Check out our New Publishers' Select for Free Articles

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.137-140, #1-12)

Introduction to the proceedings of the twenty-eighth symposium on biotechnology for fuels and chemicals by Jonathan R. Mielenz (pp. iii-viii).

Introduction to session 1A by Michael E. Himmel (pp. 1-2).

Grass lignocellulose by Danny E. Akin (pp. 3-15).

Grass lignocelluloses are limited in bioconversion by aromatic constituents, which include both lignins and phenolic acids esters. Histochemistry, ultraviolet absorption microspectrophotometry, and response to microorganisms and specific enzymes have been used to determine the significance of aromatics toward recalcitrance. Coniferyl lignin appears to be the most effective limitation to biodegradation, existing in xylem cells of vascular tissues; cell walls with syringyl lignin, for example, leaf sclerenchyma, are less recalcitrant. Esterified phenolic acids, i.e., ferulic and p-coumaric acids, often constitute a major chemical limitation in nonlignified cell walls to biodegradation in grasses, especially warm-season species. Methods to improve biodegradation in grasses, especially warm-season species. Methods to improve biodegradability through modification of aromatics include: plant breeding, use of lignin-degrading white-rot fungi, and addition of esterases. Plant breeding for new cultivars has been especially effective for nutritionally improved forages, for example, bermudagrasses. In laboratory studies, selective white-rot fungi that lack cellulases delignified the lignocellulosic materials and improved fermentation of residual carbohydrates. Phenolic acid esterases released p-coumaric and ferulic acids for potential coproducts, improved the available sugars for fermentation, and improved biodegradation. The separation and removal of the aromatic components for coproducts, while enhancing the availability of sugars for bioconversion, could improve the economics of bioconversion.

Keywords: Lignin; microspectrophotometry; phenolic acid esters; plant breeding; white-rot fungi

Enzymatic microreactors for the determination of ethanol by an automatic sequential injection analysis system by Eliana M. Alhadeff; Andrea M. Salgado; Oriol Cos; Nei Pereira; BelkisValdman; Francisco Valero (pp. 17-25).

A sequential injection analysis system with two enzymatic microreactors for the determination of ethanol has been designed. Alcohol oxidase and horseradish peroxidase were separately immobilized on glass aminopropyl beads, and packed in 0.91-mL volume microreactors, working in line with the sequential injection analysis system. A stop flow of 120 s was selected for a linear ethanol range of 0.005–0.04 g/L ± 0.6% relative standard deviation with a throughput of seven analyses per hour. The system was applied to measure ethanol concentrations in samples of distilled and nondistilled alcoholic beverages, and of alcoholic fermentation with good performance and no significant difference compared with other analytical procedures (gas chromatography and high-performance liquid chromatography).

Keywords: Alcohol oxidase; ethanol; horseradish peroxidase; immobilized enzymes; sequential injection analysis; biosensors

Optimization of cyclodextrin glucanotransferase production from Bacillus clausii E16 in submerged fermentation using response surface methodology by Heloiza Ferreira Alves-Prado; Daniela Alonso Bocchini; Eleni Gomes; Luis Carlos Baida; Jonas Contiero; Inêes Conceição Roberto; Roberto Da Silva (pp. 27-40).

Cyclodextrin glucanotransferase production from Bacillus clausii E16, a new bacteria isolated from Brazilian soil samples was optimized in shake-flask cultures. A 2⁴ full-factorial central composite design was performed to optimize the culture conditions, using a response surface methodology. The combined effect among the soluble starch concentration, the peptone concentration, the yeast extract concentration, and the initial pH value of the culture medium was investigated. The optimum concentrations of the components, determined by a 2⁴ full-factorial central composite design, were 13.4 g/L soluble starch, 4.9 g/L peptone, 5.9 g/L yeast extract, and initial pH 10.1. Under these optimized conditions, the maximum cyclodextrin glucanotransferase activity was 5.9 U/mL after a 48-h fermentation. This yield was 68% higher than that obtained when the microorganism was cultivated in basal culture medium.

Keywords: Alkalophillus Bacillus clausii ; CGTase production; cyclodextrin glucanotransferase; experimental design; submerged fermentation; response surface methodology

Purification and characterization of a cyclomaltodextrin glucanotransferase from Paenibacillus campinasensis strain H69-3 by Heloiza Ferreira Alves-Prado; Eleni Gomes; Roberto da Silva (pp. 41-55).

A cyclomaltodextrin glucanotransferase (E.C. 2.4.1.19) from a newly isolated alkalophilic and moderately thermophilic Paenibacillus campinasensis strain H69-3 was purified as a homogeneous protein from culture supernatant. Cyclomaltodextrin glucanotransferase was produced during submerged fermentation at 45°C and purified by gel filtration on Sephadex G50 ion exchange using a Q-Sepharose column and ion exchange using a Mono-Q column. The molecular weight of the purified enzyme was 70 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the pI was 5.3. The optimum pH for enzyme activity was 6.5, and it was stable in the pH range 6.0–11.5. The optimum temperature was 65°C at pH 6.5, and it was thermally stable up to 60°C without substrate during 1 h in the presence of 10 mM CaCl₂. The enzyme activity increased in the presence of Co²⁺, Ba²⁺, and Mn²⁺. Using maltodextrin as substrate, the K _m and K _cat were 1.65 mg/mL and 347.9 µmol/mg·min, respectively.

Keywords: CGTase characterization; CGTase purification; cyclomaltodextrin glucanotransferase; thermostable CGTase

Acetone powder from dormant seeds of Ricinus communis L by Elisa D. C. Cavalcanti; Fábio M. Maciel; Pierre Villeneuve; Regina C. A. Lago; Olga L. T. Machado; Denise M. G. Freire (pp. 57-65).

The influence of several factors on the hydrolytic activity of lipase, present in the acetone powder from dormant castor seeds (Ricinus communis) was evaluated. The enzyme showed a marked specificity for short-chain substrates. The best reaction conditions were an acid medium, Triton X-100 as the emulsifying agent and a temperature of 30°C. The lipase activity of the acetone powder of different castor oil genotypes showed great variability and storage stability of up to 90%. The toxicology analysis of the acetone powder from genotype Nordestina BRS 149 showed a higher ricin (toxic component) content, a lower 2S albumin (allergenic compound) content, and similar allergenic potential compared with untreated seeds.

Keywords: 2S albumin; castor seeds; lipase; ricin; Ricinus communis ; acetone powder

Immobilization of Candida antarctica lipase B by covalent attachment to green coconut fiber by Ana I. S. Brígda; Álvaro D. T. Pinheiro; Andrea L. O. Ferreira; Gustavo A. S. Pinto; Luciana R. B. Gonçalves (pp. 67-80).

The objective of this study was to covalently immobilize Candida antarctica type B lipase (CALB) onto silanized green coconut fibers. Variables known to control the number of bonds between enzyme and support were evaluated including contact time, pH, and final reduction with sodium borohydride. Optimal conditions for lipase immobilization were found to be 2 h incubation at both pH 7.0 and 10.0. Thermal stability studies at 60°C showed that the immobilized lipase prepared at pH 10.0 (CALB-10) was 363-fold more stable than the soluble enzyme and 5.4-fold more stable than the biocatalyst prepared at pH 7.0 (CALB-7). CALB-7 was found to have higher specific activity and better stability when stored at 5°C. When sodium borohydride was used as reducing agent on CALB-10 there were no improvement in storage stability and at 60°C stability was reduced for both CALB-7 and CALB-10.

Keywords: Coconut fiber; covalent attachment; enzyme immobilization; lipase; hydrolysis; esterification

Pretreatment of corn stover by soaking in aqueous ammonia at moderate temperatures by Tae Hyun Kim; Y. Y. Lee (pp. 81-92).

Soaking in aqueous ammonia at moderate temperatures was investigated as a method of pretreatment for enzymatic hydrolysis as well as simultaneous saccharification and cofermentation (SSCF) of corn stover. The method involves batch treatment of the feedstock with aqueous ammonia (15–30 wt%) at 40–90°C for 6–24 h. The optimum treatment conditions were found to be 15 wt% of NH₃, 60°C, 1 : 6 of solid-to-liquid ratio, and 12 h of treatment time. The treated corn stover retained 100% glucan and 85% of xylan, but removed 62% of lignin. The enzymatic digestibility of the glucan content increased from 17 to 85% with 15 FPU /g-glucan enzyme loading, whereas the digestibility of the xylan content increased to 78%. The treated corn stover was also subjected to SSCF test using Spezyme-CP and recombinant Escherichia coli (KO11). The SSCF of the soaking in aqueous ammonia treated corn stover resulted in an ethanol concentration of 19.2 g/L from 3% (w/v) glucan loading, which corresponds to 77% of the maximum theoretical yield based on glucan and xylan.

Keywords: Biofuel; bioethanol; biomass conversion; simultaneous saccharification and cofermentation; hemicellulose; lignin

β-d-Xylosidase from Selenomonas ruminantium of glycoside hydrolase family 43 by Douglas B. Jordan; Xin-Liang Li; Christopher A. Dunlap; Terence R. Whitehead; Michael A. Cotta (pp. 93-104).

β-d-Xylosidase from the ruminal anaerobic bacterium, Selenomonas ruminantium (SXA), catalyzes hydrolysis of β-1,4-xylooligosacharides and has potential utility in saccharification processes. The enzyme, heterologously produced in Escherichia coli and purified to homogeneity, has an isoelectric point of approx 4.4, an intact N terminus, and a Stokes radius that defines a homotetramer. SXA denatures between pH 4.0 and 4.3 at 25°C and between 50 and 60°C at pH 5.3. Following heat or acid treatment, partially inactivated SXA exhibits lower k _cat values, but similar K _m values as untreated SXA. d-Glucose and d-xylose protect SXA from inactivation at high temperature and low pH.

Keywords: Fuel ethanol; glycohydrolase; hemicellulose; protein stability; saccharification; arabinofuranosidase; inhibitors; catalysis

Biodiesel fuel production by the transesterification reaction of soybean oil using immobilized lipase by Otávio L. Bernardes; Juliana V. Bevilaqua; Márcia C. M. R. Leal; Denise M. G. Freire; Marta A. P. Langone (pp. 105-114).

The enzymatic alcoholysis of soybean oil with methanol and ethanol was investigated using a commercial, immobilized lipase (Lipozyme RMIM). The effect of alcohol (methanol or ethanol), enzyme concentration, molar ratio of alcohol to soybean oil, solvent, and temperature on biodiesel production was determined. The best conditions were obtained in a solvent-free system with ethanol/oil molar ratio of 3.0, temperature of 50°C, and enzyme concentration of 7.0% (w/w). Three-step batch ethanolysis was most effective for the production of biodiesel. Ethyl esters yield was about 60% after 4 h of reaction.

Keywords: Enzyme; ethanol; immobilized lipase; methanol; solvent; soybean oil

Thermoinactivation mechanism of glucose isomerase by Leng Hong Lim; Bradley A. Saville (pp. 115-130).

In this article, the mechanisms of thermoinactivation of glucose isomerase (GI) from Streptomyces rubiginosus (in soluble and immobilized forms) were investigated, particularly the contributions of thiol oxidation of the enzyme’s cysteine residue and a “Maillard-like” reaction between the enzyme and sugars in high fructose corn syrup (HFCS). Soluble GI (SGI) was successfully immobilized on silica gel (13.5 µm particle size), with an activity yield between 20 and 40%. The immobilized GI (IGI) has high enzyme retention on the support during the glucose isomerization process. In batch reactors, SGI (half-life =145 h) was more stable than IGI (half-life =27 h) at 60°C in HFCS, whereas at 80°C, IGI (half-life =12 h) was more stable than SGI (half-life =5.2 h). IGI was subject to thiol oxidation at 60°C, which contributed to the enzyme’s deactivation. IGI was subject to thiol oxidation at 80°C, but this did not contribute to the deactivation of the enzyme. SGI did not undergo thiol oxidation at 60°C, but at 80°C SGI underwent severe precipitation and thiol oxidation, which caused the enzyme to deactivate. Experimental results show that immobilization suppresses the destabilizing effect of thiol oxidation on GI. A “Maillard-like” reaction between SGI and the sugars also caused SGI thermoinactivation at 60, 70, and 80°C, but had minimal effect on IGI. At 60 and 80°C, IGI had higher thermostability in continuous reactors than in batch reactors, possibily because of reduced contact with deleterious compounds in HFCS.

Keywords: Deactivation; immobilized enzyme; kinetics; silica gel; thermostability; glucose isomerase

Measuring cellulase activity by Tor Soren Nordmark; Alan Bakalinsky; Michael H. Penner (pp. 131-139).

An approach is presented for obtaining relative filter paper activities for enzyme preparations having activities below that required for application of the traditional International Union of Pure and Applied Chemistry filter paper assay. The approach involves the utilization of protein stabilizers to retard the time-dependent enzyme inactivation that may occur under traditional filter paper assay conditions. Enzyme stabilization allows extended reaction times and the calculation of relative activities based on the time required for saccharification of 3.6% of the traditional substrate, making results proportional to those obtained in the traditional International Union of Pure and Applied Chemistry assay. The assay is demonstrated using a commercial cellulase preparation along with KCl and bovine serum albumin as protein stabilizers.

Keywords: Assay; cellulase; filter paper

Enzymatic hydrolysis optimization to ethanol production by simultaneous saccharification and fermentation by Mariana Peñuela Vásquez; Juliana Nascimento C. da Silva; Maurício Bezerra de Souza; Nei Pereira (pp. 141-153).

There is tremendous interest in using agro-industrial wastes, such as cellulignin, as starting materials for the production of fuels and chemicals. Cellulignin are the solids, which result from the acid hydrolysis of the sugarcane bagasse. The objective of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cellulignin, and to study its fermentation to ethanol using Saccharomyces cerevisiae. Cellulose conversion was optimized using response surface methods with pH, enzyme loading, solid percentage, and temperature as factor variables. The optimum conditions that maximized the conversion of cellulose to glucose, calculated from the initial dried weight of pretreated cellulignin, (43°C, 2%, and 24.4 FPU/g of pretreated cellulignin) such as the glucose concentration (47°C, 10%, and 25.6 FPU/g of pretreated cellulignin) were found. The desirability function was used to find conditions that optimize both, conversion to glucose and glucose concentration (47°C, 10%, and 25.9 FPU/g of pretreated cellulignin). The resulting enzymatic hydrolyzate was fermented yielding a final ethanol concentration of 30.0 g/L, in only 10 h, and reaching a volumetric productivity of 3.0 g/L·h, which is close to the values obtained in the conventional ethanol fermentation of sugar cane juice (5.0–8.0 g/L·h) in Brazil.

Keywords: Cellulignin; enzymatic hydrolysis; ethanol production; sugarcane bagasse; cellulases; simultaneous saccharification; saccharomyces cerevisiae

Filter paper degrading ability of a Trichoderma strain with multinucleate conidia by Hideo Toyama; Makiko Yano; Takeshi Hotta; Nobuo Toyama (pp. 155-160).

The multinucleate conidia were produced from the green mature conidia of Trichoderma reesei Rut C-30 strain by colchicine treatment. The strain with higher Filter paper degrading ability was selected among those conidia using a double layer selection medium. The selected strain, JS-2 was able to collapse the filter paper within 15 min but the original strain took 25 min to collapse it completely. Moreover, the amount of reducing sugar in the L-type glass tube of the strain, JS-2 was greater than that of the original strain. The Avicel, CMC-Na, and Salicin hydrolyzing activity of the strain, JS-2, increased 2.1 times, 1.2 times, and 3.6 times higher than that of the original strain.

Keywords: Cellulase; cellulose; conidia; nuclei; Trichoderma ; filter paper

Use of glucose oxidase in a membrane reactor for gluconic acid production by Luiz Carlos Martins das Neves; Michele Vitolo (pp. 161-170).

This article aims at the evaluation of the catalytic performance of glucose oxidase (GO) (EC.1.1.3.4) for the glucose/gluconic acid conversion in the ultrafiltration cell type membrane reactor (MB-CSTR). The reactor was coupled with a Millipore ultrafiltration-membrane (cutoff of 100 kDa) and operated for 24 h under agitation of 100 rpm, pH 5.5, and 30°C. The experimental conditions varied were the glucose concentration (2.5, 5.0, 10.0, 20.0, and 40.0 mM), the feeding rate (0.5, 1.0, 3.0, and 6.0/h), dissolved oxygen (8.0 and 16.0 mg/L), GO concentration (2.5, 5.0, 10.0, and 20.0 U_GO/mL), and the glucose oxidase/catalase activity ratio (U_GO/U_CAT)(1:0, 1:10, 1:20, and 1:30). A conversion yield of 80% and specific reaction rate of 40 × 10⁻⁴ mmol/h·U_GO were attained when the process was carried out under the following conditions: D =3.0/h, dissolved oxygen =16.0 mg/L, [G] =40 mM, and (U_GO/U_CAT) = 1:20. A simplified model for explaining the inhibition of GO activity by hydrogen peroxide, formed during the glucose/gluconic acid conversion, was presented.

Keywords: Catalase; gluconic acid; glucose oxidase; membrane reactor; glucose; continuous process

Enzyme production by industrially relevant fungi cultured on coproduct from corn dry grind ethanol plants by Eduardo A. Ximenes; Bruce S. Dien; Michael R. Ladisch; Nathan Mosier; Michael A. Cotta; Xin-Liang Li (pp. 171-183).

Distillers dried grain with solubles (DDGS) is the major coproduct produced at a dry grind ethanol facility. Currently, it is sold primarily as a ruminant animal feed. DDGS is low cost and relatively high in protein and fiber contents. In this study, DDGS was investigated as carbon source for extracellular hydrolytic enzyme production. Two filamentous fungi, noted for their high cellulolytic and hemicellulolytic enzyme titers, were grown on DDGS: Trichoderma reesei Rut C-30 and Asper gillus niger NRRL 2001. DDGS was either used as delivered from the plant (untreated) or after being pretreated with hot water. Both microorganisms secreted a broad range of enzymes when grown on DDGS. Higher xylanase titers were obtained when cultured on hot water DDGS compared with growth on untreated DDGS. Maximum xylanase titers were produced in 4 d for A. niger and 8 d for T. reesei in shake flask cultures. Larger amounts of enzymes were produced in bioreactors (5 L) either equipped with Rushton (for T. reesei) or updraft marine impellers (A. niger). Initial production titers were lower for bioreactor than for flask cultures, especially for T. reesei cultures. Improvement of enzyme titers were obtained using fed-batch feeding schemes.

Keywords: Aspergillus niger ; biomass; DDGS; cellulases; hemicellulases; Trichoderma reesei

Production of biosurfactant by Pseudomonas aeruginosa grown on cashew apple juice by Maria V. P. Rocha; Maria C. M. Souza; Sofia C. L. Benedicto; Márcio S. Bezerra; Gorete R. Macedo; Gustavo A. Saavedra Pinto; Luciana R. B. Gonçalves (pp. 185-194).

In this work, the ability of biosurfactant production by Pseudomonas aeruginosa in batch cultivation using cashew apple juice (CAJ) and mineral media was evaluated. P. aeruginosa was cultivated in CAJ, which was supplemented with peptone (5.0 g/L) and nutritive broth. All fermentation assays were performed in Erlenmeyer flasks containing 300 mL, incubated at 30°C and 150 rpm. Cell growth (biomass and cell density), pH, and superficial tension were monitored vs time. Surface tension was reduced by 10.58 and 41% when P. aeruginosa was cultivated in nutrient broth and CAJ supplemented with peptone, respectively. These results indicated that CAJ is an adequate medium for growth and biosurfactant production. Best results of biosurfactant production were obtained when CAJ was supplemented with peptone.

Keywords: Biosurfactant; cashew apple juice; Pseudomonas aeruginosa ; raw materials; rhamnolipid; fermentation

Thermoascus aurantiacus CBHI/Cel7A production in Trichoderma reesei on alternative carbon sources by Zsuzsa Benkő; Eszter Drahos; Zsolt Szengyel; Terhi Puranen; Jari Vehmaanperä; Kati Réczey (pp. 195-204).

To develop functional enzymes in cellulose hydrolysis at or above 70°C the cellobiohydrolase (CBHI/Cel7A) of Thermoascus aurantiacus was cloned and expressed in Trichoderma reesei Rut-C30 under the strong cbh1 promoter. Cellulase production of the parental strain and the novel strain (RF6026) was examined in submerged fermentation experiments using various carbon sources, which were lactose, Solka Floc 200 cellulose powder, and steam pretreated corn stover. An industrially feasible production medium was used containing only distiller’s spent grain, KH₂PO₄, and (NH₄)₂SO₄. Enzyme production was followed by measurements of protein concentration, total cellulase enzyme activity (filter paper activity), β-glucosidase activity, CBHI activity, and endogenase I (EGI) activity. The Thermoascus CBHI/Cel7A activity was taken as an indication of the heterologous gene expression under the cbh1 promoter.

Keywords: Cellulose; fermentor; lactose; shake flask; Solka Floc 200; steam pretreated corn stover

Introduction to session 1B by Mariam B. Sticklen (pp. 205-205).

Heterologous Acidothermus cellulolyticus 1,4-β-endoglucanase E1 produced within the corn biomass converts corn stover into glucose by Callista Ransom; Venkatesh Balan; Gadab Biswas; Bruce Dale; Elaine Crockett; Mariam Sticklen (pp. 207-219).

Commercial conversion of lignocellulosic biomass to fermentable sugars requires inexpensive bulk production of biologically active cellulase enzymes, which might be achieved through direct production of these enzymes within the biomass crops. Transgenic corn plants containing the catalytic domain of Acidothermus cellulolyticus E1 endo-1,4-β glucanase and the bar bialaphos resistance coding sequences were generated after Biolistic® (BioRad Hercules, CA) bombardment of immature embryo-derived cells. E1 sequences were regulated under the control of the cauliflower mosaic virus 35S promoter and tobacco mosaic virus translational enhancer, and E1 protein was targeted to the apoplast using the signal peptide of tobacco pathogenesis-related protein to achieve accumulation of this enzyme. The integration, expression, and segregation of E1 and bar transgenes were demonstrated, respectively, through Southern and Western blotting, and progeny analyses. Accumulation of up to 1.13% of transgenic plant total soluble proteins was detected as biologically active E1 by enzymatic activity assay. The corn-produced heterologous E1 could successfully convert ammonia fiber explosion-pretreated corn stover polysaccharides into glucose as a fermentable sugar for ethanol production, confirming that the E1 enzyme is produced in its active form.

Keywords: Ammonia fiber explosion; biomass conversion; cellulose; ethanol; transgenic maize; endoglucanase

The impact of enzyme characteristics on corn stover fiber degradation and acid production during ensiled storage by Haiyu Ren; Tom L. Richard; Kenneth J. Moore (pp. 221-238).

Ensilage can be used to store lignocellulosic biomass before industrial bioprocessing. This study investigated the impacts of seven commercial enzyme mixtures derived from Aspergillus niger, Trichoderma reesei, and T. longibrachiatum. Treatments included three size grades of corn stover, two enzyme levels (1.67 and 5 IU/g dry matter based on hemicellulase), and various ratios of cellulase to hemicellulase (C:H). The highest C: H ratio tested, 2.38, derived from T. reesei, resulted in the most effective fermentation, with lactic acid as the dominant product. Enzymatic activity during storage may complement industrial pretreatment; creating synergies that could reduce total bioconversion costs.

Keywords: Biomass; cellulase; hemicellulase; silage; wet storage; pretreatment

Fractionation of Cynara cardunculus (cardoon) biomass by dilute-acid pretreatment by Mercedes Ballesteros; M. José Negro; Paloma Manzanares; Ignacio Ballesteros; Felicia Sáez; J. Miguel Oliva (pp. 239-252).

Cynara cardunculus L. (cardoon) is a Mediterranean perennial herb offering good potential as substrate for sustainable production of bioethanol. In this work the first approach to the study of dilute-acid pretreatment of cardoon biomass for biological conversion was made. The influence of temperature (160–200°C), acid concentration (0–0.2% [w/w]), and solid concentration (5–10% [w/v]) in the formation of free sugars and sugar decomposition products in the prehydrolyzate was studied using a response surface methodology. Results show a negative interaction effect between acid concentration and temperature in xylose recovery yield in prehydrolyzate, whereas dry matter concentration does not exert a significant effect. Xylose recovery yield reaches a maximum of about 80% of the content in dry untreated raw material at 180°C and 0.1 or 0.2% acid addition. At these conditions the ratio of monomers found in prehydrolyzate in relation to total sugar yield for xylose is close to 100%. Furfural concentration, the major furan determined in the prehydrolyzate, increases as pretreatment severity rises. Maximum furfural yield of 4.2 g/100 g dry untreated raw material was found at 200°C and 0.2% acid concentration. The yield of furfural at the conditions in which maximum xylose recovery is attained is substantially lower, less than 2 g/100 g dry untreated raw material. This fact supports the idea of using moderate temperatures in dilute-acid processes, which at the same time provides reasonably high sugar recovery yield and avoids high inhibitory products formation.

Keywords: Acid prehydrolyzate; furfural; pretreatment optimization; surface-response methodology; xylose recovery; herbaceous crop

Heat extraction of corn fiber hemicellulose by Zsuzsa Benkő; Alexandra Andersson; Zsolt Szengyel; Melinda Gáspár; Kati Réczey; Henrik Stålbrand (pp. 253-265).

Water-soluble hemicellulose was extracted from corn fiber with microwave-assisted heat treatment. The effects of treatment temperature and initial pH of the aqueous extraction media were investigated regarding hemicellulose recovery and molecular mass of the isolated polysaccharides. In treatments carried out at neutral pH (simple water extraction), it has been demonstrated that hemicellulose recovery could be increased by applying higher treatment temperatures. However, the molecular weight of isolated hemicellulose gets significantly lower. For example, 10% of the raw materials’ xylan was extracted at 160 °C and about 30% recovery was reached at 210°C. However, the molecular mass of the isolated polysaccharide at 210°C (5.82 × 10⁴) was about half of that measured at 160°C (1.37 × 10⁵). Reducing the pH with sulfuric acid resulted in shorter polymer chains (1.7 × 10⁴) and lower hemicellulose yields (2.2%). Application of sodium hydroxide in the treatment showed that, compared with acid, considerably higher yields (11%) with longer polysaccharide chains (1.3 × 10⁵) could be obtained.

Keywords: Alkaline extraction; carbohydrate analysis; microwave-assisted fractionation; size exclusion chromatography; weight-average molecular weight; maize

An evaluation of british columbian beetle-killed hybrid spruce for bioethanol production by Alex Berlin; Claudio Muñoz; Neil Gilkes; Sepideh Massoumi Alamouti; Pablo Chung; Kyu-Young Kang; Vera Maximenko; Jaime Baeza; Juanita Freer; Regis Mendonça; Jack Saddler (pp. 267-280).

The development of bioconversion technologies for production of fuels, chemicals, and power from renewable resources is currently a high priority for developed nations such as the United States, Canada, and the European Union as a way to improve national energy security and reduce greenhouse gas emissions. The widespread implementation of such technologies will require a sustainable supply of biomass from forestry and agriculture. Forests are a major source of feedstocks for biofuels production in Canada. Woody biomass includes residues from logging and forest thinning, and from wood processing and pulp production.More recently, damaged wood caused by beetle infestations has become available on a large scale in Western Canada. This study evaluates beetle-killed British Columbian hybrid spruce (HS) (Picea glauca × P. engelmannii) as a feedstock for the production of bioethanol. In the past 30 yr, attack by the beetle Dendroctonus rufipennis and associated fungi has resulted in estimated losses of more than three billion board feet in British Columbia alone. Here we describe the chemical and some physical characteristics of both healthy (HHS) and beetle-killed (BKHS) British Columbian HS and evaluate the technical feasibility of using these feedstocks as a source of biomass for bioethanol production. Untreated HHS and BKHS did not differ significantly in chemical composition except for the moisture content, which was significantly lower in BKHS (approx 10%) compared with HHS (approx 18%). However, the yields of carbohydrates in hydrolyzable and fermentable forms were higher at mild pretreatment conditions (H-Factor <1000) for BKHS compared with HHS. At medium (H-Factor 1000–2000) and severe (H-Factor >2000) pretreatment conditions HHS and BKHS behaved similarly. Organosolv pretreated HHS and BKHS demonstrated good ethanol theoretical yields, approx 70 and 80%, respectively.

Keywords: Cellulase; enzyme; ethanol; hydrolysis; lignocellulose; fermentation

Production of cellulolytic and hemicellulolytic enzymes from Aureobasidium pulluans on solid state fermentation by Rodrigo Simões Ribeiro Leite; Daniela Alonso Bocchini; Eduardo Da Silva Martins; Dêenis Silva; Eleni Gomes; Roberto Da Silva (pp. 281-288).

This article investigates a strain of the yeast Aureobasidium pullulans for cellulase and hemicellulase production in solid state fermentation. Among the substrates analyzed, the wheat bran culture presented the highest enzymatic production (1.05 U/mL endoglucanase, 1.3 U/mL β-glucosidase, and 5.0 U/mL xylanase). Avicelase activity was not detected. The optimum pH and temperature for xylanase, endoglucanase and β-glucosidase were 5.0 and 50, 4.5 and 60, 4.0 and 75°C, respectively. These enzymes remained stable between a wide range of pH. The β-glucosidase was the most thermostable enzyme, remaining 100% active when incubated at 75°C for 1 h.

Keywords: Cellulases; endoglucanase; hemicellulases; solid state fermentation; β-glucosidase

The effect of particle size on hydrolysis reaction rates and rheological properties in cellulosic slurries by Rajesh K. Dasari; R. Eric Berson (pp. 289-299).

The effect of varying initial particle sizes on enzymatic hydrolysis rates and rheological properties of sawdust slurries is investigated. Slurries with four particle size ranges (33 µm<x≤75 µm, 150 µm<x≤180 µm, 295 µm<x≤425 µm, and 590 µm<x≤850 µm) were subjected to enzymatic hydrolysis using an enzyme dosage of 15 filter paper units per gram of cellulose at 50°C and 250 rpm in shaker flasks. At lower initial particle sizes, higher enzymatic reaction rates and conversions of cellulose to glucose were observed. After 72 h 50 and 55% more glucose was produced from the smallest size particles than the largest size ones, for initial solids concentration of 10 and 13% (w/w), respectively. The effect of initial particle size on viscosity over a range of shear was also investigated. For equivalent initial solids concentration, smaller particle sizes result in lower viscosities such that at a concentration of 10% (w/w), the viscosity decreased from 3000 cP for 150 µm<x≤180 µm particle size slurries to 61.4 cP for 33 µm<x≤75 µm particle size slurries. Results indicate particle size reduction may provide a means for reducing the long residence time required for the enzymatic hydrolysis step in the conversion of biomass to ethanol. Furthermore, the corresponding reduction in viscosity may allow for higher solids loading and reduced reactor sizes during large-scale processing.

Keywords: Biomass; enzymatic hydrolysis; non-Newtonian; particle suspension; red oak wood; sawdust slurry; viscosity

Effect of dissolved carbon dioxide on accumulation of organic acids in liquid hot water pretreated biomass hydrolyzates by G. Peter Van Walsum; Maurilio Garcia-Gil; Shou-Feng Chen; Kevin Chambliss (pp. 301-311).

Liquid hot water pretreatment has been proposed as a possible means of improving rates of enzymatic hydrolysis of biomass while maintaining low levels of inhibitory compounds. Supplementation of liquid hot water pretreatment with dissolved carbon dioxide, yielding carbonic acid, has been shown to improve hydrolysis of some biomass substrates compared with the use of water alone. Previous studies on the application of carbonic acid to biomass pretreatment have noted a higher pH of hydrolyzates treated with carbonic acid as compared with the samples prepared with water alone. This study has applied recently developed analytical methods to quantify the concentration of organic acids in liquid hot water pretreated hydrolyzates, prepared with and without the addition of carbonic acid. It was observed that the addition of carbon dioxide to liquid hot water pretreatment significantly changed the accumulated concentrations of most measured compounds. However, the measured differences in product concentrations resulting from addition of carbonic acid did not account for the measured differences in hydrolyzate pH.

Keywords: Analysis; aspen wood; carbonic acid; corn stover; pretreatment; organic acids

Separation of glucose and pentose sugars by selective enzyme hydrolysis of AFEX-treated corn fiber by Robert J. Hanchar; Farzaneh Teymouri; Chandra D. Nielson; Darold McCalla; Mark D. Stowers (pp. 313-325).

A process was developed to fractionate corn fiber into glucose- and pentose-rich fractions. Corn fiber was ammonia fiber explosion treated at 90°C, using 1 g anhydrous ammonia pergram of drybiomass, 60% moisture, and 30-min residence time. Twenty four hour hydrolysis of ammonia fiber explosion-treated corn fiber with cellulase converted 83% of available glucanto-glucose. In this hydrolysis the hemicellulose was partially broken down with 81% of the xylan and 68% of the arabinan being contained in the hydrolysate after filtration to remove lignin and other insoluble material. Addition of ethanol was used to precipitate and recover the solubilized hemicellulose from the hydrolysate, followed by hydrolysis with 2% (v/v) sulfuric acid to convert the recovered xylan and arabinan to monomeric sugars. Using this method, 57% of xylose and 54% of arabinose available in corn fiber were recovered in a pentose-rich stream. The carbohydrate composition of the pentose-enriched stream was 5% glucose, 57% xylose, 27% arabinose, and 11% galactose. The carbohydrate composition of the glucose-enriched stream was 87% glucose, 5% xylose, 6% arabinose, and 1% galactose, and contained 83% of glucose available from the corn fiber.

Keywords: Ammonia fiber explosion; arabinose; lignocellulose; sugar separation; xylose; glucose

The potential in bioethanol production from waste fiber sludges in pulp mill-based biorefineries by Anders Sjöde; Björn Alriksson; Leif J. Jönsson; Nils-Olof Nilvebrant (pp. 327-337).

Industrial production of bioethanol from fibers that are unusable for pulp production in pulp mills offers an approach to product diversification and more efficient exploitation of the raw material. In an attempt to utilize fibers flowing to the biological waste treatment, selected fiber sludges from three different pulp mills were collected, chemically analyzed, enzymatically hydrolyzed, and fermented for bioethanol production. Another aim was to produce solid residues with higher heat values than those of the original fiber sludges to gain a better fuel for combustion. The glucan content ranged between 32 and 66% of the dry matter. The lignin content varied considerably (1–25%), as did the content of wood extractives (0.2–5.8%). Hydrolysates obtained using enzymatic hydrolysis were found to be readily fermentable using Saccharomyces cerevisiae. Hydrolysis resulted in improved heat values compared with corresponding untreated fiber sludges. Oligomeric xylan fragments in the solid residue obtained after enzymatic hydrolysis were identified using matrix-assisted laser desorption ionization-time of flight and their potential as a new product of a pulp mill-based biorefinery is discussed.

Keywords: Bioethanol; biorefinery; fiber sludge; lignocellulose; Saccharomyces cerevisiae ; xylan

Dilute sulfuric acid pretreatment of agricultural and agro-industrial residues for ethanol production by Carlos Martin; Björn Alriksson; Anders Sjöde; Nils-Olof Nilvebrant; Leif J. Jönsson (pp. 339-352).

The potential of dilute-acid prehydrolysis as a pretreatment method for sugarcane bagasse, rice hulls, peanut shells, and cassava stalks was investigated. The prehydrolysis was performed at 122°C during 20, 40, or 60 min using 2% H₂SO₄ at a solid-to-liquid ratio of 1:10. Sugar formation increased with increasing reaction time. Xylose, glucose, arabinose, and galactose were detected in all of the prehydrolysates, whereas mannose was found only in the prehydrolysates of peanut shells and cassava stalks. The hemicelluloses of bagasse were hydrolyzed to a high-extent yielding concentrations of xylose and arabinose of 19.1 and 2.2 g/L, respectively, and a xylan conversion of more than 80%. High-glucose concentrations (26–33.5 g/L) were found in the prehydrolysates of rice hulls, probably because of hydrolysis of starch of grain remains in the hulls. Peanut shells and cassava stalks rendered low amounts of sugars on prehydrolysis, indicating that the conditions were not severeenough to hydrolyze the hemicelluloses in these materials quantitatively. All prehydrolysates were readily fermentable by Saccharomyces cerevisiae. The dilute-acid prehydrolysis resulted in a 2.7- to 3.7-fold increase of the enzymatic convertibility of bagasse, but was not efficient for improving the enzymatic hydrolysis of peanut shells, cassava stalks, or rice hulls.

Keywords: Bagasse; ethanol; acid hydrolysis; pretreatment; enzymatic hydrolysis; agricultural residues

Xylanase contribution to the efficiency of cellulose enzymatic hydrolysis of barley straw by María P. García-Aparicio; Mercedes Ballesteros; Paloma Manzanares; Ignacio Ballesteros; Alberto González; M. José Negro (pp. 353-365).

In this study, different enzyme preparations available from Novozymes were assessed for their efficiency to hydrolyze lignocellulosic materials. The enzyme mixture was evaluated on a pretreated cellulose-rich material, and steam-exploded barley straw pretreated under different temperatures (190, 200, and 210°C, respectively) in order to produce fermentable sugars. Results show that xylanase supplementation improves initial cellulose hydrolysis effectiveness of water-insoluble solid fraction from all steam-exploded barley straw samples, regardless of the xylan content of substrate. The mixture constituted by cellulase: β-glucosidase: endoxylanase of the new kit for lignocellulose conversion at a ratio 10:1:5% ([v/w], enzyme [E]/substrate [S]) provides the highest increment of cellulose conversion in barley straw pretreated at 210°C, for 10 min.

Keywords: Agricultural residues; cellulose conversion; lignocellulose; pretreatment; xylanase supplementation; steam explosion

Effect of organosolv ethanol pretreatment variables on physical characteristics of hybrid poplar substrates by Xuejun Pan; Dan Xie; Kyu-Young Kang; Seung-Lak Yoon; Jack N. Saddler (pp. 367-377).

Hybrid poplar (Populus nigra × P. maximowiczii) chips were pretreated using an organosolv ethanol process. The effect of pretreatment conditions (temperature, time, catalyst, and ethanol concentration) on the substrate characteristics, including fiber size, crystallinity, and degree of polymerization of cellulose, was investigated using an experimental matrix designed with response surface methodology. The conditions ranged 155–205°C, 26–94 min, 0.83–1.67% catalyst (H₂SO₄) on oven-dry wood chip (w/w), and 25–75% ethanol concentration (v/v). The results indicated that the substrate characteristics are controllable and predictable. Desirable substrates can be prepared by fine-tuning the processing parameters. The regression models developed, allowed the quantitative prediction of the substrate characteristics from the pretreatment conditions used.

Keywords: Crystallinity; degree of polymerization; fiber size; hybrid poplar; organosolv ethanol pretreatment; substrate characteristics

Liquid hot water pretreatment of olive tree pruning residues by Cristóbal Cara; Inmaculada Romero; Jose Miguel Oliva; Felicia Sáez; Eulogio Castro (pp. 379-394).

Olive tree pruning generates an abundant, renewable lignocellulose residue, which is usually burnt on fields to prevent propagation of vegetal diseases, causing economic costs and environmental concerns. As a first step in an alternative use to produce fuel ethanol, this work is aimed to study the pretreatment of olive tree pruning residues by liquid hot water. Pretreatment was carried out at seven temperature levels in the range 170–230°C for 10 or 60 min. Sugar recoveries in both solid and liquid fractions resulting from pretreatment as well as enzymatic hydrolysis yield of the solid were used to evaluate pretreatment performance. Results show that the enzyme accessibility of cellulose in the pretreated solid fraction increased with pretreatment time and temperature, although sugar degradation in the liquid fraction was concomitantly higher.

Keywords: Biomass; enzymatic hydrolysis; ethanol; liquid hot water pretreatment; olive tree residues; glucose

Ammonia fiber expansion pretreatment and enzymatic hydrolysis on two different growth stages of reed canarygrass by Tamika C. Bradshaw; Hasan Alizadeh; Farzaneh Teymouri; Venkatesh Balan; Bruce E. Dale (pp. 395-405).

Plant materials from the vegetative growth stage of reed canarygrass and the seed stage of reed canarygrass are pretreated by ammonia fiber expansion (AFEX) and enzymatically hydrolyzed using 15 filter paper units (FPU) cellulase/g glucan to evaluate glucose and xylose yields. Percent conversions of glucose and xylose, effects of temperature and ammonia loading, and hydrolysis profiles are analyzed to determine the most effective AFEX treatment condition for each of the selected materials. The controls used in this study were untreated samples of each biomass material. All pretreatment conditions tested enhanced enzyme digestibility and improved sugar conversions for reed canarygrass compared with their untreated counterparts. Based on 168 h hydrolysis results using 15 FPU Spezyme CP cellulase/g glucan the most effective AFEX treatment conditions were determined as: vegetative growth stage of reed canarygrass—100°C, 60% moisture content, 1.2: 1 kg ammonia/kg of dry matter (86% glucose and 78% xylose) and seed stage of reed canarygrass—100°C, 60% moisture content, 0.8 : 1 kg ammonia/kg of dry matter (89% glucose and 81% xylose). Supplementation by commercial Multifect 720 xylanase along with cellulase further increased both glucose and xylose yields by 10–12% at the most effective AFEX conditions.

Keywords: Ammonia fiber expansion; biomass; enzymatic hydrolysis; pretreatment; reed canary grass; cellulosic ethanol

Mitigation of cellulose recalcitrance to enzymatic hydrolysis by ionic liquid pretreatment by Anantharam P. Dadi; Constance A. Schall; Sasidhar Varanasi (pp. 407-421).

Efficient hydrolysis of cellulose-to-glucose is critically important in producing fuels and chemicals from renewable feedstocks. Cellulose hydrolysis in aqueous media suffers from slow reaction rates because cellulose is a water-insoluble crystalline biopolymer. The high-crystallinity of cellulose fibrils renders the internal surface of cellulose inaccessible to the hydrolyzing enzymes (cellulases) as well as water. Pretreatment methods, which increase the surface area accessible to water and cellulases are vital to improving the hydrolysis kinetics and conversion of cellulose to glucose. In a novel technique, the microcrystalline cellulose was first subjected to an ionic liquid (IL) treatment and then recovered as essentially amorphous or as a mixture of amorphous and partially crystalline cellulose by rapidly quenching the solution with an antisolvent. Because of their extremely low-volatility, ILs are expected to have minimal environmental impact. Two different ILs, 1-n-butyl-3-methylimidazolium chloride (BMIMC1) and 1-allyl-3-methylimidazolium chloride (AMIMC1) were investigated. Hydrolysis kinetics of the IL-treated cellulose is significantly enhanced. With appropriate selection of IL treatment conditions and enzymes, the initial hydrolysis rates for IL-treated cellulose were up to 90 times greater than those of untreated cellulose. We infer that this drastic improvement in the “overall hydrolysis rates” with IL-treated cellulose is mainly because of a significant enhancement in the kinetics of the “primary hydrolysis step” (conversion of solid cellulose to soluble oligomers), which is the rate-limiting step for untreated cellulose. Thus, with IL-treated cellulose, primary hydrolysis rates increase and become comparable with the rates of inherently faster “secondary hydrolysis” (conversion of soluble oligomers to glucose).

Keywords: Cellulose; enzymatic hydrolysis; ionic-liquid; pretreatment crystallinity index; initial rates; reducing sugars

Evaluation of different biomass materials as feedstock for fermentable sugar production by Yi Zheng; Zhongli Pan; Ruihong Zhang; John M. Labavitch; Donghai Wang; Sarah A. Teter; Bryan M. Jenkins (pp. 423-435).

Saline crops and autoclaved municipal organic solid wastes were evaluated for their potential to be used as feedstock for fermentable sugar production through dilute acid pretreatment and enzymatic hydrolysis. The saline crops included two woods, athel (Tamarix aphylla L) and eucalyptus (Eucalyptus camaldulensis), and two grasses, Jose tall wheatgrass (Agropyron elongatum), and creeping wild rye (Leymus triticoides). Each of the biomass materials was first treated with dilute sulfuric acid under selected conditions (acid concentration =1.4% (w/w), temperature =165°C, and time =8 min) and then treated with the enzymes (cellulases and β-glucosidase). The chemical composition (cellulose, hemicellulose, and lignin contents) of each biomass material and the yield of total and different types of sugars after the acid and enzyme treatment were determined. The results showed that among the saline crops evaluated, the two grasses (creeping wild rye and Jose tall wheatgrass) had the highest glucose yield (87% of total cellulose hydrolyzed) and fastest reaction rate during the enzyme treatment. The autoclaved municipal organic solid wastes showed reasonable glucose yield (64%). Of the two wood species evaluated, Athel has higher glucose yield (60% conversion of cellulose) than eucalyptus (38% conversion of cellulose).

Keywords: Dilute acid pretreatment; enzymatic hydrolysis; ethanol potential; municipal solid waste; saline crops; fermentable sugar

Techno-economic analysis of biocatalytic processes for production of alkene epoxides by Abhijeet P. Borole; Brian H. Davison (pp. 437-449).

A techno-economic analysis of two different bioprocesses was conducted, one for the conversion of propylene to propylene oxide (PO) and other for conversion of styrene to styrene epoxide (SO). The first process was a lipase-mediated chemo-enzymatic reaction, whereas the second one was a one-step enzymatic process using chloroperoxidase. The PO produced through the chemo-enzymatic process is a racemic product, whereas the latter process (based on chloroperoxidase) produces an enantio-pure product. The former process thus falls under the category of high-volume commodity chemical (PO); whereas the latter is a low-volume, high-value product (SO).A simulation of the process was conducted using the bioprocess engineering software SuperPro Designer v6.0 (Intelligen, Inc., Scotch Plains, NJ) to determine the economic feasibility of the process. The purpose of the exercise was to compare biocatalytic processes with existing chemical processes for production of alkene expoxides. The results show that further improvements are needed in improving biocatalyst stability to make these bioprocesses competitive with chemical processes.

Keywords: Bioprocess; chloroperoxidase; economics; enzymatic; epoxides; lipase

Evaluation of liquid-liquid extraction process for separating acrylic acid produced from renewable sugars by M. E. T. Alvarez; E. B. Moraes; A. B. Machado; R. Maciel Filho; M. R. Wolf-Maciel (pp. 451-461).

In this article, the separation and the purification of the acrylic acid produced from renewable sugars were studied using the liquid-liquid extraction process. Nonrandom two-liquids and universal quasi-chemical models and the prediction method universal quasi-chemical functional activity coefficients were used for generating liquid-liquid equilibrium diagrams for systems made up of acrylic acid, water, and solvents (diisopropyl ether, isopropyl acetate, 2-ethyl hexanol, and methyl isobutyl ketone) and the results were compared with available liquid-liquid equilibrium experimental data. Aspen Plus (Aspen Technology, Inc., version 2004.1) software was used for equilibrium and process calculations. High concentration of acrylic acid was obtained in this article using diisopropyl ether as solvent.

Keywords: Acrylic acid recovery; liquid-liquid extraction; separation; simulation; thermodynamic characterization; renewable sugars

Enhancement of rhamnoplipid production in residual soybean oil by an isolated strain of Pseudomonas aeruginosa by C. J. B. De Lima; F. P. França; E. F. C. Sérvulo; M. M. Resende; V. L. Cardoso (pp. 463-470).

In the present work, the production of rhamnolipid from residual soybean oil (RSO) from food frying facilities was studied using a strain of Pseudomonas aeruginosa of contaminated lagoon, isolated from a hydrocarbon contaminated soil. The optimization of RSO, ammonium nitrate, and brewery residual yeast concentrations was accomplished by a central composite expeirmental design and surface response analysis. The experiments were performed in 500-mL Erlenmeyer flasks containing 50 mL of mineral medium, at 170 rpm and 30 ± 1°C, for a 48-h fermentation period. Rhamnolipid production has been monitored by measurements of surface tension, rhamnose concentration, and emulsifying activity. The best-planned results, located on the central point, have corresponded to 22 g/L of RSO, 5.625 g/L of NH₄NO₃, and 11.5 g/L of brewery yeast. At the maximum point the values for rhamnose and emulsifying index were 2.2 g/L and 100%, respectively.

Keywords: Biosurfactant; experimental design; Pseudomonas aeruginosa ; rhamnolipids; surface tension; soybean oil

Optimizing carbon/nitrogen ratio for biosurfactant production by a Bacillus subtilis strain by R. R. Fonseca; A. J. R. Silva; F. P. De França; V. L. Cardoso; E. F. C. Sérvulo (pp. 471-486).

A Bacillus subtilis strain isolated from contaminated soil from a refinery has been screened for biosurfactant production in crystal sugar (sucrose) with different nitrogen sources (NaNO₃, (NH₄)₂SO₄, urea, and residual brewery yeast). The highest reduction in surface tension was achieved with a 48-h fermentation of crystal sugar and ammonium nitrate. Optimization of carbon/nitrogen ratio (3, 9, and 15) and agitation rate (50, 150, and 250 rpm) for biosurfactant production was carried out using complete factorial design and response surface analysis. The condition of C/N 3 and 250 rpm allowed the maximum increase in surface activity of biosurfactant. A suitable model has been developed, having presented great accordance experimental data. Preliminary characterization of the bioproduct suggested it to be a lipopeptide with some isomers differing from those of a commercial surfactin.

Keywords: Biosurfactant/production; crystal sugar; lipopeptide; surfaceactive substances; surfactin; Bacillus subtilis

A new process for acrylic acid synthesis by fermentative process by B. H. Lunelli; E. R. Duarte; E. C. Vasco de Toledo; M. R. Wolf Maciel; R. Maciel Filho (pp. 487-499).

With the synthesis of chemical products through biotechnological processes, it is possible to discover and to explore innumerable routes that can be used to obtain products of high added value. Each route may have particular advantages in obtaining a desired product, compared with others, especially in terms of yield, productivity, easiness to separate the product, economy, and environmental impact. The purpose of this work is the development of a deterministic model for the biochemical synthesis of acrylic acid in order to explore an alternative process. The model is built-up with the tubular reactor equations together with the kinetic representation based on the structured model. The proposed process makes possible to obtain acrylic acid continuously from the sugar cane fermentation.

Keywords: Acrylic acid; biotechnological processes; bidimensional model; dynamic reduced model; modeling; Saccharomyces cerevisiae ; tubular bioreactor

Ethanol/water pulps from sugar cane straw and their biobleaching with xylanase from Bacillus pumilus by Regina Y. Moriya; Adilson R. Gonçalves; Marta C. T. Duarte (pp. 501-513).

The influence of independent variables (temperature and time) on the cooking of sugar cane straw with ethanol/water mixtures was studied to determine operating conditions that obtain pulp with high cellulose contents and a low lignin content. An experimental 2² design was applied for temperatures of 185 and 215°C, and time of 1 and 2.5 h with the ethanol/water mixture concentration and constant straw-to-solvent ratio. The system was scaled-up at 200°C cooking temperature for 2 h with 50% ethanol-water concentration, and 1:10 (w/v) straw-to-solvent ratio to obtain a pulp with 3.14 cP viscosity, 58.09 kappa-number, and the chemical composition of the pulps were 3.2% pentosan and 31.5% lignin. Xylanase from Bacillus pumilus was then applied at a loading of 5–150 IU/g dry pulp in the sugar cane straw ethanol/water pulp at 50°C for 2 and 20 h. To ethanol/water pulps, the best enzyme dosage was found to be 20 IU/g dry pulp at 20 h, and a high enzyme dosage of 150 IU/g dry pulp did not decrease the kappa-number of the pulp.

Keywords: Bacillus pumilus ; biobleaching; ethano/water pulp; organosolv pulping; sugarcane straw; xylanase

Nisin production utilizing skimmed milk aiming to reduce process cost by Angela Faustino Jozala; Maura Sayuri de Andrade; Luciana Juncioni de Arauz; Adalberto Pessoa; Thereza Christina Vessoni Penna (pp. 515-528).

Nisin is a natural additive for conservation of food, pharmaceutical, and dental products and can be used as a therapeutic agent. Nisin inhibits the outgrowth of spores, the growth of a variety of Gram-positive and Gram-negative bacteria. This study was performed to optimize large-scale nisin production in skimmed milk and subproducts aiming at low-costs process and stimulating its utilization. Lactococcus lactis American Type Culture Collection (ATCC) 11454 was developed in a rotary shaker (30°C/36 h/100 rpm) in diluted skimmed milk and nisin activity, growth parameters, and media components were also studied. Nisin activity in growth media was expressed in arbitrary units (AU/mL) and converted to standard nisin concentration (Nisaplin®, 25 mg of pure nisin is 1.0×10⁶ AU/mL). Nisin activity in skimmed milk 2.27 g_{total solids} was up to threefold higher than transfers in skimmed milk 4.54 g_{total solids} and was up to 85-fold higher than transfers in skimmed milk 1.14 g_{total solids}. L. lactis was assayed in a New Brunswick fermentor with 1.5 L of diluted skimmed milk (2.27 g_{total solids}) and airflow of 1.5 mL/min (30°C/36/200 rpm), without pH control. In this condition nisin activity was observed after 4 h (45.07 AU/mL) and in the end of 36 h process (3312.07 AU/mL). This work shows the utilization of a low-cost growth medium (diluted skimmed milk) to nisin production with wide applications. Furthermore, milk subproducts (milk whey) can be exploited in nisin production, because in Brazil 50% of milk whey is disposed with no treatment in rivers and because of high organic matter concentrations it is considered an important pollutant. In this particular case an optimized production of an antimicrobial would be lined up with industrial disposal recycling.

Keywords: Artificial compounds; EDTA; fermentation processes; Gram-negative; Gram-positive; Lactococcus lactis ; nisin

Bacterial cellulose production by Gluconacetobacter sp. PKY5 in a rotary biofilm contactor by Yong-Jun Kim; Jin-Nam Kim; Young-Jung Wee; Don-Hee Park; Hwa-Won Ryu (pp. 529-537).

A rotary biofilm contactor (RBC) inoculated with Gluconacetobacter sp. RKY5 was used as a bioreactor for improved bacterial cellulose production. The optimal number of disk for bacterial cellulose production was found to be eight, at which bacterial cellulose and cell concentrations were 5.52 and 4.98 g/L. When the aeration rate was maintained at 1.25 vvm, bacterial cellulose and cell concentrations were maximized (5.67 and 5.25 g/L, respectively). The optimal rotation speed of impeller in RBC was 15 rpm. When the culture pH in RBC was not controlled during fermentation, the maximal amount of bacterial cellulose (5.53 g/L) and cells (4.91 g/L) was obtained. Under the optimized culture conditions, bacterial cellulose and cell concentrations in RBC reached to 6.17 and 5.58 g/L, respectively.

Keywords: Bacterial cellulose; bioreactor; fermentation; Gluconacetobacter ; optimization; rotary biofilm contactor

Biosurfactant production by cultivation of Bacillus atrophaeus ATCC 9372 in semidefined glucose/casein-based media by Luiz Carlos Martins das Neves; Kátia Silva de Oliveira; Márcio Junji Kobayashi; Thereza Christina Vessoni Penna; Attilio Converti (pp. 539-554).

Biosurfactants are proteins with detergent, emulsifier, and antimicrobial actions that have potential application in environmental applications such as the treatment of organic pollutants and oil recovery. Bacillus atrophaeus strains are nonpathogenic and are suitable source of biosurfactants, among which is surfactin. The aim of this work is to establish a culture medium composition able to stimulate biosurfactants production by B. atrophaeus ATCC 9372. Batch cultivations were carried out in a rotary shaker at 150 rpm and 35°C for 24 h on glucose-and/or casein-based semidefined culture media also containing sodium chloride, dibasic sodium phosphate, and soy flour. The addition of 14.0 g/L glucose in a culture medium containing 10.0 g/L of casein resulted in 17 times higher biosurfactant production (B _max=635.0 mg/L). Besides, the simultaneous presence of digested casein (10.0 g/L), digested soy flour (3.0 g/L), and glucose (18.0 g/L) in the medium was responsible for a diauxic effect during cell growth. Once the diauxie started, the average biosurfactants concentration was 16.8% less than that observed before this phenomenon. The capability of B. atrophaeus strain to adapt its own metabolism to use several nutrients as energy sources and to preserve high levels of biosurfactants in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

Keywords: Bacillus atrophaeus ; batch cultivation; biosurfactant production; casein; glucose; kinetics

Evaluation of the pH- and thermal stability of the recombinant green fluorescent protein (GFP) in the presence of sodium chloride by Marina Ishii; Juliana Sayuri Kunimura; Hélio Tallon Jeng; Thereza Christina Vessoni Penna; Olivia Cholewa (pp. 555-571).

The thermal stability of recombinant green fluorescent protein (GFP) in sodium chloride (NaCl) solutions at different concentrations, pH, and temperatures was evaluated by assaying the loss of fluorescence intensity as a measure of denaturation. GFP, extracted from Escherichia coli cells by the three-phase partitioning method and purified through a butyl hydrophobic interaction chromatography (HIC) column, was diluted in water for injection (WFI) (pH 6.0–7.0) and in 10 mM buffer solutions (acetate, pH 5.0; phosphate, pH 7.0; and Tris-EDTA, pH 8.0) with 0.9–30% NaCl or without and incubated at 80–95°C. The extent of protein denaturation was expressed as a percentage of the calculated decimal reduction time (D-value). In acetate buffer (pH 4.84±0.12), the mean D-values for 90% reduction in GFP fluorescence ranged from 2.3 to 3.6 min, independent of NaCl concentration and temperature. GFP thermal stability diluted in WFI (pH 5.94±0.60) was half that observed in phosphate buffer (pH 6.08±0.60); but in both systems, D-values decreased linearly with increasing NaCl concentration, with D-values (at 80°C) ranging from 3.44, min (WFI) to 6.1 min (phosphate buffer), both with 30% NaCl. However, D-values in Tris-EDTA (pH 7.65±0.17) were directly dependent on the NaCl concentration and 5–10 times higher than D-values for GFP in WFI at 80°C. GFP pH- and thermal stability can be easily monitored by the convenient measure of fluorescence intensity and potentially be used as an indicator to monitor that processing times and temperatures were attained.

Keywords: Bioindicator; D-value; green fluorescent protein; pH-stability; sodium chloride; thermal stability

Carboxymethylcellulose obtained by ethanol/water organosolv process under acid conditions by Denise S. Ruzene; Adilson R. Gonçalves; José A. Teixeira; Maria T. Pessoa de Amorim (pp. 573-582).

Sugar cane bagasse pulps were obtained by ethanol/water organosolv process under acid and alkaline conditions. The best condition of acid pulping for the sugarcane bagasse was 0.02 mol/L sulfuric acid at 160°C, for 1 h, whereas the best condition for alkaline pulping was 5% sodium hydroxide (base pulp) at 160°C, for 3 h. For the residual lignin removal, the acid and alkaline pulps were submitted to a chemical bleaching using sodium chlorite. Pulps under acid and alkaline conditions bleached with sodium chlorite presented viscosities of 3.6 and 7.8 mPa·s, respectively, and μ-kappa numbers of 1.1 and 2.4, respectively. The pulp under acid condition, bleached with sodium chlorite was used to obtain carboxymethylcellulose (CMC). CMC yield was 35% (pulp based), showing mass gain after the carboxymethylation reaction corresponding to 23.6% of substitution or 0.70 groups −CH₂ COONa per unit of glucose residue. The infrared spectra showed the CMC characteristic bands and by the infrared technique it was possible to obtain a substitution degree (0.63), similar to the substitution degree calculated by mass gain (0.70).

Keywords: Acid and alkaline catalyzed ethanol pulping; carboxymethylcellulose; chemical bleaching; infrared spectra; sugarcane bagasse; Organosolv pulping

Response surface methodological approach for optimization of free fatty acid removal in feedstock by Gwi-Taek Jeong; Do-Heyoung Kim; Don-Hee Park (pp. 583-593).

Fatty acid methyl esters, also referred to as biodiesel, have been determined to have a great deal of potential as substitutes for petro-diesel. In order to optimize conversion yield in the biodiesel production process, feedstocks were previously recommended to be anhydrous, with a free fatty acid content of less than 0.5%. In this study, we removed free fatty acid from feedstock through the use of solid catalysts and response surface methodology. In order to optimize free fatty acid removal, response surface methodology was applied to delineate the effects of five-level-four-factors and their reciprocal interactions on free fatty acid removal. A total of 30 individual experiments were conducted, each of which was designed to study reaction temperature, reaction time, catalyst amounts, or methanol amounts. A statistical model was used to estimate that the optimal free fatty acid removal yield would be 100%, under the following optimized reaction conditions: a reaction temperature of 66.96°C, a catalyst amount of 12.66% (w/v), and a reaction time of 37.65 min. Using these optimal factor values under experimental conditions in three independent replicates, an average conversion yield was well achieved within the values predicted by the model.

Keywords: Biodiesel; free fatty acid; optimization; response surface methodology

Optimization of lipase-catalyzed synthesis of sorbitan acrylate using response surface methodology by Gwi-Taek Jeong; Don-Hee Park (pp. 595-609).

In this study, we have synthesized sorbitan acrylate through response surface methodology, using sorbitan and vinyl acrylate that catalyze immobilized lipase. In order to optimize the enzymatic synthesis of the sorbitan acrylate, we applied response surface techniques to determine the effects of five-level-four-factors and their reciprocal interactions with the biosynthesis of sorbitan acrylate. Our statistical model predicted that the highest conversion yield of sorbitan acrylate would be approx 100%, under the following optimized reaction conditions: a reaction temperature of 40.1°C, a reaction time of 237.4 min, an enzyme concentration of 8%, and a 4.49 : 1 acyl donor/acceptor molar ratio. Using these optimal conditions in three independent replicates, the conversion yield reached 97.6±1.3%.

Keywords: Bioconversion; central composite rotatable design; esterification; lipase; optimization; response surface methodology; sorbitan ester

Renewable energy in the United States by Alvin O. Converse (pp. 611-624).

A review of studies of biomass potential in the United States finds a wide variation in the estimates. A number of specific policy-relevant questions about the potential of biofuels in the United States are answered. A recently published global analysis of the potential conflict between land needed for bioenergy and land needed for food is extended to the situation in the United States. A renewable energy supply scenario, capable of meeting the 2001 US energy demand, indicates that there is enough land to support a renewable energy system but that the utilization of biomass would be limited by its land requirement.

Keywords: Agriculture; forest; postfossil; renewable energy; scenario; land requirements

Optimizing the logistics of anaerobic digestion of manure by Emad Ghafoori; Peter C. Flynn (pp. 625-637).

Electrical power production from the combustion of biogas from anaerobic digestion (AD) of manure is a means of recovering energy from animal waste. We evaluate the lowest cost method of moving material to and from centralized AD plants serving multiple confined feeding operations. Two areas are modeled, Lethbridge County, Alberta, Canada, an area of concentrated beef cattle feedlots, and Red Deer County, Alberta, a mixed-farming area with hog, dairy, chicken and beef cattle farms, and feedlots. We evaluate two types of AD plant: ones that return digestate to the source confined feeding operation for land spreading (current technology), and ones that process digestate to produce solid fertilizer and a dischargeable water stream (technology under development). We evaluate manure and digestate trucking, trucking of manure with return of digestate by pipelines, and pipelining of manure plus digestate. We compare the overall cost of power from these scenarios to farm or feedlot-based AD units. For a centralized AD plant with digestate return for land spreading the most economical transport option for manure plus digestate is by truck for the mixed-farming area and by pipelines for the concentrated feedlot area. For a centralized AD plant with digestate processing, the most economical transport option is trucking of manure for both cases.However, for the concentrated feedlot area, pipeline transport of manure is close in cost to trucking, and the impact of truck congestion would likely lead to selection of pipeline transport. For the mixed-farming area, centralized AD is more economical than for any individual farm or feedlot unit. For the concentrated feedlot area, a centralized AD plant is less economical than a feedlot-based AD unit more than 55,000 head (digestate return) and 300,000 head (digestate processing). The study demonstrates the viability of centralized AD plants vs farm-based units in most farming environments, and that careful analysis of the cost of pipeline vs truck transport of manure and digestate is required on a case-by-case basis.

Keywords: Anaerobic digestion; biogas plant; digestate processing; manure; manure pipeline; optimum size; trucking

The relative cost of biomass energy transport by Erin Searcy; Peter Flynn; Emad Ghafoori; Amit Kumar (pp. 639-652).

Logistics cost, the cost of moving feedstock or products, is a key component of the overall cost of recovering energy from biomass. In this study, we calculate for small- and large-project sizes, the relative cost of transportation by truck, rail, ship, and pipeline for three biomass feedstocks, by truck and pipeline for ethanol, and by transmission line for electrical power. Distance fixed costs (loading and unloading) and distance variable costs (transport, including power losses during transmission), are calculated for each biomass type and mode of transportation. Costs are normalized to a common basis of a giga Joules of biomass. The relative cost of moving products vs feedstock is an approximate measure of the incentive for location of biomass processing at the source of biomass, rather than at the point of ultimate consumption of produced energy. In general, the cost of transporting biomass is more than the cost of transporting its energy products. The gap in cost for transporting biomass vs power is significantly higher than the incremental cost of building and operating a power plant remote from a transmission grid. The cost of power transmission and ethanol transport by pipeline is highly dependent on scale of project. Transport of ethanol by truck has a lower cost than by pipeline up to capacities of 1800 t/d. The high cost of transshipment to a ship precludes shipping from being an economical mode of transport for distances less than 800 km (woodchips) and 1500 km (baled agricultural residues).

Keywords: Biomass transportation; ethanol transport; pipeline transport; power transmission; rail transport; ship transport; transportation cost; truck transport

The effect of initial cell concentration on xylose fermentation by Pichia stipitis by Frank K. Agbogbo; Guillermo Coward-Kelly; Mads Torry-Smith; Kevin Wenger; Thomas W. Jeffries (pp. 653-662).

Xylose was fermented using Pichia stipitis CBS 6054 at different initial cell concentrations. A high initial cell concentration increased the rate of xylose utilization, ethanol formation, and the ethanol yield. The highest ethanol concentration of 41.0 g/L and a yield of 0.38 g/g was obtained using an initial cell concentration of 6.5 g/L. Even though more xylitol was produced when the initial cell concentrations were high, cell density had no effect on the final ethanol yield. A two-parameter mathematical model was used to predict the cell population dynamics at the different initial cell concentrations. The model parameters, a and b correlate with the initial cell concentrations used with an R ² of 0.99.

Keywords: Cell concentration; fermentation; model; Pichia stipitis ; xylose

Construction and evaluation of a Clostridium thermocellum ATCC 27405 whole-genome oligonucleotide microarray by Steven D. Brown; Babu Raman; Catherine K. McKeown; Shubha P. Kale; Zhili He; Jonathan R. Mielenz (pp. 663-674).

Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.

Keywords: Biomass; cellulose; ethanol; fermentation; transcriptomics

Tannase production by solid state fermentation of cashew apple bagasse by Tigressa H. S. Rodrigues; Maria Alcilene A. Dantas; Gustavo A. S. Pinto; Luciana R. B. Gonçalves (pp. 675-688).

The ability of Aspergillus oryzae for the production of tannase by solid state fermentation was investigated using cashew apple bagasse (CAB) as substrate. The effect of initial water content was studied and maximum enzyme production was obtained when 60 mL of water was added to 100.0 g of CAB. The fungal strain was able to grow on CAB without any supplementation but a low enzyme activity was obtained, 0.576 U/g of dry substrate (g_ds). Optimization of process parameters such as supplementation with tannic acid, phosphorous, and different organic and inorganic nitrogen sources was studied. The addition of tannic acid affected the enzyme production and maximum tannase activity (2.40 U/g_ds) was obtained with 2.5% (w/w) supplementation. Supplementation with ammonium nitrate, peptone, and yeast extract exerted no influence on tannase production. Ammonium sulphate improved the enzyme production in 3.75-fold compared with control. Based on the experimental results, CAB is a promising substrate for solid state fermentation, enabling A. oryzae growth and the production of tannase, with a maximum activity of 3.42 U/g_ds and enzyme productivity of 128.5×10⁻³ U · g _ds ⁻¹ · h⁻¹.

Keywords: Cashew apple bagasse; solid state fermentation; tannase; Aspergillus oryzae ; tannic acid; ammonium sulfate

Studying pellet formation of a filamentous fungus Rhizopus oryzae to enhance organic acid production by Wei Liao; Yan Liu; Shulin Chen (pp. 689-701).

Using pelletized fungal biomass can effectively improve the fermentation performance for most of fugal strains. This article studied the effects of inoculum and medium compositions such as potato dextrose broth (PDB) as carbon source, soybean peptone, calcium carbonate, and metal ions on pellet formation of Rhizopus oryzae. It has been found that metal ions had significantly negative effects on pellet formation whereas soybean peptone had positive effects. In addition PDB and calcium carbonate were beneficial to R. oryzae for growing small smooth pellets during the culture. The study also demonstrated that an inoculum size of less than 1.5×10⁹ spores/L had no significant influence on pellet formation. Thus, a new approach to form pellets has been developed using only PDB, soybean peptone, and calcium carbonate. Meanwhile, palletized fungal fermentation significantly enhanced organic acid production. Lactic acid concentration reached 65.0 g/L in 30 h using pelletized R. oryzae NRRL 395, and fumeric acid concentration reached 31.0 g/L in 96 h using pelletized R. oryzae ATCC 20344.

Keywords: Calcium carbonate; fumaric acid; fungal pellet; inoculum size; lactic acid; metal ions; potato dextrose broth; Rhizopus oryzae

Comparison of multiple gene assembly methods for metabolic engineering by Chenfeng Lu; Karen Mansoorabadi; Thomas Jeffries (pp. 703-710).

A universal, rapid DNA assembly method for efficient multigene plasmid construction is important for biological research and for optimizing gene expression in industrial microbes. Three different approaches to achieve this goal were evaluated. These included creating long complementary extensions using a uracil-DNA glycosylase technique, overlap extension polymerase chain reaction, and a SfiI-based ligation method. SfiI ligation was the only successful approach for assembling large DNA fragments that contained repeated homologous regions. In addition, the SfiI method has been improved over a similar, previous published technique so that it is more flexible and does not require polymerase chain reaction to incorporate adaptors. In the present study, Saccharomyces cerevisiae genes TAL1, TKL1, and PYK1 under control of the 6-phosphogluconate dehydrogenase promoter were successfully ligated together using multiple unique SfiI restriction sites. The desired construct was obtained 65% of the time during vector construction using four-piece ligations. The SfiI method consists of three steps: first a SfiI linker vector is constructed, whose multiple cloning site is flanked by two three-base linkers matching the neighboring SfiI linkers on SfiI digestion; second, the linkers are attached to the desired genes by cloning them into SfiI linker vectors; third, the genes flanked by the three-base linkers, are released by SfiI digestion. In the final step, genes of interest are joined together in a simple one-step ligation.

Keywords: Gene expression; cloning; ligation; optimization; xylose; yeast

Fed-Batch production of glucose 6-Phosphate dehydrogenase using recombinant saccharomyces cerevisiae by Luiz Carlos Martins das Neves; Adalberto Pessoa; Michele Vitolo (pp. 711-720).

The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC 181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in the formation of glucose 6-phosphate dehydrogenase (EC.1.1.1.49). Two liters of culture medium (10.0 g/L glucose, 3.7 g/L yeast nitrogen broth (YNB), 0.02 g/L l-tryptophan, 0.02 g/L l-histidine, 0.02 g/L uracil, and 0.02 g/L adenine) were inoculated with 1.5 g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30°C, and agitation of 400 rpm. After glucose concentration in the medium was lower than 1.0 g/L, the cell culture was fed with a solution of glucose (10.0 g/L) or micronutrients (l-tryptophan, l-histidine, uracil, and adenine each one at a concentration of 0.02 g/L) following the constant, linear, or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2 L up to 3 L during 5 h. The highest glucose 6-phosphate dehydrogenase activity (350 U/L; 1 U=1 µmol of NADP/min) occurred when the glucose solution was fed into the fermenter through the decreasing linear mode.

Keywords: Fed-batch; glucose 6-phosphate dehydrogenase; glucose; recombinant strain; Saccharomyces cerevisiae ; fermentation

Conversion of aqueous ammonia-treated corn stover to lactic acid by simultaneous saccharification and cofermentation by Yongming Zhu; Y. Y. Lee; Richard T. Elander (pp. 721-738).

Treatment of corn stover with aqueous ammonia removes most of the structural lignin, whereas retaining the majority of the carbohydrates in the solids. After treatment, both the cellulose and hemicellulose in corn stover become highly susceptible to enzymatic digestion. In this study, corn stover treated by aqueous ammonia was investigated as the substrate for lactic acid production by simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Spezyme-CP) and Lactobacillus pentosus American Type Culture Collection (ATCC) 8041 (Spanish Type Culture Collection [CECT]-4023) were used for hydrolysis and fermentation, respectively. In batch SSCF operation, the carbohydrates in the treated corn stover were converted to lactic acid with high yields, the maximum lactic acid yield reaching 92% of the stoichiometric maximum based on total fermentable carbohydrates (glucose, xylose, and arabinose). A small amount of acetic acid was also produced from pentoses through the phosphoketolase pathway. Among the major process variables for batch SSCF, enzyme loading and the amount of yeast extract were found to be the key factors affecting lactic acid production. Further tests on nutrients indicated that corn steep liquor could be substituted for yeast extract as a nitrogen source to achieve the same lactic acid yield. Fed-batch operation of the SSCF was beneficial in raising the concentration of lactic acid to a maximum value of 75.0 g/L.

Keywords: Aqueous ammonia pretreatment; biomass; cofermentation; lactic acid; simultaneous saccharification; response surface method

Introduction to session 5 by Luca Zullo; Seth W. Snyder (pp. 739-740).

Economic evaluation of isolation of hemicelluloses from process streams from thermomechanical pulping of spruce by Tobias Persson; Anna-Karin Nordin; Guido Zacchi; Ann-Sofi Jönsson (pp. 741-752).

Hemicelluloses, which are abundant in nature and have potential use in a wide variety of applications, may make an important contribution in helping relieve society of its dependence on petrochemicals. However, cost-efficient methods for the isolation of hemicelluloses are required. This article presents an economic evaluation of a full-scale process to isolate hemicelluloses from process water from a thermomechanical pulp mill. Experimental data obtained in laboratory scale were used for the scale up of the process by computer simulation. The isolation method consisted of two process steps. The suspended matter in the process water was removed by microfiltration and thereafter the hemicelluloses were concentrated by ultrafiltration, and at the same time, separated from smaller molecules and ions in the process water. The isolated hemicelluloses were intended for the production of oxygen barriers for food packaging, an application for which they have been shown to have suitable properties. The solution produced contained 30 g hemicelluloses/L with a purity (defined as the ratio between the hemicelluloses and the total solids) of approx 80%. The evaluation was performed for a plant with a daily production of 4 metric tonnes (t) of hemicelluloses, which is the estimated future need of barrier films at Tetra Pak (Lund, Sweden). The production cost was calculated to be € 670/t of hemicelluloses. This is approx 9 times lowr than the price of ethylene vinyl alcohol, which is produced by petrochemicals and is currently used as an oxygen barrier in fiberbased packaging materials. This indicates that it is possible to produce oxygen barriers made of hemicelluloses at a price that is competitive with the materials used today.

Keywords: Barrier film; economic evaluation; galactoglucomannan; hemicelluloses; ultrafiltration; thermomechanical pulp

Estimation of temperature dependent parameters of a batch alcoholic fermentation process by Rafael Ramos de Andrade; Elmer Ccopa Rivera; Aline C. Costa; Daniel I. P. Atala; Francisco Maugeri Filho; Rubens Maciel Filho (pp. 753-763).

In this work, a procedure was established to develop a mathematical model considering the effect of temperature on reaction kinetics. Experiments were performed in batch mode in temperatures from 30 to 38°C. The microorganism used was Saccharomyces cerevisiae and the culture media, sugarcane molasses. The objective is to assess the difficulty in updating the kinetic parameters when there are changes in fermentation conditions. We conclude that, although the re-estimation is a time-consuming task, it is possible to accurately describe the process when there are changes in raw material composition if a re-estimation of parameters is performed.

Keywords: Alcoholic fermentation; kinetic parameters estimation; mathematical modeling; Quasi-Newton algorithm; Saccharomyces cerevisiae ; ethanol production

Thermophilic anaerobic digester performance under different feed-loading frequency by John Bombardiere; Teodoro Espinosa-Solares; Max Domaschko; Mark Chatfield (pp. 765-775).

The effect of feed-loading frequency on digester performance was studied on a thermophilic anaerobic digester with a working volume of 27.43 m³. The digester was fed 0.93 m³ of chicken-litter slurry/d, containing 50.9 g/L chemical oxygen demand. The treatments were loading frequencies of 1, 2, 6, and 12 times/d. The hourly pH, biogas production, and methane percent of the biogas were less stable at lower feed frequencies. There was no statistical difference among treatments in methanogenic activity. The feed-loading frequency of six times per day treatment provided the greatest biogas production.

Keywords: Biogas; chicken litter; digester stability; methane; methanogenic activity; thermophilic

A proposed mechanism for detergent-assisted foam fractionation of lysozyme and cellulase restored with β-cyclodextrin by Vorakan Burapatana; Elizabeth A. Booth; Ian M. Snyder; Ales Prokop; Robert D. Tanner (pp. 777-791).

Foam fractionation by itself cannot effectively concentrate hydrophilic proteins such as lysozyme and cellulase. However, the addition of a detergent to a protein solution can increase the foam volume, and thus, the performance of the foam fractionation process. In this article, we propose a possible protein concentration mechanism of this detergent-assisted foam fractionation: A detergent binds to an oppositely charged protein, followed by the detergent-protein complex being adsorbed onto a bubble during aeration. The formation of this complex is inferred by a decrease in surface tension of the detergent-protein solution. The surface tension of a solution with the complex is lower than the surface tension of a protein or a detergent solution alone. The detergent can then be stripped from the adsorbed protein, such as cellulase, by an artificial chaperone such as β-cyclodextrin. Stripping the detergent from the protein allows the protein to return to its original conformation and to potentially retain all of its original activity following the foam fractionation process. Low-cost alternatives to β-cyclodextrin such as corn dextrin were tested experimentally to restore the protein activity through detergent stripping, but without success.

Keywords: Artificial chaperones; detergent stripping; protein refolding; protein renaturation; surface tension

Study on the production of biodiesel by magnetic cell biocatalyst based on lipase-producing Bacillus subtilis by Ming Ying; Guanyi Chen (pp. 793-803).

Production of biodiesel from waste cooking oils by a magnetic cell biocatalyst (MCB) immobilized in hydrophobic magnetic polymicrosphere is studied here. The cells of lipase-producing Bacillus subtilis were encapsulated within the net of hydrophobic carrier with magnetic particles (Fe₃O₄), and the secreted lipase can be conjugated with carboxyl at the magnetic polymicrosphere surface. Environmental scanning electron microscope, transmission electron microscope, and vibrating magnetometer, and so on were used to characterize the MCB. The MCB was proved to be superparamagnetic; and could be recovered by magnetic separation; moreover it could be regenerated under 48 h of cultivation. When methanolysis is carried out using MCB with waste cooking oils under stepwise additions of methanol, the methyl esters in the reaction mixture reaches about 90% after 72 h reaction in a solvent-free system. The process presented here is environmentally friendly and simple without purification and immobilized process required by the current lipase-catalyzed process. Therefore, the process is very promising for development of biodiesel fuel industry.

Keywords: Bacillus subtilis ; biodiesel; magnetic cell biocatalyst; magnetic polymicrosphere; waste cooking oils

Production of ω-3 polyunsaturated fatty acids from cull potato using an algae culture process by Zhanyou Chi; Bo Hu; Yan Liu; Craig Frear; Zhiyou Wen; Shulin Chen (pp. 805-815).

Algal cultivation for converting cull potato to docosahexaenoic acid (DHA) was studied. Schizochytrium limacinum SR21 was selected as the better producing strain, compared with Thraustochytrium aureum because of higher cell density and DHA content. Used as both carbon and nitrogen source, an optimal ratio of hydrolyzed potato broth in the culture medium was determined as 50%, with which the highest production of 21.7 g/L dry algae biomass and 5.35 g/L DHA was obtained, with extra glucose supplemented. Repeat culture further improved the cell density but not fed batch culture, suggesting limited growth was most likely caused by metabolites inhibition.

Keywords: Docosahexaenoic acid; microalgae; omega-3 fatty acid; Schizochytrium ; cull potato; fish oil

Hybrid neural network model of an industrial ethanol fermentation process considering the effect of temperature by Ivana C. C. Mantovanelli; Elmer Ccopa Rivera; Aline C. da Costa; Rubens Maciel Filho (pp. 817-833).

In this work a procedure for the development of a robust mathematical model for an industrial alcoholic fermentation process was evaluated. The proposed model is a hybrid neural model, which combines mass and energy balance equations with functional link networks to describe the kinetics. These networks have been shown to have a good nonlinear approximation capability, although the estimation of its weights is linear. The proposed model considers the effect of temperature on the kinetics and has the neural network weights reestimated always so that a change in operational conditions occurs. This allow to follow the system behavior when changes in operating conditions occur.

Keywords: Alcoholic fermentation; functional link networks; kinetic parameters estimation; mathematical modeling; process simulation; bioreactors

Characterization of thermostructural damages observed in a seaweed used for biosorption of cadmium by Antonio Carlos Augusto da Costa; Aderval S. Luna; Robson Pafumé (pp. 835-845).

The effect of drying Sargassum filipendula on the kinetics and uptake of cadmium was studied. The maximum uptake was not reduced when ovendried biomass was used for cadmium concentrations from 10.0 to 500.0 mg/L. Kinetics indicated better performance of the in natura biomass. Drying at 333 K affected the uptake capacity. Results fit the Langmuir model better than the Freundlich. This process followed pseudo-second-order kinetics. Thermogravimetric and infrared analysis confirmed that no structural damage occurred after drying, and no differences between the biomasses were observed. Temperatures from 303 to 328 K affected cadmium uptake capacity.

Keywords: Cadmium; kinetics; Sargassum filipendula ; thermal effects; uptake capacity; biosorption

Ethanol fermentation of various pretreated and hydrolyzed substrates at low initial pH by Zsófia Kádár; San Feng Maltha; Zsolt Szengyel; Kati Réczey; Wim de Laat (pp. 847-858).

Lignocellulosic materials represent an abundant feedstock for bioethanol production. Because of their complex structure pretreatment is necessary to make it accessible for enzymatic attack. Steam pretreatment with or without acid catalysts seems to be one of the most promising techniques, which has already been applied for large variety of lignocellulosics in order to improve enzymatic digestibility. During this process a range of toxic compounds (lignin and sugar degradation products) are formed which inhibit ethanol fermentation. In this study, the toxicity of hemicellulose hydrolysates obtained in the steam pretreatment of spruce, willow, and corn stover were investigated in ethanol fermentation tests using a yeast strain, which has been previously reported to have a resistance to inhibitory compounds generated during steam pretreatment. To overcome bacterial contamination, fermentations were carried out at low initial pH. The fermentability of hemicellulose hydrolysates of pretreated lignocellulosic substrates at low pH gave promising results with the economically profitable final 5 vol% ethanol concentration corresponding to 85% of theoretical. Adaptation experiments have shown that inhibitor tolerance of yeast strain can be improved by subsequent transfer of the yeast to inhibitory medium.

Keywords: Inhibitors; lignocellulose; Saccharomyces cerevisiae ; toxicity; yeast adaptation; bioethanol

The effects of engineering design on heterogeneous biocatalysis in microchannels by Frank Jones; Robert Bailey; Stephanie Wilson; James Hiestand (pp. 859-873).

The results of a numerical study of the fundamental interactions of engineering design and micromixing on conversion in packed microchannels are presented. Previously, channel-based microreactors made of molded silicon plastic were designed, fabricated, and experimentally tested. These reactors have enzymes immobilized on the channel walls by various methods including layer-by-layer nano self-assembly techniques. They also contain molded packing features to add reactive surface area and to redistribute the fluid. An arbitrary but intuitively sensible packing arrangement was initially chosen and used in experimental studies. The current computer simulation study was undertaken to understand how static laminar mixing affects the conversion efficiency. The reactors previously used experimentally have been simulated using CFD-ACE+multiphysics software (ESI CFD Inc., Huntsville, AL). It is found that packing significantly increases conversion when compared with empty channels over the entire flow rate range of the study (0.25<Re<62.5). The boost in conversion has an optimal point near Re=20 for the particular geometry examined.

Keywords: Catalase; enzyme; micromixing; microreactor; numerical simulation; heterogeneous catalysis

Utilization of condensed distillers solubles as nutrient supplement for production of nisin and lactic acid from whey by Chuanbin Liu; Bo Hu; Shulin Chen; Richard W. Glass (pp. 875-884).

The major challenge associated with the rapid growth of the ethanol industry is the usage of the coproducts, i.e., condensed distillers solubles (CDS) and distillers dried grains, which are currently sold as animal feed supplements. As the growth of the livestock industries remains flat, alternative usage of these coproducts is urgently needed. CDS is obtained after the removal of ethanol by distillation from the yeast fermentation of a grain or a grain mixture by condensing the thin stillage fraction to semisolid. In this work, CDS was first characterized and yeast biomass was proven to be the major component of CDS. CDS contained 7.50% crude protein but with only 42% of that protein being water soluble. Then, CDS was applied as a nutrient supplement for simultaneous production of nisin and lactic acid by Lactococcus lactis subsp. lactis (ATCC 11454). Although CDS was able to support bacteria growth and nisin production, a strong inhibition was observed when CDS was overdosed. This may be caused by the existence of the major ethanol fermentation byproducts, especially lactate and acetate, in CDS. In the final step, the CDS based medium composition for nisin and lactic acid production was optimized using response surface methodology.

Keywords: Condensed distillers solubles; nutrient supplement; fermentation; lactic acid; Lactococcus lactis ; nisin

Optimization of tocopherol concentration process from soybean oil deodorized distillate using response surface methodology by Vanessa Mayumi Ito; César Benedito Batistella; Maria Regina Wolf Maciel; Rubens Maciel Filho (pp. 885-896).

Soybean oil deodorized distillate is a product derived from the refining process and it is rich in high value-added products. The recovery of these unsaponifiable fractions is of great commercial interest, because of the fact that in many cases, the “valuable products” have vitamin activities such as tocopherols (vitamin E), as well as anticarcinogenic properties such as sterols. Molecular distillation has large potential to be used in order to concentrate tocopherols, as it uses very low temperatures owing to the high vacuum and short operating time for separation, and also, it does not use solvents. Then, it can be used to separate and to purify thermosensitive material such as vitamins.In this work, the molecular distillation process was applied for tocopherol concentration, and the response surface methodology was used to optimize free fatty acids (FFA) elimination and tocopherol concentration in the residue and in the distillate streams, both of which are the products of the molecular distiller. The independent variables studied were feed flow rate (F) and evaporator temperature (T) because they are the very important process variables according to previous experience. The experimental range was 4–12 mL/min for F and 130–200°C for T. It can be noted that feed flow rate and evaporator temperature are important operating variables in the FFA elimination. For decreasing the loss of FFA, in the residue stream, the operating range should be changed, increasing the evaporator temperature and decreasing the feed flow rate; D/F ratio increases, increasing evaporator temperature and decreasing feed flow rate. High concentration of tocopherols was obtained in the residue stream at low values of feed flow rate and high evaporator temperature. These results were obtained through experimental results based on experimental design.

Keywords: Deodorized distillate; free fatty acids; molecular distillation; response surface methodology; soya sludge; tocopherol

Semicontinuous production of lactic acid from cheese whey using integrated membrane reactor by Yebo Li; Abolghasem Shahbazi; Sekou Coulibaly; Michele M. Mims (pp. 897-907).

Semicontinuous production of lactic acid from cheese whey using free cells of Bifidobacterium longum with and without nanofiltration was studied. For the semicontinuous fermentation without membrane separation, the lactic acid productivity of the second and third runs is much lower than the first run. The semicontinuous fermentation with nanoseparation was run semicontinuously for 72 h with lactic acid to be harvested every 24 h using a nanofiltration membrane unit. The cells and unutilized lactose were kept in the reactor and mixed with newly added cheese whey in the subsequent runs. Slight increase in the lactic acid productivity was observed in the second and third runs during the semicontinuous fermentation with nanofiltration. It can be concluded that nanoseparation could improve the lactic acid productivity of the semicontinuous fermentation process.

Keywords: Cheese whey; fermentation; lactic acid; lactose; membrane; nanofiltration; semicontinuous

Functional stability of a mixed microbial consortium producing PHA from waste carbon sources by Erik R. Coats; Frank J. Loge; William A. Smith; David N. Thompson; Michael P. Wolcott (pp. 909-925).

Polyhydroxyalkanoates (PHAs) represent an environmentally effective alternative to synthetic thermoplastics; however, current production practices are not sustainable. In this study, PHA production was accomplished in sequencing batch bioreactors utilizing real wastewaters and mixed microbial consortia from municipal activated sludge as inoculum. Polymer production reached 85, 53, and 10% of the cell dry weight from methanol-enriched pulp and paper mill foul condensate, fermented municipal primary solids, and biodiesel wastewater, respectively. Using denaturing gradient gel electrophoresis of 16S-rDNA from polymerase chain reaction-amplified DNA extracts, distinctly different communities were observed between and within wastewaters following enrichment. Most importantly, functional stability was maintained despite differing and contrasting microbial populations.

Keywords: Activated sludge; denaturing gradient gel electrophoresis; polyhydroxyalkanoates; wastewater; primary solids fermentate; foul condensate; environmental biotechnology

Separating a mixture of egg yolk and egg white using foam fractionation by Tiffany M. Ward; Ross A. Edwards; Robert D. Tanner (pp. 927-934).

A mixture created by blending with a spatula, an egg yolk and an egg white from the same egg can serve as a binary system for testing to see how well foam fractionation can be used to separate two different groups of proteins naturally found together. This mixture of two phases is particularly attractive for such a study because the two phases can be visualized distinctly when in their separated states. It has been shown that air alone at a low flow rate and with little or no water added can effect visually clean separations of egg yolk from egg white, making this a “green” separation process. The white precedes the yolk in the process, which takes less than 10 min at a laboratory scale.

Keywords: Egg albumin; egg protein; egg white; egg yolk; foam fractionation; protein separation; serum albumin

Synthesis of poly(sorbitan methacrylate) hydrogel by free-radical polymerization by Gwi-Taek Jeong; Kyoung-Min Lee; Hee-Seung Yang; Seok-Hwan Park; Jae-Hee Park; Changshin Sunwoo; Hwa-Won Ryu; Doman Kim; Woo-Tae Lee; Hae-Sung Kim; Wol-Seog Cha; Don-Hee Park (pp. 935-946).

Hydrogels are materials with the ability to swell in water through the retention of significant fractions of water within their structures. Owing to their relatively high degree of biocompatibility, hydrogels have been utilized in a host of biomedical applications. In an attempt to determine the optimum conditions for hydrogel synthesis by the free-radical polymerization of sorbitan methacrylate (SMA), the hydrogel used in this study was well polymerized under the following conditions: 50% (w/v) SMA as monomer, 1% (w/w) α, α′-azo-bis(isobutyro-nitrile) as thermal initiator, and 1% (w/w) ethylene glycol dimethacrylate as cross-liking agent. Under these conditions, the moisture content of the polymerized SMA hydrogel was higher than in the other conditions. Moreover, the moisture content of the poly(SMA) hydrogel was also found to be higher than that of the poly(methyl methacrylate [MMA]) hydrogel. When the Fourier transform-infrared spectrum of poly(SMA) hydrogel was compared with that of poly(MMA) hydrogel, we noted a band at 1735–1730/cm, which did not appear in the Fourier transform-infrared spectrum of poly(MMA). The surface of the poly(SMA) hydrogel was visualized through scanning electron microscopy, and was uniform and clear in appearance.

Keywords: Free radical hydrogel; moisture content; polymerization; sorbitan methacrylate

Hybrid thermochemical/biological processing by Robert C. Brown (pp. 947-956).

The conventional view of biorefineries is that lignocellulosic plant material will be fractionated into cellulose, hemicellulose, lignin, and terpenes before these components are biochemically converted into market products. Occasionally, these plants include a thermochemical step at the end of the process to convert recalcitrant plant components or mixed waste streams into heat to meet thermal energy demands elsewhere in the facility. However, another possibility for converting high-fiber plant materials is to start by thermochemically processing it into a uniform intermediate product that can be biologically converted into a bio-based product. This alternative route to bio-based products is known as hybrid thermochemical/biological processing. There are two distinct approaches to hybrid processing: (a) gasification followed by fermentation of the resulting gaseous mixture of carbon monoxide (CO), hydrogen (H₂), and carbon dioxide (CO₂) and (b) fast pyrolysis followed by hydrolysis and/or fermentation of the anhydrosugars found in the resulting bio-oil. This article explores this “cart before the horse” approach to biorefineries.

Keywords: Biochemical processing; biorefinery; gasification; lignocellulose; pyrolysis; thermochemical processing

Physical and chemical properties of bio-oils from microwave pyrolysis of corn stover by Fei Yu; Shaobo Deng; Paul Chen; Yuhuan Liu; Yiqin Wan; Andrew Olson; David Kittelson; Roger Ruan (pp. 957-970).

This study was aimed to understand the physical and chemical properties of pyrolytic bio-oils produced from microwave pyrolysis of corn stover regarding their potential use as gas turbine and home heating fuels. The ash content, solids content, pH, heating value, minerals, elemental ratio, moisture content, and viscosity of the bio-oils were determined. The water content was approx 15.2 wt%, solids content 0.22 wt%, alkali metal content 12 parts per million, dynamic viscosity 185 mPa·s at 40°C, and gross high heating value 17.5 MJ/kg for a typical bio-oil produced. Our aging tests showed that the viscosity and water content increased and phase separation occurred during the storage at different temperatures. Adding methanol and/or ethanol to the bio-oils reduced the viscosity and slowed down the increase in viscosity and water content during the storage. Blending of methanol or ethanol with the bio-oils may be a simple and cost-effective approach to making the pyrolytic bio-oils into a stable gas turbine or home heating fuels.

Keywords: Aging; chemical behavior; microwave pyrolysis; physical behavior; stability; bio-oils

Comparison of diafiltration and size-exclusion chromatography to recover hemicelluloses from process water from thermomechanical pulping of spruce by Alexandra Andersson; Tobias Persson; Guido Zacchi; Henrik Stålbrand; Ann-Sofi Jönsson (pp. 971-983).

Hemicelluloses constitute one of the most abundant renewable resources on earth. To increase their utilization, the isolation of hemicelluloses from industrial biomass side-streams would be beneficial. A method was investigated to isolate hemicelluloses from process water from a thermomechanical pulp mill. The method consists of three steps: removal of solids by microfiltration, preconcentration of the hemicelluloses by ultrafiltration, and purification by either size-exclusion chromatography (SEC) or diafiltration. The purpose of the final purification step is to separate hemicelluloses from small oligosaccharides, monosaccharides, and salts. The ratio between galactose, glucose, and mannose in oligo- and polysaccharides after preconcentration was 0.8: 1: 2.8, which is similar to that found in galactoglucomannan. Continuous diafiltration was performed using a composite fluoro polymer membrane with cutoff of 1000 Da. After diafiltration with four diavolumes the purity of the hemicelluloses was 77% (gram oligo- and polysaccharides / gram total dissolved solids) and the recovery was 87%. Purification by SEC was performed with 5, 20, and 40% sample loadings, respectively and a flow rate of 12 or 25 mL/min (9 or 19 cm/h). The purity of hemicelluloses after SEC was approx 82%, and the recovery was above 99%. The optimal sample load and flow rate were 20% and 25 mL/min, respectively. The process water from thermomechanical pulping of spruce is inexpensive. Thus, the recovery of hemicelluloses is not of main importance. If the purity of 77%, obtained with diafiltration, is sufficient for the utilization of the hemicelluloses, diafiltration probably offers a less expensive alternative in this application.

Keywords: Galactoglucomannan; diafiltration; microfiltration; polysaccharides; ultrafiltration; SEC; gelfiltration

Sections

Personal tools

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.137-140, #1-12)

Sections

Personal tools

Local resources

Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.137-140, #1-12)