry sample), respectively, were obtained in 1%(v/v) hydrogen peroxide and 1 wt.% sodium hydroxide treatment at 90 °C for 60 min. Therefore, it was concluded that alkaline peroxide pretreatment is an effective and environmentally friendly method for the enzyme saccharification of bamboo.

High-solid paper sludge was investigated for its potential to produce renewable biofuel. As a pretreatment method without consuming a large amount of energy, mechanical grinding for a comparatively short time followed by chemical swelling was used for optimal enzyme saccharification and ethanol production from paper sludge. Chemical swelling by phosphoric acid was more favorable than ball mill grinding. However, sequential pretreatment system using ball milling for 2 min and then phosphoric acid swelling for 1 h enhanced the enzyme saccharification rate and the reducing sugar productivity, 84.1% and 28.1 mg/g/h, respectively. The simultaneous saccharification and fermentation (SSF) from the sequential pretreatment system resulted to 81.5% ethanol conversion rate, the productivity being 1.27 g/L/h compared to untreated raw paper sludge which gave 54.3% ethanol conversion rate and 0.424 g/L/h productivity. Our work shows that consecutive mechanical grinding and chemical swelling are effective pretreatment methods for the enhancement of enzyme saccharification and efficient ethanol production from paper sludge.

During past decades, the considerable efforts have been made to utilize lignocellulose as biomass feedstock for the optimal production of bio-ethanol as an alternative source of fuel. In this work, the effective bioconversion of paper sludge to ethanol was investigated by using Zymomonas mobilis NBRC 13756. Major components of the raw paper sludge were carbohydrates and ash. Fermentation utilizing strains of Z. mobilis instead of traditional yeasts and the use of simultaneous saccharification and fermentation (SSF) process have been proposed due to their high ethanol yields with cost-effectiveness. Free cells of Z. mobilis resulted in no ethanol production even after 24 h of incubation because cells’ growth was inhibited by metal ions contained in the paper sludge. The application of SSF with Ca-alginate-immobilized cells of Z. mobilis was performed and 18 g/L of ethanol was obtained after 48 h of incubation at an initial paper sludge concentration of 200 g/L. In addition, the repeated batch fermentation of Ca-alginate-immobilized Z. mobilis cells was attempted for producing ethanol up to run 4. Our work suggests that Ca-alginate-immobilized cells of Z. mobilis could effectively produce ethanol from paper sludge not only under the batch fermentation but also under the repeated batch fermentation.

Antioxidant compounds were extracted from various parts of Sasa palmata (Bean) Nakai, a bamboo plant whose leaves are commonly used to wrap foodstuffs such as Sushi in Japan. Highest concentrations of antioxidant compounds existed in the leaf part of S. palmata. Steam explosion treatment followed by hot water and methanol extractions was used for separating the antioxidant compounds from S. palmata leaf. The steam explosion treatment is the physical–chemical treatment which crushes a sample by sudden reduction of the pressure in reactor to atmospheric pressure after steaming the sample at high temperature and pressures. Sasa palmata leaf was hydrolyzed by steaming and crushed by the rapid decompression. The optimal condition of steam explosion for the effective extraction of antioxidant compounds from S. palmata was determined as a steam of temperature of 250 °C and a steaming time of 1 min. In these conditions 217.41 mg/(g-Sasa leaf) of phenolic compounds and 142.81 mg/(g-Sasa leaf) of radical scavenging activity, that was expressed as butylated hydroxyanisole (BHA), were obtained.

The present study focused on the continuous prefermentation process affecting curd formation, and the subsequent pH changes in the first stage of cheese manufacture. Two typical continuous prefermentation systems in cheese manufacture were analyzed based on a kinetic model and the process control theory. A previously proposed mathematical model, which considers the effect of pH, adequately simulated the dynamic behavior of pH in the two continuous prefermentation processes. In the stability analysis of steady state using the Routh-Hurwitz criterion, we found that the continuous feed of a cheese starter culture prevented wash-out and stabilized the system independent of the fraction of seed feed rate. When no cheese starter culture was fed, the stabilities of steady states were dependent on the partial differentials of the maximum specific growth rate to lactic acid concentration. The feeding of cheese starter culture was effective to improve prefermentation process performance.

It is known empirically that the lightning stimulates plant growth in realm of agriculture. Furthermore, growth stimulations of pulsed power for mushrooms have been reported as well. Therefore, the application of pulsed power technology could enhance the growth of agricultural crops easily. This study examined the effects of electrical stimulation on mycelium of a fungus. Sparassis crispa. S. crispa contains a large amount of β-1, 3-glucan which has anticancer action. While there have been several experimental studies producing fruit body of fungi, we selected an experiment for mycelium of S. crispa. In case of mycelium cultivation, the culture period is shorter than fruit body and, the culture operation is relatively facile. A Blumlein-type pulse forming network (B-PFN) was used to generate electrical pulsed discharges of approximately 20 kV and 450 ns on the surface of the culture solution containing the mycelium of S. crispa. The length of discharges was approximately 14 mm. During the 10 day culture period, electrical pulse discharge treatment was applied on the second, fourth, and sixth day. The mycelium pellets were observed and metabolites were evaluated with a high-performance liquid chromatography (HPLC). The observed amount of β-1, 3-glucan in pulse-treated samples was significantly 1.22 times higher than in control samples.

The optimal culture condition of basidiomycete Sparassis crispa mycelia was determined for producing a large amount of antineoplastic constituent, i.e. β-1,3-glucan. Furthermore, a new extraction method with steam explosion as a pretreatment was attempted to increase the amount of antineoplastic constituent extracted from S. crispa mycelia. The shake-flask liquid culture of fungal mycelia could shorten a culture time up to about 1/3 compared with the conventional stationary liquid culture. The productivity of S. crispa mycelia reached its maximum at an initial glucose concentration of 30 g/L, a culture temperature of 25–30 °C, and pH 5. Steam explosion at a steam temperature of 225 °C for 5 min not only enhanced the amount of antineoplastic constituent extracted from the mycelia but also shortened the extraction time significantly.

A novel treatment system of wastewater contaminated with copper was developed by using some mosses that are demonstrably metal tolerant and accumulate heavy metals into the cells. Scopelophila cataractae could remove copper more efficiently than other mosses, i.e. Physcomitrella patens and Polytrichum formosum . One hundred milligram per liter of copper ion was removed completely for 9 d using the suspended cultivation system flowing air coupled with intermittent mechanical disruption of the protonema filaments of S. cataractae by a homogenizer.

Methane fermentation of woody waste was carried out using various pretreatment methods for delignification in order to evaluate the efficient pretreatment method for producing methane gas from woody waste. Japanese cedar (Cryptomeria japonica) chips were used as sample of woody waste. As pretreatment methods, refiner treatment, steaming treatment, steaming treatment followed by refiner treatment, basidiomycete fungi treatment, and steam explosion treatment were attempted and the methane gas evolved from treated wood chips were compared. The steam explosion treatment resulted in the most effective pretreatment method for methane fermentation from woody waste among the pretreatment methods. The steam explosion at a steam pressure of 4.51 MPa and a steaming time of 5 min was the optimal pretreatment method for obtaining a large amount of methane gas in energy saving manner. The strong correlation between the amount of methane gas evolved and the ratio of amount of Klason lignin was observed. The study confirmed the necessity of pretreatment for delignification of cedar wood waste.

The chemical characteristics, enzymatic saccharification, and ethanol fermentation of autohydrolyzed lignocellulosic material that was exposed to steam explosion were investigated using bagasse as the sample. The effects of the steam explosion on the change in pH, organic acids production, degrees of polymerization and crystallinity of the cellulose component, and the amount of extractive components in the autohydrolyzated bagasse were examined. The steam explosion decreased the degree of polymerzation up to about 700 but increased the degree of crystallinity and the micelle width of the cellulose component in the bagasse. The steam explosion, at a pressure of 2.55 MPa for 3 mins, was the most effective for the delignification of bagasse. 40 g/L of glucose and 20 g/L of xylose were produced from 100 g/L of the autohydrolyzed bagasse by the enzymatic saccharification using mixed cellulases, acucelase and meicelase. The maximum ethanol concentration, 20 g/L, was obtained from the enzymatic hydrolyzate of 100 g/L of the autohydrolyzed bagasse by the ethanol fermentation usingPichia stipitis CBS 5773; the ethanol yield from sugars was 0.33 g/g sugars.

A waste reduction system for the production of useful materials from un-utilized plant biomass was developed for zero emission using a steam explosion, extraction and separation, and various conversion methods. This system utilizes a significant conversion approach without generating pollutants, i.e., waste gas, wastewater, and solid waste materials. The conversion of un-utilized bamboo (Phyllostachys pubescens) into useful materials was studied for the effective utilization of its components such as holocellulose, water-soluble material, methanol-soluble lignin, and Klason lignin. These components were converted into useful materials, i.e., lactic acid, antibacterial violet pigment, methane gas, monosaccharides and oligosaccharides, epoxy resin without estrogenic activity, or activated carbon. The amount of useful materials and products produced from each component was estimated, and the reduction in the amount of pollutants generated in their production process and waste treatment and the detoxification of the lignin resin were confirmed.

金沢大学自然計測応用研究センターエコテクノロジー研究部門, 金沢大学工学部, Phytoremediation is an innovative technology that utilizes the natural properties of plants to remediate hazardous waste sites. For more cost-effective phytoremediation, it is important to utilize a hyperaccumulating plant after phytoremediation, i.e. the recovery of valuable metals and the production of useful materials. In this work, the determination of metals in plant component polymers in a fern, Athyrium yokoscense, as a hyper-accumulating plant was established using steam explosion, Wayman's extraction method, and ICP emission spectrometry. After A. yokoscense plants were treated by steam explosion, the steam-exploded A. yokoscense were separated into four plant component polymers, i.e. water-soluble material fraction, holocellulose fraction, methanol-soluble lignin fraction, and residual lignin fraction. The concentrations of Cu, Pb, Fe, and Zn in these plant component polymers and the dry weights of plant component polymers were measured. These analytical process determining metals in the plants will contribute to not only the evaluation and the efforts of phytoremediation using a hyperaccumulating plant, but also to the development of more effective phytoremediation. 2005 © The Japan Society for Analytical Chemistry.

A novel pulping method without generating pollutants consists of a sodium hydroxide (NaOH) treatment followed by steam explosion was proposed and evaluated in this investigation. The cellulose component (degree of glucose polymerization: about 500) from Eucalyptus globlus chips treated with 10 g·dm–3 NaOH and then exploded with steam at 2.5 MPa for a steaming time of 5 min, was converted into pulp with physical properties almost the same as those of conventional hardwood pulp.

A mathematical model of direct ethanol production from starch in immobilized recombinant amylase-producing yeast culture was proposed for estimating the dynamic behavior of cell growth, starch degradation, glucose accumulation, ethanol production, and glucoamylase synthesis by immobilized yeast. The application of the model was compared to the experiments of ethanol production from starch in batch and continuous cultures. The calculated values agreed well with experimental data, regarding concentration of cells, starch, ethanol, and glucoamylase. The profiles of concentration of starch, glucoamylase, cells, and ethanol in the gel bead for estimating the efficient ethanol production were calculated by the model. By comparing the ethanol productivity and the washout dilution rate in an immobilized cell culture with those in a free cell culture, it was confirmed that the immobilized cell culture had a higher ethanol productivity than the free cell culture and produced ethanol continuously with a dilution rate of 0.5 h −1 , which is about 10 times larger than the washout rate in a free cell culture.

The bioremediation of water system contaminated with phenolic compounds having endocrine-disrupting activity,i.e. 2,4-dichlorophenol, 2,4-dichlorophenoxy acetic acid (2,4-D), and 2,4,5-trichlorophenoxy acetic acid (2,4,5-T), was investigated by using ozone oxidation and activated sludge treatment. Ozone oxidation (ozonation time: 30 min) followed by activated sludge treatment (incubation time: 5 days) was an efficient treatment method for the conversion of phenolic compounds in water into carbon dioxide and decreased the value of total organic carbon (TOC) up to about 10% of initial value. Furthermore, 2,4-D was dissolved in water containing salt,i.e. artificial seawater (ASW), and this water was used as model coastal water contaminated with phenolic compounds. The activated sludge treatment (incubation time: 5 days) could consume significantly organic acids produced from 2,4-D in the model costal water by the ozone oxidation (ozonation time: 30 min) and decrease the value of TOC up to about 35% of initial value.

In order to clarify the effect of repressor gene on direct conversion process of starch into ethanol using a recombinant yeast, the stability of steady states and dynamic behaviors of cell growth, substrate consumption, and ethanol production were studied in a continuous culture using two recombinant yeasts, Saccharomyces cerevisiae SR93 and SR96. S. cerevisiae SR93 was constructed by integrating a glucoamylase gene (STA1) into chromosome of efficient ethanol-fermenting yeast, S. cerevisiae SH1089. S. cerevisiae SR96 could produce glucoamylase more than S. cerevisiae SR93 by the disruption of the MAT locus repressing the expression of glucoamylase gene. The validity of the authors previously proposed model was verified in a continuous culture as a result of close agreement between calculated values and experimental data using both recombinant yeasts. The stability of steady states was analytically discussed based on the eigenvalues using this model. The stabilities of steady states were classified into three categories: (i) a stable steady state where ethanol was produced, (ii) an unstable steady state where ethanol was produced, and (iii) a washout state. The number and stability of steady states changed remarkably with operational conditions such as dilution rate and feed starch concentration. The inactivation of repressor gene could increase the regions that stable steady state where ethanol was produced exists, and decrease the region that only washout exists.

This research involved field surveys in different geographical locations in Tanzania for collection of fungi species, identification of the isolates, and screening for lignin-degrading enzyme production. Out of 18 saprophytic fungi (both wood and non-wood inhabiting) isolates, 17 were positive in the rhemazol brilliant blue-R (RBBR) decolorization test; 15 of the isolates oxidized guaiacol while 7 and 12 oxidized α-naphthol and pyrogallol, respectively. The cultures with 1% glucose concentration showed the fastest growth of mycelia and highest MnP, LiP, and Lac activities. MnP was found to be dominant among the tested isolates and Laetiporus sulphurae had the highest LiP activity of 65.2 U/ml. Most of the fungi isolates produced more than two extracellular enzymes, suggesting that these isolates possess the ability to degrade lignin or related polyphenolic compounds, and thus they can be used in upgrading low quality animal feed. The selected fungi isolates reduced cellulose and hemicellulose contents in rice straw by an average of 10% and 35%, respectively, delignification ratio of 23% and increased its relative crude protein content by more than 50%.

Endocrine-disrupting phenolic compounds in the water were degraded by laccase fromTrametes sp. followed by activated sludge treatment. The effect of temperature on the degradation of phenolic compounds and the production of organic compounds were investigated using endocrine-disrupting chemicals such as bisphenol A, 2,4-dichlorophenol, and diethyl phthalate. Bisphenol A and 2,4-dichlorophenol disappeared completely after the laccase treatment, but no disappearance of diethyl phthalate was observed. The Michaelis-Menten type equation was proposed to represent the degradation rate of bisphenol A by the lacasse under various temperatures. After the laccase treatment of endocrine-disrupting chemicals, the activated sludge treatment was attempted and it could convert about 85 and 75% of organic compounds produced from bisphenol A and 2,4-dichlorophenol into H2O and CO2, respectively.

Anaerobic fermentation was attempted to produce methane from the wood chip (Eucalyptus globulus). By the pretreatment of the wood chip using hot water with high temperature, NaOH, and steam explosion, the production of methane gas was enhanced. The pretreatment using steam explosion resulted in more amount of methane gas produced than the treatment using either hot water or 1% (w/w) NaOH with high temperature, and the steam explosion at a steam pressure of 25 atm and a steaming time of 3 min was the most effective for the methane production. The amount of methane gas produced depended on the ratio of weight of Klason lignin, a high molecular weight lignin, in the treated wood chip.

Solubilization of domestic household waste through steam explosion with subsequent ethanol production by the microbial saccharification and fermentation of the exploded product was studied. The effects of steam explosion on the changes of the density, viscosity, pH, and amounts of extractive components in artificial household waste were determined. The composition of artificial waste used was similar to leftover waste discharged from a typical home in Japan. Consecutive microbial saccharification and fermentation, and simultaneous microbial saccharification and fermentation of the steam-exploded product were attempted usingAspergillus awamori, Trichoderma viride, andSaccharomyces cerevisiae, the ethanol yields of each process were compared. The highest ethanol yield was obtained with simultaneous microbial saccharification and fermentation of exploded product at a steam pressure of 2 MPa and a steaming time of 3 min.

The antibacterial action of violet pigment, a mixture of violacein and deoxyviolacein, isolated from phychrotrophic bacterium RT102 strain was examined, and the operational conditions for the effective production of violet pigment were studied. The antibacterial activity of the violet pigment was confirmed for several bacteria such asBacillus licheniformis, Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, andPseudomonas aeruginosa, and the high concentration of violet pigment, above about 15 mg/L, caused not only growth inhibition but also death of cells. The growth properties of RT102 strain were clarified under various incubation conditions such as pH, temperature, and dissolved oxygen concentration. The maximum violet pigment concentration,i.e. 3.7 g/L, and the maximum productivity of violet pigment,i.e. 0.12 g L−1h−1, were obtained in a batch culture of pH 6, 20°C, and 1 mg/L of dissolved oxygen concentration.

A mathematical model for direct alcohol fermentation from starch was proposed using an amylase-producing recombinant yeast, Saccharomyces cerevisiae SR93. This model consisted of the reaction rate equations for glucoamylase synthesis in the recombinant yeast, starch degradation by a glucoamylase, cell growth, production of glucose, and production of ethanol. The rate of glucoamylase synthesis was expressed on the basis of the diauxic growth model that represents catabolite repression and enzyme induction. The rate of starch degradation was expressed on the basis of the enzymatic hydrolysis model representing the change of structure resulting from starch degradation. The calculated values were in satisfactory agreement with the experimental data in a batch culture of direct alcohol fermentation from starch using S cerevisiae SR93. Furthermore, the calculated values obtained by changing only one parameter concerning the synthesis rate of glucoamylase were in satisfactory agreement with the experimental data using another recombinant yeast, S cerevisiae SR96. Copyright © 2003 Society of Chemical Industry

To produce glucoamylase efficiently as a recombinant protein, E. coli was grown with 20 g (NH4)2SO4 l−1 which removed proteolytic activity but did not effect cell growth. Growth in M9 medium with 20 g (NH4)2SO4 l−1 produced 11 U glucoamylase ml−1 compared to 7 U ml−1 without addition. Furthermore, the glucoamylase activity was maintained at about 9 U ml−1.

The identification of a psychrotrophic bacterium, which produced a violet pigment, isolated from the organic residue of a water tank keeping rainbow trout and the antibacterial effect of the violet pigment were investigated experimentally. The psychrotrophic bacterium was found to be a new species very closely related to Janthinobacterium lividum. 1 H NMR, 13 C NMR, and FT-MS spectra analyses results showed that the chemical structure of the violet pigment was a mixture of violacein and deoxyviolacein. The antibacterial effect of the violet pigment was confirmed for putrefactive bacteria such as Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, and Pseudomonas aeruginosa. The growth-inhibitory and lethal effects of the violet pigment on the putrefactive bacteria were evaluated by increasing the concentration of the violet pigment, ranging from 5 to 20 mg dm−3. It was found that higher concentrations of the violet pigment caused not only growth inhibition but also the death of the putrefactive bacteria.

Continuous production of lignin-degrading enzymes by Bjerkandera adusta immobilized on polyurethane foam gave maximum activities of 220 U lignin peroxidase ml−1, 150 U manganese peroxidase ml−1, 50 U laccase ml−1 and 6.2 U protease ml−1 at the retention time of 24 h for 60 days. Protease secretion destabilized the produced lignin peroxidase, manganese peroxidase and laccase.

固定化菌による銅イオン含有フェノールの処理の効果をゲルビーズ内へのフェノールおよび銅イオンの浸透の動的挙動などから検討した。ゲルビーズ内のフェノールと銅イオンの濃度分布を測定した結果, ゲルビーズ中心部では銅イオン濃度が低いためにフェノール菌の増殖阻害のない領域が存在し, この領域ではフェノールが比較的容易に処理できることがわかった。固定化菌は1～10mg/lの比較的高濃度の銅イオン含有フェノールをほぼ完全に分解した。固定化ゲルビーズ内の菌の増殖やフェノールの処理を表す数式モデルが提出され, 回分処理における計算値が実験的に検証された。固定化菌は回分処理だけでなく連続処理でも銅イオン含有フェノールを効果的に処理できることが示唆された。

In order to develop a method for converting raw starch into ethanol efficiently, direct fermentation of ozonized raw starch using a recombinant yeast was investigated. Ozonolysis was carried out as a pretreatment to convert raw starch into ethanol rapidly and efficiently, and then the effect of the ozone degradation conditions on the degree of polymerization and the amount of amylose in a raw starch was determined. Since the degree of polymerization was low and the amount of amylose was high, raw starch treated with an ozone concentration of 40 gm−3 and an ozonation time of 30 min was the material chosen for alcohol fermentation. Though the recombinant yeast could not convert the untreated raw starch, it converted the soluble starch and the ozonized raw starch at a comparatively high yield into ethanol. About 56% of the ozonized raw starch decomposed, and the ethanol concentration obtained from the ozonized raw starch was markedly greater than that obtained from untreated raw starch. The dynamic behavior of cell growth, substrate degradation, and ethanol production was examined in a continuous culture under various dilution rates, and the optimal dilution rate, ie 0.15 h−1, was determined for maximizing the ethanol productivity (amount of ethanol produced per unit time). © 2002 Society of Chemical Industry

This study investigated the effect of high-temperature saturated steam treatments on the chemical characteristics and enzymatic saccharification of softwood and hardwood. The weight loss and chemical modification of cedar and beech wood pieces treated at 25, 35, and 45 atm for 5 min were determined. Fourier transform infrared and X-ray diffraction analyses indicated that solubilization and removal of hemicellulose and lignin occurred by the steam treatment. The milling treatment of steam-treated wood enhanced its enzymatic saccharification. Maximum enzymatic saccharification (i.e., 94% saccharification rate of cellulose) was obtained using steam-treated beech at 35 atm for 5 min followed by milling treatment for 1 min. However, the necessity of the milling treatment for efficient enzymatic saccharification is dependent on the wood species.

Arbutin is a tyrosinase inhibitor and is extensively used as a human skin-whitening agent. This study investigated the optimum conditions for extracting arbutin by ultrasonic homogenization from discarded branches pruned from Japanese pear (Pyrus pyrifolia cv. Kousui) trees. The arbutin content was measured in the branches and also in the leaves, stems, fruit peel, and fruit flesh.

In order to use effectively the core after utilizing the bark in a kenaf plant, the degradation characteristics and enzymatic saccharification of steam exploded core were studied experimentally. The effects of the steam explosion on the modification of kenaf core, i.e. shape and size, pH, amounts of organic acids produced, and amounts of extracted components, were examined under various conditions. The saccharification of exploded kenaf core at a steam pressure of 2.5 MPa and a steaming time of 3 min was about 0.6; this suggests that about 85% of the holocellulose (hemicellulose and cellulose) contained in the kenaf core was converted into reducing sugars.

The rational operational condition for maximizing the pretreatment effect on plant biomass while minimizing heat required was investigated. Eucalyptus globulus chips were used to evaluate the operational method for the most efficient conversion of plant biomass into useful materials by steam explosion. The energy consumption required to carry out the steam explosion was calculated by considering the mass balances of the water, the wood component, and the heat balance in the steam explosion apparatus. The energy consumption increased significantly with the increase of steam pressure and steaming time, and decreased rapidly with increase of the thickness of the heat-insulating material in the steam explosion apparatus. The amount of methanol-soluble lignin, a low molecular weight lignin, was measured experimentally under various operational conditions such as steam pressure and steaming time. The steam explosion at the steam pressure of 3.9 MPa and steaming time of 1.1 min was the most effective method for maximizing the delignification with low energy consumption. © 2001 Society of Chemical Industry

クラフトパルプ排水中に含まれるリグニンの効率的分解のためにオゾン分解後に微生物分解する処理方法を提案した。リグニン芳香核の分解に及ぼすpH, 温度などのオゾン分解条件の影響を実験的に明らかにした。オゾン分解によってリグニン芳香核はムコン酸, マレイン酸, シュウ酸などの種々の低分子有機酸に分解された。シュウ酸分解菌A28菌を用いたオゾン分解液の微生物分解では, 菌の増殖は酢酸を消費した後にシュウ酸を分解する典型的なジオキシ増殖を示した。結果として, オゾン分解と微生物分解から成る処理方法はクラフトパルプ排水中のリグニンの完全分解のための効果的方法であることがわかった。 The efficient degradation of lignin in kraft pulp wastewater by ozonolysis and microbial degradation was presented. The effects of operational conditions such as pH and temperature on the degradation of lignin aromatic ring by ozone were experimentally clarified. The lignin aromatic ring was degraded by the ozonolysis into various low-molecular weight organic acids such as muconic acid, malefic acid, and oxalic acid. In the microbial degradation of ozonolyzed wastewater using an oxalic acid degrading microorganism, strain A28, cells grew in a typical diauxic growth, i.e. the oxalic acid was degraded only after the exhaustion of acetic acid. As a result, it was found that after ozonolysis of kraft pulp wastewater microbial degradation by strain A28 was effective method for the complete degradation of lignin in it.

A mathematical model for estimating the dynamic behavior of ethanol production from mixed sugars such as glucose and xylose is presented. This model was constructed by introducing the term for ethanol production into the diauxic growth model proposed previously by the authors. It was assumed that the first substrate, glucose, is metabolized by a constitutive enzyme and the second substrate, xylose, is utilized by an inducible enzyme. The synthesis of the inducible enzyme is controlled by the catabolite repression caused by glucose and with the induction caused by xylose as an inducer, and the xylose is then converted into cell mass and ethanol by the inducible enzyme. The parameters of the model were estimated from the experimental data in the medium containing glucose and xylose, singly or in combination in a batch culture. The application of the model was examined for experiments in both batch and continuous culture, with glucose and xylose as carbon sources. The calculated values, according to the model, corresponded satisfactorily with experimental data, such as cell growth, substrate consumption, and ethanol production, especially in the estimation of the lag times between the first log phase and the second log phase in cell growth and ethanol production. © 2001 Society of Chemical Industry

Alcohol fermentation of an enzymatic hydrolyzate of exploded rice straw was studied experimentally. Rice straw was treated under variable conditions, such as steam pressure and steaming time. The exploded rice straw was separated into water-soluble material, methanol-soluble lignin, Klason lignin, and a mixture of cellulose and a low molecular weight substance. The effects of steam explosion on the characteristics of the exploded rice straw were clarified from the point of view of the amounts of extractive components. Steam explosion was found to be effective for the delignification of rice straw and for increasing its susceptibility to enzyme hydrolysis and alcohol fermentation. The polysaccharides (cellulose and hemicellulose) in the rice straw treated at a steam pressure of 3.5 MPa with a steaming time of 2 min were hydrolyzed almost completely into monosaccharides, (ie glucose and xylose) by a mixture of Trichoderma viride cellulase (Meicelase) and Aspergillus aculeatus cellulase (Acucelase). The enzymatic hydrolyzate of exploded rice straw was converted into ethanol efficiently by Pichia stipitis and the ethanol yield from sugar was about 86%(w/w) of the theoretical value. The ethanol concentration in a membrane bioreactor coupled with a pervaporation system reached 50 gdm−3 and was about five times higher than that in the culture broth. The energy efficiency (ratio of combustion energy of ethanol produced to energy for steam explosion) reached a maximum value at a pressure of 3.5 MPa for 2 min. © 2001 Society of Chemical Industry

The chemical characteristics of methanol soluble lignin separated from Eucalyptus globulus by steam explosion were clarified by measurement of number-averaged molecular weight, weight-averaged molecular weight, and phenolic hydroxyl group. It was found that the methanol soluble lignin separated from the exploded wood at a steam pressure of 3.5 MPa and a steaming time of 5 min was the most adequate raw material for the synthesis of epoxidized lignin, i.e. lignin epoxy resin. The thermosetting behavior of epoxidized lignin was almost the same as that of commercial bisphenol A resin. According to E-Screen assay, the methanol soluble lignin, a raw material of epoxidized lignin, did not show estrogenic activity. Since the methanol soluble lignin was easily degraded by the lignin-degrading enzymes, the epoxidized lignin seems to be a biodegradable resin in waste treatment after its use.

A microbial growth model was presented for estimating the dynamic behavior of cell growth and substrate consumption in the biodegradation of phenol containing a heavy metal, such as zinc or copper ions. The application of the model for experiments in a batch culture and a continuous culture was examined. The values calculated according to the model corresponded satisfactorily with experimental data, such as the optical density of cells and the concentration of phenol. Using the model, the stability of steady states in a continuous culture was analytically evaluated based on the eigenvalues. The steady states were separated into three categories: (i) a stable steady state where phenol was consumed, (ii) an unstable steady state where phenol was consumed, and (iii) a washout state where phenol was not consumed. The number, kind, and stability of steady states varied significantly with operational conditions such as dilution rate, feed concentration of phenol, and feed concentration of heavy metal ions. The operational conditions required to obtain the stable steady state where phenol was consumed are shown graphically. © 2000 Society of Chemical Industry

The effective culture method to produce bacterial cellulose from fruit juices by Acetobacter xylinum NBRC 13693 was examined. Bacterial cellulose production from various fruit juices including orange, pineapple, apple, Japanese pear and grape were investigated, and the possibility of producing bacterial cellulose from those juices was suggested. The yields of the bacterial cellulose were increased by addition of the nitrogen source to the fruit juices. In addition, it was confirmed that the orange and Japanese pear juices were suitable medium for a bacterial cellulose production. The bacterial cellulose was produced from the various component of orange such as a peel and squeeze residue, and the bacterial cellulose of 0.65 g (dry weight) was produced from the orange of 100 g, and the solid residue from the orange was about 17.2 g.

The breeding of a recombinant yeast having glucoamylase activity, Saccharomyces cerevisiae SR93, has already been reported by the authors, and further improvement of this yeast is attempted to significantly increase the conversion rate of starch into ethanol. It is generally known that the disruption of the MAT locus, which produces a repressor protein, enhances the expression of glucoamylase gene. A glucoamylase-producing yeast with the disrupted MAT locus, Saccharomyces cerevisiae SR96, is bred to convert starch into ethanol rapidly. Disruption of the MAT locus is performed by inserting the LEU2 gene into the MAT locus of S. cerevisiae SR93 and was confirmed by Southern blot analysis. The induction effect of starch and the repression effect of glucose on the glucoamylase synthesis are examined experimentally. The glucoamylase activity per unit cell concentration increases about 1.6-fold due to the disruption of the MAT locus. The specific growth rate, the glucoamylase synthesis rate, and the ethanol production rate of S. cerevisiae SR96 are much higher than S. cerevisiae SR93, and the direct alcohol fermentation of starch using S. cerevisiae SR96 gave the highest ethanol production rate in the various incubation systems.

フェノール分解微生物, Acinetobacter calcoaceticus AH株に及ぼす亜鉛イオンと銅イオンの増殖阻害と死滅の影響が回分培養において菌体光学密度, 総菌数, 生菌数と細胞1個当たりの高分子含有量を測定することによって検討された。比増殖速度や生菌数変化速度に及ぼす重金属イオン濃度の影響が明らかにされ, 銅イオンの阻害効果は亜鉛イオンよりも非常に強いことと銅イオンは菌の増殖阻害だけでなく菌の死滅も引き起こすことがわかった。菌の担体としてアルギン酸カルシウムのゲルビーズを用いた固定化培養は従来までの液体培養よりも高濃度の重金属イオン存在下のフェノールを迅速かつ高効率で分解できた。固定化菌を用いた反復回分培養は10回の回分培養の間30mg/lの亜鉛イオンを含む100mg/lのフェノールを逐次的に分解でき, 重金属イオン存在下のフェノールの効果的処理への適用を可能とした。

Phenol is an organic compound widely used as a solvent. It is discharged by various industries into rivers and then accumulates in lakes and seawater. It is difficult to treat phenol in seawater by biological means because the high concentrations of dissolved mineral salts in seawater inhibit the growth of most microorganisms. We investigated the bioremediation of phenol in seawater using a novel microorganism isolated from the intestine of a marine slug. A novel bacterium was isolated from the intestine of the sea slug (Aplysia kurodai) using enrichment culture with a high concentration of phenol. From experimental research on the bacterium's morphological, and chemotaxonomic characteristics, and using molecular (16S rDNA) techniques, it was found that it belongs to the genus Serratia. Serratia sp. could degrade phenol completely; this is in contrast to activated sludge, which degraded only about 35% of phenol in seawater. This novel microorganism seems to have the potential for the efficient treatment of organic pollutants in seawater.

ナホトカ号重油, その漂着油および油砂について石川県沿岸の現場海水を用いて微生物分解実験やバイオレメデェーションの基礎的実験を行い, また, 同時に沿岸海域における石油分解細菌の分布密度を調査し, 以下の結果を得た。 (1) 石油分解細菌の密度は102～101MPN/100mlであり, (2) 石油の分解率は海水中の窒素やリン源の濃度によって大きく変化し, 窒素やリンの添加によって約2倍まで増加した。 (3) 重油成分ごとの分解性では, ジクロロメタン抽出物はn-ヘキサン抽出物に比べて分解されにくい。個々の成分における分解性は炭素数が24以下のn-パラフィン有機硫黄化合物 (ジベンゾチオフェン) , ベンゾ (a) ピレン, ピレン, フルオランテンが分解されやすく, クリセンやベンゾ (k) フルオランテンは分解されにくく, 全パラフィン, ジベンゾチオフェン, ベンゾ (a) ピレン, ピレン, フルオランテンの培養38日における分解率はそれぞれ62%, 100%, 63%, 68%, 81%であった.

Kraft pulp wastewater was treated with a combination of ozone and activated sludge. The effects of pH on the degradation of lignin and the production of organic acids were examined experimentally in the ozonolysis of wastewater. The strong alkaline condition enhanced not only the degradation of lignin but also the production of organic acids. The dynamic behaviours of microbial growth and substrate consumption were investigated in the biodegradation of organic acids using activated sludge. Maleic acid and oxalic acid in the ozonized wastewater were degraded completely by the activated sludge. The immobilized activated sludge culture using polyurethane foam was effective in degrading organic acids.

Production of lignin peroxidase by a white-rot basidiomycete, Phanerochaete chrysosporium, was investigated experimentally using polyurethane foam as a carrier of immobilized fungal mycelia. The immobilized cell culture using polyurethane foam as a carrier of mycelia yielded significantly greater lignin peroxidase activity than the conventional stationary liquid culture. The effects of operational conditions, such as the kind and number of polyurethane foam cubes, glucose concentration and temperature, on the lignin peroxidase production were examined. Addition of 0.05% Tween 80, 1 mM veratryl alcohol and 1 mM FeSO4-·7H2O greatly improved the production of lignin peroxidase up to 2,700 units/ml culture medium. The lignin peroxidase activity in this culture was about three times larger than that obtained from the culture cultivated in the absence of these additives. Step change incubation lowering the temperature from 37°C to 30°C over an incubation time of three days was carried out for the large scale production of lignin peroxidase, and this incubation gave the highest lignin peroxidase activity 3,800 units/ml culture medium.