Continuous sucrose hydrolysis by an immobilized whole yeast cell biocatalyst

An immobilized biocatalyst with invertase activity prepared by immobilization of whole yeast cells without use of any insoluble carrier was tested in tubular fixed-bed reactors from the point of view of possible application for continuous full-scale sucrose hydrolysis. At inlet sucrose concentration above 60% (w/w) and reaction temperature 60–70°C, total sucrose hydrolysis was achieved at a flow rate of 0.6–1.5 bed volumes per hour. At a flow rate about 10 bed volumes per hour, the conversion was still 0.5. The specific productivity of the biocatalyst was 3–25 h⁻¹; the productivity of the reactor was 1–9 kg l⁻¹ h⁻¹. The half-life of the biocatalyst invertase activity was 815 h at 70°C. The specific pressure drop over the biocatalyst bed was less than 23 kPa m⁻¹. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Immobilized cells of recombinant Escherichia coli strain for continuous production of L-aspartic acid

For L-aspartic acid biosynthesis, high production cells of Escherichia coli mutant B-715 and P1 were immobilized in chitosan gel using a technique developed in our laboratory. The immobilization process reduced initial activity of the intact cells, however, the biocatalyst produced was very stabile for long-term use in multi-repeated batch or continuous processes. Temperature influence on the conversion of ammonium fumarate to L-aspartic acid was investigated. In long-term experiments, over 603 hours, the temperature 40 degrees C was found to be the best for both biocatalyst stability and high conversion rate. The optimum substrate concentration was 1.0 M. Continuous production of L-aspartic acid was investigated in three types of column bioreactors characterized by different volumes as well as different high to biocatalyst bed volume rations (Hz/Vz). The highest conversion rate, 99.8%, and the productivity 6 g/g/h (mass of L-aspartic acid per dry mass of cells in biocatalyst per time unit) was achieved in the bioreactor with the highest value Hz/Vz = 3.1, and liquid hour space velocity value of 5.2, defined as the volume of feeding substrate passed per volume of catalyst in bioreactor per one hour.     

Corn Residue Supply in the Irrigated Corn Belt

The present study quantifies the profit-maximizing supply of corn stover per unit of land under alternative crop rotations in irrigated cropland with and without limits to irrigation. The model is parameterized based on growing conditions in Nebraska, USA. Our analysis quantifies a trilemma between stover supply, groundwater consumption, and food/feed supply per unit of land. In the absence of water conservation policies, higher stover prices are associated with higher supply and increased irrigation application. If an upper limit is imposed on irrigation at the baseline level (for groundwater conservation purposes), an increase in net revenue from stover sale from $15 to $25/metric ton is associated with an increase in stover supply of 5 metric tons/ha and a 2-metric ton reduction in corn yield under continuous corn. Under corn/soybean rotation, such an increase in net revenue is associated with an increase in stover supply of 10 metric tons/ha, and reductions of 0.5 metric tons in corn yield and 0.7 metric tons in soybean yield.     

Improvement of productivity and efficiency in ethanol production with ca-alginate immobilized zymomonas mobilis

Summary A three-stage reactor system was used to obtain high ethanol concentration (74 g/1) with immobilized Zymomonas mobilis DSM 424. The catalyst efficiency was improved by formation of small diameter beads and arrangement of the biocatalyst in a reactor configuration adapted to the kinetics of ethanol production. A higher productivity than reported before was obtained with 56,5 g/lh based on total working volume at a sugar conversion rate of 98%. The bioreactor was run continuously for 65d.     

Optimization of reaction conditions and stabilization of phenylalanine ammonia lyase-containing Rhodotorula glutinis cells during bioconversion of trans-cinnamic acid to L-phenylalanine

Studies were performed to elucidate the optimal reaction conditions (pH, temperature, ammonia concentration and biocatalyst loading) for bioconversion of trans-cinnamic acid (t-CA) to L-phenylalanine (L-Phe) by L-phenylalanine ammonia lyase (PAL) containing Rhodotorula glutinis cells. All treatments with permeabilizing agents stimulated L-Phe production and also enhanced instability of the catalyst, except Triton X-100 which gave a superior (56%) increase in conversion as compared to the control and a significant stabilization of PAL enzyme. Inclusion of several activity modifiers and stabilizer additives in reaction mixtures were shown to enhance the yield of L-Phe and maintained PAL stability over several successive incubations during the bioconversion process. Maximum stabilization of PAL and enhancement of L-Phe production was achieved with addition of 20% polyhydric alcohol (glycerol). The production of L-Phe continued to the fifth cycle and the total yield increased 2.3 times compared to the yield produced by the control (without glycerol addition) during the repeated batch process. Reducing agents such as 2-mercaptoethanol and thioglycolic acid were added to the bioconversion mixture in order to reduce the effects of oxygen on PAL catalyst life. Production of L-Phe by addition of 400 mgL(-1) of thioglycolic acid was maximized over the control by 55%. When both 20% glycerol and 400 mgL(-1) thioglycolic acid were simultaneously present in the reaction mixture, reuseability and stability of biocatalyst (PAL) were extended to eight consecutive cycles and conversion rate and overall productivity of L-Phe were higher than that of the control. These results may lead to improvements in the production of the essential amino acid L-Phe.     

Selective conversion of bio-oil to light olefins: controlling catalytic cracking for maximum olefins

Light olefins are the basic building blocks for the petrochemical industry. In this work, selective production of light olefins from catalytic cracking of bio-oil was performed by using the La/HZSM-5 catalyst. With a nearly complete conversion of bio-oil, the maximum yield reached 0.28±0.02 kg olefins/(kg bio-oil), which was close to that from methanol. Addition of La into zeolite efficiently changed the total acid amount of HZSM-5, especially the acid distribution among the strong, medium and weak acid sites. A moderate increase of the number of the medium acid sites effectively enhanced the olefins selectivity and improved the catalyst stability. The comparison between the catalytic cracking and pyrolysis of bio-oil was studied. The mechanism of the conversion of bio-oil to light olefins was also discussed.     

Nitrile hydratases (NHases): At the interface of academia and industry

Nitrile hydratase (NHase, EC 4.2.1.84) is one of the key enzymes of nitrile metabolism in a large number of microbes that catalyses the hydration of nitriles to corresponding amides, and has been successfully adopted in chemical industry for production of acrylamide, nicotinamide and 5-cyanovaleramide. However, NHase is still under active consideration of enzymologists to expand its potential for synthesis of various amides. Most of the NHases have been reported for their limited substrates acceptability, low enantioselectivity and thermostability and therefore a considerable improvement is required for developing as robust biocatalyst for synthesis of a range of organic amides. Studies on biochemical properties, gene configuration, active-site chemical models and site-directed mutagenesis have given the insight into the structural and functional characteristics of NHase. Keeping in view, the present review critically describes the available information on natural sources (based on activity and phylogenetic analysis), biochemical properties, catalysis–structure relationship, molecular expression and potential applications of this enzyme.     

Recent developments in manufacturing and marketing carrageenan

Carrageenan has annual sales of over US$ 200 million, about 15% of the world use of food hydrocolloids. The market for carrageenan has grown exponentially at 5% per year for at least 25 years: 5 500 metric tons in 1970, and over 20 000 metric tons expected in 1995. The industry has become dominated by very large, multi-product companies with carrageenan factories in Europe and the US, but factories are now springing up in the Philippines and Chile, where red seaweeds grow in abundance. About 80 000 tons of dry red seaweeds are needed to produce 20 000 tons of carrageenan. About 40 000 tons comes from the Philippines, 15 000 tons from Indonesia, 15 000 tons from Chile, and 10 000 tons from elsewhere. Carrageenan growth depends on food fads like the McLean hamburger and food winners like processed pork and turkey. Carrageenan is a regulated food additive, and current health concerns focus on the minimum safe molecular weight for carrageenan when eaten. The most innovative development in carrageenans in recent years has been the introduction of a food grade version of lower cost natural grade carrageenan. Its acceptance, however, has been hampered by strong resistance from conventional carrageenan producers.     

A fermentation process for producing both ethanol and lysine-enriched yeast.

In 18 batch-fermentation experiments, baker's yeast was grown in an enriched mineral medium, containing 10% by weight glucose, at various pH and temperature levels. The pH and temperature are just two representative engineering variables which can be easily varied at negligible cost. The commercial yeast inoculum, 20% by weight or about .16% viable cells, was selected to represent industrial (nonsterile) conditions. Free L-lysine, ethanol, and cell growth were followed in time for each batch run held at a fixed pH and temperature. The maximum free lysine level reached at either 10 1/2 or 24 hr occurred at a pH of 5 and 32 degrees C. At 24 hr, the peak free lysine level, 120 mg/liter, is three times as great as the uncontrolled pH counterpart. In terms of total L-lysine (free plus protein-bound) the peak represents a 25% improvement over the uncontrolled case, based on an average 3.5% lysine level per cell weight. The greatest measured cell level, .9% by weight in the fermentation broth, or a 5 1/2-fold increase over th inoculum, was reached during the 36 degrees C and pH 3 run, while the largest measured ethanol value (3%, or 30% conversion by weight from glucose) was achieved during the 28 degrees C and pH 6 experiment. The optimal lysine run product, however, no less than 15% of the maximum cell and 30% of the maximum ethanol levels.     

A novel hydantoinase process using recombinant Escherichia coli cells with dihydropyrimidinase and L-N-carbamoylase activities as biocatalyst for the production of L-homophenylalanine

A dihydropyrimidinase gene (pydB) was cloned from the moderate thermophilic Brevibacillus agri NCHU1002 and expressed in Escherichia coli. The purified dihydropyrimidinase exhibited strict d-enantioselectivity for D,L-p-hydroxyphenylhydantoin and D,L-5-[2-(methylthio)ethyl]hydantoin, and non-enantiospecificity for D,L-homophenylalanylhydantoin (D,L-HPAH). The hydrolytic activity of PydB was enhanced notably by Mn2+, with a maximal activity at 60 degrees C and pH 8.0. This enzyme was completely thermostable at 50 degrees C for 20 days. A whole cell biocatalyst for the production of L-homophenylalanine (L-HPA) from D,L-HPAH by coexpression of the pydB gene and a thermostable L-N-carbamoylase gene from Bacillus kaustophilus CCRC11223 in E. coli JM109 was developed. The expression levels of dihydropyrimidinase and L-N-carbamoylase in the recombinant E. coli cells were estimated to be about 20% of the respective total soluble proteins. When 1% (w/v) isopropyl-beta-D-thiogalactopyranoside-induced cells were used as biocatalysts, a conversion yield of 49% for L-HPA with more than 99% ee could be reached in 16 h at pH 7.0 from 10mM D,L-HPAH. The cells can be reused for at least eight cycles at a conversion yield of more than 43%. Our results revealed that coexpression of pydB and lnc in E. coli might be a potential biocatalyst for L-HPA production.     

Plant lipases: biocatalyst aqueous environment in relation to optimal catalytic activity in lipase-catalyzed synthesis reactions.

Adsorption and desorption isotherms of two commercial enzyme preparations of papain and bromelain were determined with a Dynamic Vapor System. The Guggenheim-Anderson-deBoer (GAB) modeling of the obtained sorption isotherms allowed the definition of different levels of hydration of those samples. Afterward, these enzyme preparations were used as biocatalysts in water and solvent-free esterification and alcoholysis reactions. The evolution of the obtained fatty acid ester level as a function of the initial hydration level of the biocatalyst, i.e., thermodynamic water activity (a(w)) and water content, was studied. The results show an important correlation between the initial hydration level of the biocatalyst and its catalytic activity during the lipase-catalyzed synthesis reactions. Thus, the Carica papaya lipase (crude papain preparation) catalytic activity is highly dependent on the biocatalyst hydration state. The optimized synthesis reaction yield is obtained when the a(w) value of the enzyme preparation is stabilized at 0.22, which corresponds to 2% water content. This optimal level of hydration occurs on the linear part of the biocatalyst's sorption isotherm, where the water molecules can form a mono- or multiple layer with the protein network. The synthesis reaction yield decreases when the a(w) of the preparation is higher than 0.22, because the excess water molecules modify the system equilibrium leading to the reverse and competitive reaction, i.e., hydrolysis. These results show also that an optimal storage condition for the highly hydrophilic crude papain preparation is a relative humidity strictly lower than 70% to avoid an irreversible structural transition leading to a useless biocatalyst. Concerning the bromelain preparation, no effect of the hydration level on the catalytic activity during esterification reactions was observed. This biocatalyst has too weak a catalytic activity which makes it difficult to observe any differences. Furthermore, the bromelain preparation is far more hydrophobic as it adsorbs only 18 g of water per 100 g of dry material at a(w) around 0.90. No deliquescence of this enzymatic preparation is observed at this a(w) value.     

The fate of a roach Rutilus rutilus stock under an extremely strong fishing pressure and its predicted development after the cessation of mass removal

The effects of very intensive exploitation on a roach stock over 5 years are estimated. The total roach catch of the mass removal carried out with trawls was 507 metric tons (195 kg ha⁻¹, 54% of the total catch). According to the virtual population analysis the biomass of the stock has decreased from 180 kg ha⁻¹ in 1989 to c . 50 kg ha⁻¹ in 1993. The production of the stock has fallen from 64 to 33 kg ha⁻¹. The stock is dominated by younger age groups than before the mass removal. The future development of the stock is predicted by considering different levels of natural and fishing mortality. If the exploitation is stopped totally after 1993, roach biomass is predicted to double in 3 years. A constant fishing mortality rate of 0.3 (yearly catches 45–50 metric tons) would prevent the recovery of the stock, providing that 1–year–old and older roaches are exploited.     

Acrylamide synthesis using agar entrapped cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34 in a partitioned fed batch reactor

The nitrile hydratase (Nhase) induced cells of Rhodococcus rhodochrous PA-34 catalyzed the conversion of acrylonitrile to acrylamide. The cells of R. rhodochrous PA-34 immobilized in 2% (w/v) agar (1.76 mg dcw/ml agar matrix) exhibited maximum Nhase activity (8.25 U/mg dcw) for conversion of acrylonitrile to acrylamide at 10°C in the reaction mixture containing 0.1 M potassium phosphate buffer (pH 7.5), 8% (w/v) acrylonitrile and immobilized cells equivalent to 1.12 mg dcw (dry cell weight) per ml. In a partitioned fed batch reaction at 10°C, using 1.12 g dcw immobilized cells in a final volume of 1 l, a total of 372 g of acrylonitrile was completely hydrated to acrylamide (498 g) in 24 h. From the above reaction mixture 87% acrylamide (432 g) was recovered through crystallization at 4°C. By recycling the immobilized biocatalyst (six times), a total of 2,115 g acrylamide was produced.     

Comparison of Catalytic Characteristics of Biomass Derivates with Different Structures Over ZSM-5

The conversion of three major biomass derivates was conducted in a quartz tubular fixed bed reactor over a ZSM-5 catalyst. As the model compounds of polyols, saturated furans and unsaturated furans, ethylene glycol (EG), tetrahydrofuran (THF) and furan were pyrolyzed to find out the influence of chemical structure on the catalytic characteristics. The effect of pyrolysis temperature (400?～?650 °C), weight hourly space velocity (2.9?～?15.5 h^?1) and partial pressure (2.12?～?20.49 Torr) on the feed conversion, product yield and selectivity were investigated. The hydrogen to carbon effective ratio (H/C_eff) was referred to, to analyze the capacity of biomass derivates being converted to chemicals (olefins and aromatics). The results showed that the existence of rings and C=C had great effect on the catalytic characteristics. The conversion of furan was much lower (mainly less than 60 %) than that of EG and TH,F which were close to 100 %. It was also found that the chemical yield of THF was slightly more than that of EG, which can be attributed to its relative higher H/C_eff of 1.5. Furan produced the highest coke yield, which was more than 15 %, whereas that of EG and THF was only around 5 %. The serious coking of furan led to the lowest chemical yield, which was less than 35 %. This study paves a way for the mechanism study on catalytic characteristics of biomass-derived feedstocks over zeolite catalysts.     

催化山梨醇脱水制备异山梨醇的固体酸催化剂

为提高目标产物异山梨醇的产率,考察多种固体酸催化剂催化山梨醇脱水的反应性能。结果表明:催化剂酸性与其催化性能之间有密切联系,酸性较强的H3PO4/Nb2O5催化剂显示出比其他催化剂更优异的催化性能。对磷酸负载量进行优化后,在n(P)/n(Nb)为0.8的H3PO4/Nb2O5催化剂上得到了100%的山梨醇转化率和63%的异山梨醇选择性。     

Biocatalytic conversion of cycloalkanes to lactones using an in‐vivo cascade in Pseudomonas taiwanensis VLB120

Chemical synthesis of lactones from cycloalkanes is a multi‐step process challenged by limitations in reaction efficiency (conversion and yield), atom economy (by‐products) and environmental performance. A heterologous pathway comprising novel enzymes with compatible kinetics was designed in Pseudomonas taiwanensis VLB120 enabling in‐vivo cascade for synthesizing lactones from cycloalkanes. The respective pathway included cytochrome P450 monooxygenase (CHX), cyclohexanol dehydrogenase (CDH), and cyclohexanone monooxygenase (CHXON) from Acidovorax sp. CHX100. Resting (non‐growing) cells of the recombinant host P. taiwanensis VLB120 converted cyclohexane, cyclohexanol, and cyclohexanone to ?‐caprolactone at 22, 80–100, and 170 U g_CDW^?1, respectively. Cyclohexane (5 mM) was completely converted with a selectivity of 65% for ?‐caprolactone formation in 2 hr without accumulation of intermediate products. Promiscuity of the whole‐cell biocatalyst gave access to analogous lactones from cyclooctane and cyclodecane. A total product concentration of 2.3 g L^?1 and a total turnover number of 36,720 was achieved over 5 hr with a biocatalyst concentration of 6.8 g_CDW L^?1.

     

Growth of water hyacinths in treated sewage effluent

Two thousand plants of the water hyacinth,Eichornia crassipes Solms., were introduced on April 11, 1971, into a series of five ponds, each 5000 sq. ft. in area and 2.6 ft. deep. Treated waste water effluent from the Ames sewage treatment plant filled the ponds and was added to pond 1 at 127 gallons per minute. By growth and vegetative reproduction, these plants increased to more than 500,000, and all five ponds were covered completely by July 26. On that date, the extrapolated estimate of total wet weight was 287 U.S. tons/ acre (645 metric tons/hectare; 64500g/m²). The estimate of oven dry weight was 13.2 U.S. tons/acre (29.7 metric tons/hectare; 2970g/m²). Ammonia and nitrate disappeared rapidly from the pond water, and phosphate concentrations were lowered appreciably. Evapotranspiration and seepage accounted for water losses of more than 0.5 inches per day. The potential economic values of this plant and its possible use in tertiary treatment to reduce N and P components in waste waters are discussed briefly.     

Continuous production of L-phenylalanine by Rhodotorula glutinis immobilized cells using a column reactor

Studies have been conducted on L-phenylalanine (L-Phe) production and phenylalanine ammonia lyase (PAL) stabilization in the presence of several optimum effectors and reducing agents under bioconversion of transcinnamic acid (t-CA) conditions during repeated batch operations. L-Phe production was maximized and reuseability of PAL catalyst was extended to eight consecutive cycles (repeated batches) in the presence of optimum effectors (glutamic acid, polyethylene glycol and glycerol), thioglycolic acid and sparging with nitrogen gas. These best optimum bioconversion conditions desensitize the PAL catalyst to substantially elevated higher substrate t-CA concentrations and inhibit inactivation of PAL enzyme over longer reaction periods compared to the control. The fed batch mode operation of bioconversion of total t-CA (300 mM) to L-Phe was superior (65.2%, conversion), comparing with conventional batch and repeated batch (58.4%, conversion) operations after 120 h. Gamma irradiation process was employed to polymerize and crosslink polyvinyl alcohol (PVA) with N,N'-methylene-bisacrylamide (BIS) agent. The use of immobilized PAL biocatalyst containing cells in PVA-BIS copolymer gel carrier produced by radiation polymerization is obviously advantageous with regards to the yield of L-Phe which was increased in average 1.2-fold when compare to those obtained with free cells during optimum bioconversion process. When comparing the magnitudes of gamma irradiation effects on immobilized entrapped yeast cells in PVA-BIS copolymer gel carrier using scanning electron microscopy it was show that yeast cells were protected and capable to overcome these conditions and had normal shape and other features as free (unirradiated) intact yeast cells. Optimum conditions for continuous production of L-Phe by PVA-BIS copolymer carrier entrapped yeast cells in a packed bed column reactor in recycle fed-batch mode were investigated. Under these optimum conditions L-Phe accumulated to concentration 240.1 mM represts a total conversion yield of 80% (w/w) from (300 mM) t-CA after 84 h of reaction process, which was higher than that obtained after 120 h of reaction, 65.2% (w/w) from (300 mM) t-CA with free cells in fed-batch mode. The results also demonstrated that during about 4 weeks of repeated continuous recycle fed batch mode experiments (using immobilized cells in packed bed reactor), the final production of L-Phe concentrations decreased gradually in eight consecutive runs with no sign of breakage or disintegration of the carrier gel beads.     

The Enzymatic Production of Adipic Acid

Mutants of Brevibacterium sp. R312 were isolated for the production of adipic acid from 1,4-dicyanobutane (adiponitrile). One mutant (Ad), with a modified cell wall showed activity against adipamide three times greater than the wild type. Another mutant (ACV2) derived from the Ad strain had 30 times more activity on 5-cyanovaleric acid, and 7 times more on adipamide than the wild type.

The nitrile hydratase from the mutant strain ACV2 was purified and compared to that from the wild type R312. The nitrile hydratase of the mutant strain is different from that of the wild type by its pHi, optimum activity pH, and its rates of hydrolysis of 5-cyanovaleramide and 5-cyanovaleric acid which were 30 and 15 folds greater.

The presence of a new amidase named “adipamidase” acting on amide intermediates in the hydrolysis of dinitriles to organic acids was demonstrated in this mutant ACV2.     

中国有机肥料养分资源潜力和环境风险分析

基于<中国农业年鉴2006)和其他文献的基础数据,计算了2005年中国人畜禽排泄物和秸秆数量及其产生的养分量.结果表明,2005年中国人畜禽排泄物总量为46.25亿t,秸秆总产量为6.43亿t.中国有机肥料养分资源潜力巨大,2005年人畜禽排泄物和秸秆共产生N、P2O5、K2O养分量分别为2824.52、1282.93、2947.99万t,分别为化肥N、P2O5、K2O投入量的1.08、0.86和4.56倍.但不同区域差异较大,其中河南、山东和四川省人畜禽排泄物产生N、P2O5、K2O量最多,均>400万t,西北地区和北京、天津、上海等地人畜禽排泄物产生的养分总量较少.秸秆中N、P2O5、K2O含量在河南和山东2个粮食主产省最高,均>150万t;西北地区秸秆养分产生量相对较少.单位农田面积人畜禽排泄物的N、P2O5和K2O养分负荷量以北京最高,达到787.26 kg·hm-2,其次是天津和上海,分别为515.31和505.35 kg·hm-2,环境风险较大.