Butyric acid production from brown algae using <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> ATCC 25755

Butyric acid fermentation by Clostridium tyrobutyricum ATCC 25755 using glucose or brown algae as a carbon source was carried out. Initially, different fermentation modes (batch, fed-batch, and semi-continuous) at pH 6 and 37°C were compared using a model medium containing glucose as a carbon source. By feeding the whole medium containing 40 ∼ 50 and 30 g/L of glucose into the fed-batch and semi-continuous fermentations, very similar butyrate yields (0.274 and 0.252 g butyrate/g glucose, respectively) and productivities (0.362 and 0.355 g/L/h, respectively) were achieved. The highest butyrate concentration was about 50 g/L, which was observed in the fed-batch fermentation with whole medium feeding. However, semi-continuous fermentation sustained a longer fermentation cycle than the fed-batch fermentation due to end-product and metabolic waste inhibition. The established conditions were then applied to the fermentation using brown algae, Laminaria japonica and Undaria pinnatifida, as substrates for butyric acid fermentation. To hydrolyze brown algae, 7.5 ∼ 10% (w/v) dried brown algae powder was suspended in 1% (w/v) NaOH or 0.5 ∼ 2.5% (w/v) H₂SO₄ and then autoclaved at 121°C for 30 ∼ 90 min. The resulting butyrate concentration was about 11 g/L, which was produced from 100 g/L of L. japonica autoclaved for 60 min in 1.5% H₂SO₄ acid solution.     

Exploring low-cost carbon sources for microbial lipids production by fed-batch cultivation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Volatile fatty acids (VFAs), acetic acid, acetates, and ethanol were used as carbon sources for the production of microbial lipids using Cryptococcus albidus in batch cultures. C. albidus utilized organic acids less than glucose in the production of lipids, resulting in a lipid yield coefficient on VFAs of 0.125 g/g. In a two-stage batch culture, the lipid content increased to 43.8% (w/w) when VFAs were used as the sole carbon source in the second stage, which was two times higher than that of the batch culture. Furthermore, a 192 h, two-stage fed-batch cultivation of C. albidus produced a dry cell weight, lipid concentration, and lipid content of 26.4 g/L, 14.5 g/L, and 55.1% (w/w), respectively. The fed-batch culture model used in this study featured pure VFA solutions, with intermittent feeding, under oxygen-enriched air supply conditions. This study investigated several alternative carbon sources to reduce the cost of microbial lipids production and proved the feasibility of using VFAs as the carbon source for the provision of a high lipid content and productivity.     

Solasodine production from cell culture of <Emphasis Type="Italic">Solanum hainanense</Emphasis> Hance

Stem explants of Solanum hainanense Hance plantlets were cultured on Murashige and Skoog solid medium, containing 3% (w/v) sucrose, supplemented with 0.1 mg/L benzylaminopurine (BAP) and 1.0 mg/L 2, 4-dichlorophenoxyacetic acid (2, 4-D) for callus production. To establish the cell suspension culture, 3 g of fresh callus were cultured in 50 mL of the same medium, but without a solid agent, at an agitation speed of 120 rpm. Every 15 mL of culture was sub-cultured in fresh MS liquid medium for maintenance. The cell biomass of S. hainanense reached a maximum value of 18.47 g after 4 weeks of culture on the same MS medium, but with the sucrose content increased to 4%, at an agitation speed of 150 rpm, with 20 mL of inoculum. Analysis via high performance liquid chromatography (HPLC) showed that the solasodine content in the cell suspension after 4-weeks old (121.01 mg/g) was higher than that of in planta 1-year old roots (20.52 mg/g) by approximately 6-fold.     

In vitro production of gymnemic acid from cell suspension cultures of Gymnema sylvestre R. Br

Gymnema sylvestre is an important medicinal plant that bears bioactive compound namely gymnemic acids. The present work deals with the optimization of a cell suspension culture system of Gymnema sylvestre for the production of biomass and gymnemic acid, which has anti‐diabetic properties. We investigated the effect of inoculum densities (2.5–20.0 g/L), the strength of the Murashige and Skoog (MS) medium (0.25–2.0), carbon source (sucrose, glucose, fructose, maltose), and the concentration of the sucrose (1–8% w/v) to determine their effects on biomass accumulation and production of gymnemic acid. Overall, 10 g/L of inoculum density, full‐strength MS medium supplemented with 2,4‐dichlorophenoxy acetic acid (2.0 mg/L) and Kinetin (0.1 mg/L), and 3% w/v sucrose was found best for the accumulation of biomass and gymnemic acid content (9.95 mg/g dry weight). The results of the current study will be useful for bioprocess and biochemical engineers for large‐scale production of gymnemic acid in cell culture.     

Enhancement of carotenoids and lipids production by oleaginous red yeast Sporidiobolus pararoseus KM281507

Bioconversion of biodiesel-derived crude glycerol into carotenoids and lipids was investigated by a microbial conversion of an oleaginous red yeast Sporidiobolus pararoseus KM281507. The methanol content in crude glycerol (0.5%, w/v) did not show a significant effect on biomass production by strain KM281507. However, demethanolized crude glycerol significantly supported the production of biomass (8.64?±?0.13?g/L), lipids (2.92?±?0.03?g/L), β-carotene (15.76?±?0.85?mg/L), and total carotenoids (33.67?±?1.28?mg/L). The optimal conditions suggested by central composite design were crude glycerol concentration (55.04?g/L), initial pH of medium (pH 5.63) and cultivation temperature (24.01°C). Under these conditions, the production of biomass, lipids, β-carotene, and total carotenoids were elevated up to 8.83?±?0.05, 4.00?±?0.06?g/L, 27.41?±?0.20, and 53.70?±?0.48?mg/L, respectively. Moreover, an addition of olive oil (0.5???2.0%) dramatically increased the production of biomass (14.47?±?0.15?g/L), lipids (6.40?±?0.09?g/L), β-carotene (54.43?±?0.95?mg/L), and total carotenoids (70.92?±?0.51?mg/L). The oleic acid content in lipids was also increased to 75.1% (w/w) of total fatty acids, indicating a good potential to be an alternative biodiesel feedstock. Meanwhile, the β-carotene content in total carotenoids was increased to 76.7% (w/w). Hence, strain KM281507 could be a good potential source of renewable biodiesel feedstock and natural carotenoids.     

Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

Ultrahigh diterpenoid tanshinone production through repeated osmotic stress and elicitor stimulation in fed-batch culture of <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis> hairy roots

Hyperosmotic stress (OS, created with 50 g/L sorbitol) and a yeast elicitor (YE, polysaccharide fraction of yeast extract) applied to Salvia miltiorrhiza hairy root cultures had a synergistic effect on the diterpenoid tanshinone production. With a single OS+YE treatment and nutrient feeding, the total tanshinone content of roots was increased by sevenfold (from 0.2 to 1.6 mg/g dry weight (dw)) and the volumetric yield by 13-fold (from 1.95 to 27.4 mg/L) compared to the batch control culture. With repeated feeding of OS and nutrient medium in an extended fed-batch culture process (i.e., 10 mL fresh medium with 50 g/L sorbitol 25 mg/L YE, every 5 days from day 21 to day 60), the total tanshinone content of roots was increased to 18.1 mg/g dw (or 1.8 wt.%) and the volumetric tanshinone yield to 145 mg/L, which were about 100-fold and 70-fold of those, respectively, in the batch control. Another interesting finding was the presence of root fragments (fine particles) with extremely high tanshinone content in the OS+YE treated cultures. It was also possible to reuse the sorbitol medium for the hairy root growth and tanshinone production to reduce the medium expenses.     

Effect of oleic acid on the production of ethanol and fructose from glucose/fructose mixtures in an immobilized cell reactor

Saccharomyces cerevisiae ATCC 39859 was immobilized onto small cubes of wood to produce ethanol and very enriched fructose syrup from glucose/fructose mixtures through the selective fermentation of glucose. A maximum ethanol productivity of 21.9 g/l-h was attained from a feed containing 9.7% (w/v) glucose and 9.9% (w/v) fructose. An ethanol concentration, glucose conversion and fructose yield of 29.6 g/l, 62% and 99% were obtained, respectively. This resulted in a final fructose/glucose ratio of 2.7. At lower ethanol productivity levels the fructose/glucose ratio increases, as does the ethanol concentration in the effluent. The addition of 30 mg/l oleic acid to the medium increased the ethanol productivity and its concentration by 13% at a dilution rate of 0.74 h^?1.     

Ethanol production from rice hull using Pichia stipitis and optimization of acid pretreatment and detoxification processes

The goal of this study was to produce ethanol from rice hull hydrolysates (RHHs) using Pichia stipitis strains and to optimize dilute acid hydrolysis and detoxification processes by response surface methodology (RSM). The optimized conditions were found as 127.14°C, solid:liquid ratio of 1:10.44 (w/v), acid ratio of 2.52% (w/v), and hydrolysis time of 22.01 min. At these conditions, the fermentable sugar concentration was 21.87 g/L. Additionally, the nondetoxified RHH at optimized conditions contained 865.2 mg/L phenolics, 24.06 g/L fermentable sugar, no hydroxymethylfurfural (HMF), 1.62 g/L acetate, 0.36 g/L lactate, 1.89 g/L glucose, and 13.49 g/L fructose + xylose. Furthermore, RHH was detoxified with various methods and the best procedures were found to be neutralization with CaO or charcoal treatment in terms of the reduction of inhibitory compounds as compared to nondetoxified RHH. After detoxification procedures, the content of hydrolysates consisted of 557.2 and 203.1 mg/L phenolics, 19.7 and 21.60 g/L fermentable sugar, no HMF, 0.98 and 1.39 g/L acetate, 0 and 0.04 g/L lactate, 1.13 and 1.03 g/L glucose, and 8.46 and 12.09 g/L fructose + xylose, respectively. Moreover, the base‐line mediums (control), and nondetoxified and detoxified hydrolysates were used to produce ethanol by using P. stipitis strains. The highest yields except that of base‐line mediums were achieved using neutralization (35.69 and 38.33% by P. stipitis ATCC 58784 and ATCC 58785, respectively) and charcoal (37.55% by P. stipitis ATCC 58785) detoxification methods. Results showed that the rice hull can be utilized as a good feedstock for ethanol production using P. stipitis. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:872–882, 2016     

Optimization of a liquid medium for beauvericin production in <Emphasis Type="Italic">Fusarium redolens</Emphasis> Dzf2 mycelial culture

Beauvericin (BEA) is a proven and potent antibiotic compound useful for bio-control and a potential antifungal and anticancer agent for human. This study was to evaluate and optimize the nutrient medium for BEA production in mycelial liquid culture of a high BEA-producing fungus Fusarium redolens Dzf2 isolated from a medicinal plant. Among various organic and inorganic carbon and nitrogen sources, glucose and peptone were found the most favorable for the F. redolens Dzf2 mycelial growth and BEA production. Through a Plackett-Burman screening test on a basal medium, glucose, peptone, and medium pH were identified as the significant factors for mycelial growth and BEA production. These factors were optimized through central composite design of experiments and response surface methodology, as 49.0 g/L glucose, 13.0 g/L peptone and pH 6.6, yielding 198 mg/L BEA (versus 156 mg/L in the basal medium). The BEA yield was further increased to 234 mg/L by feeding 10 g/L glucose to the culture during exponential phase. The results show that F. redolens Dzf2 mycelial fermentation is a feasible and promising process for production of BEA.     

Plant regeneration from protoplasts isolated from embryogenic calli of the forage legume<Emphasis Type="Italic"> Astragalus melilotoides</Emphasis> Pall.

An efficient and reproducible protocol is described for the regeneration of Astragalus melilotoides protoplasts isolated from hypocotyl-derived embryogenic calli. Maximum protoplast yield (11.74±0.6×10⁵/g FW) and viability (87.07±2.8%) were achieved using a mixture of 2% (w/v) Cellulase Onozuka R10, 0.5% (w/v) Cellulase Onozuka RS, 0.5% (w/v) Macerozyme R10, 0.5% (w/v) Hemicellulase, and 1% (w/v) Pectinase, all dissolved in a cell protoplast wash (CPW) salt solution with 13% (w/v) sorbitol. First divisions occurred 3–7 days following culture initiation. The highest division frequency (9.86±0.68%) and plating efficiency (1.68±0.05%) were obtained in solid-liquid medium (KM8P) supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid, 0.5 mg/l 6-benzylaminopurine (BA), 0.2 mg/l kinetin, 0.2 M glucose, 0.3 M mannitol and 500 mg/l casein hydrolysate. Upon transfer to MS medium with 0.5 mg/l -naphthaleneacetic acid and 1-2 mg/l BA, the protoplast-derived calli produced plantlets via somatic embryogenesis (56.3±4.1%) and organogenesis (21.6±0.6%). Somatic embryos or adventitious shoots developed into well-rooted plantlets on MS medium without any plant growth regulators or supplemented with 3.0 mg/l indole-3-butyric acid, respectively. About 81% of the regenerants survived in soil, and all were normal with respect to morphology and growth characters.Abbreviations BA: 6-Benzylaminopurine - CH: Casein hydrolysate - CPW: Cell protoplast wash - 2,4-D: 2,4-Dichlorophenoxyacetic acid - FDA: Fluorescein diacetate - IBA: Indole-3-butyric acid - KIN: Kinetin - MES: 2-(N-morpholino) Ethanesulphonic acid - NAA: -Naphthaleneacetic acidCommunicated by A. Altman     

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

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

Light-susceptibility of camptothecin production from<Emphasis Type="Italic">in vitro</Emphasis> cultures of<Emphasis Type="Italic">Camptotheca acuminata</Emphasis> Decne

Production of camptothecin (CPT) from callus cultures ofCamptotheca acuminata Decne was affected by light and culture conditions. Among the culture media tested, modified B5 medium containing 3% (w/v) sucrose, 2 mg/L 2,4-D, 2 times of MS medium vitamins, 500 mg/L casein hydrolysate, 250 mg/L myo-inositol, 0.05% (w/v) activated charcoal, and 0.15% (w/v) gelite was used for callus induction. The highest cell growth and CPT production were obtained in dark and green light condition, respectively. Photoperiod has no effect on cell growth and CPT production. Both cell growth and CPT production were also influenced by combination ratio of red and blue light. Cell growth and CPT production were the highest in the ratio of red and blue light 90∶10.     

Characteristics of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) production byRalstonia eutropha NCIMB 11599 and ATCC 17699

Ralstonia eutropha NCIMB 11599 and ATCC 17699 were grown, and their productions of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] compared. In flask cultures ofR. eutropha NCIMB 11599, cell concentration, P(3HB-co-4HB) concentration and polymer content decreased considerably with increases in the γ-butyrolactone concentration, and the 4HB fraction was also very low (maximum 1.74 mol%). In fed-batch cultures ofR. eutropha NCIMB 11599, glucose and γ-butyrolactone were fed as the carbon sources, under a phosphate limitation strategy. When glucose was fed as the sole carbon source, with its concentration controlled using an on-line glucose analyzer, 86% of the P(3HB) homopolymer was obtained from 201 g/L of cells. In a two-stage fed-batch culture, where the cell concentration was increased to 104 g/L, with glucose fed in the first step and constant feeding of γ-butyrolactone, at 6 g/h, in the second, final cell concentration at 67 h was 106 g/L, with a polymer content of 82%, while the 4HB fraction was only 0.7 mol%. When the same feeding strategy was applied to the fedbatch culture ofR. eutropha ATCC 17699, where the cell concentration was increased to 42 g/L, by feeding fructose in the first step and γ-butyrolactone (1.5 g/h) in the second, the final cell concentration, polymer content and 4HB fraction at 74 h were 51 g/L, 35% and 32 mol%, respectively. In summary,R. eutropha ATCC 17699 was better thanR. eutropha NCIMB 11599 in terms of P(3HB-co-4HB) production with various 4HB fractions.     

Absence of Food Aversion Learning in the Polyphagous Noctuid, <Emphasis Type="Italic">Spodoptera litura</Emphasis> (F.) Following Intoxication by Deleterious Chemicals

The objective of this study was to determine whether the presence of a deleterious chemical in a preferred host plant could alter the feeding preference of a polyphagous insect. The preference of the Asian armyworm, Spodoptera litura (F.) (Lepidoptera: Noctuidae) for castor, Ricinus communis (L.)(family: Euphorbiaceae) relative to cabbage, Brassica oleracea (L.) (family: Brassicaceae) was quantified by two separate two-way choice tests (without treatment and with treatment of the test chemicals used in the present study) with naive third instar larvae each time. This was followed, by continuous feeding (48 h) on the preferred host treated with the test chemicals and using naive third instar larvae for conditioning. Each treatment consisted of one of nine compounds, including seven naturally occurring allelochemicals (viz. (–)--pinene, (–)--pinene, -myrcene, {D}-limonene, cineole, rutin, and ajwain oil) and two synthetic insecticides (viz. alphamethrin and malathion). Following this, a two-way choice test was repeated with the same batch of larvae without any test chemical. Larvae continued to maintain preference for castor despite exposure to the deleterious chemicals. Among the test chemicals, {D}-limonene and alphamethrin caused significant reduction in growth. Preference for castor was not overcome by exposure to novel deleterious chemicals, suggesting that aversion, though experienced is not learned.     

Improved production of curdlan with concentrated cells ofAgrobacterium sp.

The addition of a limited concentration of yeast extract to a minimal salt medium (MSM) enhanced cell growth and increased the production of curdlan whereas nitrogenlimitation was found to be essential for the higher production of curdlan byAgrobacterium sp. ATCC 31749. As the amount of the inoculum increased, the cell growth as well as the production of curdlan also increased in the MSM without a nitrogen source. The cell growth and production of curdlan increased as the initial pH of the medium decreased as low as 5.0. The conversion rate and concentration of curdlan from 2% (w/v) glucose in the MSM with concentrated cells under nitrogen deletion was 67% and 13.4 g/L, respectively. The highest conversion rate of curdlan under the conditions optimized in this study was 71% when the glucose concentration was 1% (w/v).     

Ethanol production by Zymomonas mobilis in fed-batch fermentations

The production of ethanol by Zymomonas mobilis NRRL B-4286 was studied in fed-batch cultures. Initial percent (w/v) glucose, rate of feed, and quantity of 50%; (w/v) glucose feed were varied. Glucose inhibition of growth rate occurred at concentrations greater than 8% (w/) Feed was begun after 4 h incubation. Feed volume was ca. 36%; of starting batch volume to get ca. 10%; (w/v) ethanol at harvest. The range of feed rates studied varied from 16%; batch volume/h (glucose concentration increased to an inhibitory level) to 4%; batch volume/h (glucose concentration dropped rapidly to zero and was limiting). Increasing feed volume to 46%; of starting volume at the best feed rate (ca. 10%; feed volume/h) increased final ethanol concentration to 11.3%; (w/v). However, the resultant increase in fermentation time from ca. 21 to 29 h decreased ethanol volumetric productivity from 5.2 to 4.6 g/L h.     

Use of carbohydrate-supplemented distillery spent wash as a medium for ethanol production by a thermotolerant strain of yeast at 45°C

Summary Spent wash from the Old Bushmill's Distillery Co. Ltd. was supplemented with either glucose (10% [w/v]) or cellulose (5% [w/v]) and used as a medium for the thermotolerant yeast strain Kluyveromyces marxianus IMB3. There was no significant difference in ethanol production during growth on these media at 45° C, compared with that produced during growth on conventional, pre-defined laboratory media. On glucose supplemented spent wash ethanol yields were in the region of 45 g/L, representing 87% of the maximum theoretical yield. Analysis of spent media from the glucose-containing fermentations demonstrated that the total organic carbon (TOC) content was reduced by 36%. The results suggest a novel means of utilizing whiskey distiller spent wash.     

Growth and butyric acid production by Clostridium populeti

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

The immobilization of enzymes and cells of Bacillus stearothermophilus onto poly(maleic anhydride/styrene)-Co-polyethylene and poly(maleic anhydride/vinyl acetate)-Co-polyethylene

The graft copolymer, poly(maleic anhydride/styrene)-co-polyethylene was prepared. The copolymer immobilized bovine serum albumin (BSA), but the amount coupled appeared to be effected by the amount of styrene in the graft copolymer, temperature, and pH of the coupling medium. Competition existed between hydrolysis of the grafted anhydride groups and the protein. A graft copolymer with 66% add-on immobilized 4.5 mg/glucose oxidase/g copolymer, 4.6 mg alkaline phosphates/g copolymer and 0.2 mg cell of Bacillus stearothermophilus/g copolymer. A number of copolymers containing poly(maleic anhydride/vinyl acetate)-co-polyethylene were prepared to cover a range of grafting levels. These immobilized larger quantities of BSA, alkaline phosphatase, and cells of B. stearothermophilus than did the styrene graft copolymer. The copolymer was also hydrolyzed to release the hydroxyl group from the poly(vinyl acetate) component of the grafted chains. Using p-benzoquinone as the "activating agent," the copolymer coupled to BSA and to acid phosphatase. Using p-toluene-sulfonyl chloride, the copolymer was very effective in immobilizing trypsin.