Applied Biochemistry and Biotechnology (v.162, #6)

Cloning, Expression, and Characterization of a Novel Methylglyoxal Synthase from Thermus sp. Strain GH5 by Mohammad Pazhang; Khosro Khajeh; S. Mohsen Asghari; Hanieh Falahati; Hossein Naderi-Manesh (1519-1528).
A gene encoding methylglyoxal synthase from Thermus sp. GH5 (TMGS) was cloned, sequenced, overexpressed, and purified by Q-Sepharose. The TMGS gene was composed of 399 bp which encoded a polypeptide of 132 amino acids with a molecular mass of 14.3 kDa. The K m and k cat values of TMGS were 0.56 mM and 325 (s−1), respectively. The enzyme exhibited its optimum activity at pH 6 and 75 °C. Comparing the amino acid sequences and Hill coefficients of Escherichia coli MGS and TMGS revealed that the loss of Arg 150 in TMGS has caused a decrease in the cooperativity between the enzyme subunits in the presence of phosphate as an allosteric inhibitor. Gel filtration experiments showed that TMGS is a hexameric enzyme, and its quaternary structure did not change in the presence of phosphate.
Keywords: Cloning; Purification; Methylglyoxal synthase; Thermus sp. GH5; Cooperativity

Influences of Acidic Conditions on Formazan Assay: A Cautionary Note by Hisashi Johno; Shuhei Takahashi; Masanori Kitamura (1529-1535).
Formazan assay has been used for several decades to evaluate metabolic activity of eukaryotic and prokaryotic cells. In particular, it has been often applied for quantitative assessment of viable cells under acidic circumstances caused by, e.g., ischemia and hypoxia. However, little attention has been paid to the influence of acidic pH on formazan assays. We found that acidic culture conditions significantly affect outcomes of the assays. Absorbance of tetrazolium–formazan decreased in a pH-dependent manner without affecting cell viability. This nonspecific effect was ascribed to influences of acidic pH on the production of formazan. Replacement of culture media to fresh medium at physiologic pH partially overcame this problem. The influence of acidic culture conditions should be carefully considered when formazan assays are used for the assessment of viable cells under various experimental situations.
Keywords: Tetrazolium; Formazan; Acidosis; Hypoxia; WST; MTT

A new, simple, and original method is described for specific measurement of polyunsaturated fatty acid content in olive oil. This analytical system uses coupled enzymes, lipase and lipoxygenase. The system consists of lipase-catalyzed hydrolysis of triacylglycerol and subsequent lipoxygenation of liberated polyunsaturated fatty acids. The hydroperoxy-fatty acids formed were easily monitored by spectrophotometry at 234 nm. After being optimized, the method was validated in terms of linearity, precision sensitivity, and recovery. Linear calibration graph was obtained in the range 50–500 µg mL−1, with a correlation coefficient higher than 0.921 and a detection limit (S/N = 3) of 15 µg mL−1. The precision of the method (relative standard deviation) for within and between days is better than 7% and 12%, respectively. The proposed method was successfully applied to the estimation of polyunsaturated fatty acids level in olive oil samples and results obtained were in excellent agreement with those obtained by the classical official method. The proposed method is accurate, simple, cheap, and can be satisfactorily used for routine analysis of edible oils.
Keywords: Lipase; Lipoxygenase; Olive oil; Polyunsaturated fatty acids; Oxidative stability; Spectrophotometry

High Enrichment of MMP-9 and Carboxypeptidase A by Tweezing Adsorptive Bubble Separation (TABS) by Dirk Haller; Perihan Ekici; Albrecht Friess; Harun Parlar (1547-1557).
Tweezing adsorptive bubble separation (TABS) was used as a method for the enrichment of matrix metalloproteinases (92-kDa type IV, gelatinase B (MMP-9)) and carboxypeptidase A (CPA) from dilute aqueous solutions. The method is based on the chelation of metalloenzymes applying 2-(carbamoylmethyl-(carboxymethyl)amino)acetic acid (ADA) coupled with an octyl part to form a surface active unit. MMP-9 could be enriched with an enrichment ratio of 12.0 and a recovery of 87.3%, and CPA could be enriched 18.8-fold and with 95.3% recovery. Both enzymes were enriched without significant losses of enzymatic activity. To verify that the enzymes were tweezed by ADA-C8 without abstraction of the zinc ions from the active center, TABS trials were additionally conducted with zinc ions in complex with ADA-C8, which revealed only negligible enrichment ratios of the enzymes (2.2 for MMP-9 and 0.2 for CPA). The results obtained impressively demonstrate that zinc-containing proteases can be enriched selectively and efficiently by TABS.
Keywords: Tweezing adsorptive bubble separation; MMP-9; Carboxypeptidase A; Matrix metalloproteinases; Carboxypeptidases

Contribution of Tris Buffer on Xylitol Enzymatic Production by Ricardo de Freitas Branco; Silvio S. Silva (1558-1563).
Xylitol enzymatic production can be an alternative to chemical and microbial processes, because of advantages like higher conversion efficiency. However, for an adequate conversion, it is necessary to investigate the effect of many parameters, such as buffer initial concentration, pH, temperature, agitation, etc. In this context, the objective of this work was to evaluate xylitol enzymatic production under different Tris buffer initial concentrations in order to determine the best condition for this parameter to begin the reaction. The best results were obtained when Tris buffer initial concentration was 0.22 M, reaching 0.31 g L−1 h−1 xylitol volumetric productivity with 99% xylose–xylitol conversion efficiency. Although the increase in buffer concentration allowed better pH maintenance, it hindered the catalysis. The results demonstrate that this bioreaction is greatly influenced by involved ions concentrations.
Keywords: Enzymatic process; Glucose dehydrogenase; Polyalcohols; Tris buffer; Xylitol; Xylose reductase

Inhibitory Effects of Fatty Acids on the Activity of Mushroom Tyrosinase by Yun-Ji Guo; Zhi-Zhen Pan; Chao-Qi Chen; Yong-Hua Hu; Feng-Jiao Liu; Yan Shi; Jiang-Hua Yan; Qing-Xi Chen (1564-1573).
The effects of fatty acids, octanoic acid, (2E, 4E)-hexa-2,4-dienoic acid, hexanoic acid, (2E)-but-2-enoic acid, and butyric acid on the activities of mushroom tyrosinase have been investigated. The results showed that the fatty acids can potently inhibit both monophenolase activity and diphenolase activity of tyrosinase, and that the unsaturated fatty acids exhibited stronger inhibitory effect against tyrosinase than the corresponding saturated fatty acids, and the inhibitory effects were enhanced with the extendability of the fatty acid chain. For the monophenolase activity, the fatty acids could not only lengthen the lag period, but also decrease the steady-state activities. For the diphenolase activity, fatty acids displayed reversible inhibition. Kinetic analyses showed that octanoic acid and hexanoic acid were mixed-type inhibitors and (2E,4E)-hexa-2,4-dienoic acid and (2E)-but-2-enoic acid were noncompetitive inhibitors. The inhibition constants have been determined and compared.
Keywords: Tyrosinase; Fatty acid; Monophenolase activity; Diphenolase activity; Inhibition kinetics

An (S)-enantioselective esterase from Bacillus subtilis ECU0554, named BsE-NP01, has been cloned and over-expressed in a heterologous host Escherichia coli BL21. BsE-NP01 was shown to be a carboxylesterase with a molecular mass of about 32 kDa, and temperature and pH optima at 50 °C and 8.5, respectively. It could catalyze the selective hydrolysis of the (S)-enantiomer of racemic naproxen methyl ester, giving optically pure (S)-naproxen with 98% enantiomeric excess. A mechanic-grinding approach to substrate dispersion was also reported, which was considered to be an alternative to take the place of deleterious surfactants such as Tween-80, with improved performance of the hydrolysis reaction. Batch production of (S)-naproxen was repeatedly carried out in a solid-water biphasic system at 2-L scale, achieving an average total yield of about 85% after ten runs with complete recycling of (R)-substrate. Figure
Keywords: (S)-naproxen; Bacillus subtilis carboxylesterase; Enantioselective hydrolysis; Naproxen methyl ester

Expression, High Cell Density Culture and Purification of Recombinant EC-SOD in Escherichia coli by Young-Jin Son; Ji-Young Bae; Seon-Ha Chong; Hui Sun Lee; Sang Hyun Mo; Tae Yoon Kim; Han Choe (1585-1598).
Superoxide dismutase (SOD) catalyzes the dismutation of the biologically toxic superoxide anion into oxygen and hydrogen peroxide and is deployed by the immune system to kill invading microorganisms. Extracellular SOD (EC-SOD) is a copper- and zinc-containing glycoprotein found predominantly in the soluble extracellular compartment that consists of ∼30-kDa subunits. Here, we purified recombinant EC-SOD3 (rEC-SOD) from Escherichia coli BL21(DE3) expressing a pET-SOD3-1 construct. Cells were cultured by high-density fed-batch fermentation to a final OD600 of 51.8, yielding a final dry cell weight of 17.6 g/L. rEC-SOD, which was expressed as an inclusion body, comprised 48.7% of total protein. rEC-SOD was refolded by a simple dilution refolding method and purified by cation-exchange and reverse-phase chromatography. The highly purified rEC-SOD thus obtained was a mixture of monomers and dimers, both of which were active. The molecular weights of monomeric and dimeric rEC-SOD were 25,255 and 50,514 Da, respectively. The purified rEC-SOD had 4.3 EU/mg of endotoxin and the solubility of rEC-SOD was more than 80% between pH 7 and 10. In 2 L of fed-batch fermentation, 60 mg of EC-SOD (99.9% purity) could be produced and total activity was 330.24 U. The process established in this report, involving high-cell-density fermentation, simple dilution refolding, and purification with ion-exchange and reverse-phase chromatography, represents a commercially viable process for producing rEC-SOD.
Keywords: rEC-SOD; Fermentation; Refolding; Purification; MALDI-TOF

A Study of the Expression of Functional Human Coagulation Factor IX in Keratinocytes Using a Nonviral Vector Regulated by K14 Promoter by Seyed Javad Hosseini; Alireza Zomorodipour; Razieh Jalal; Farzaneh Sabouni; Fariba Ataei (1599-1611).
Ex vivo gene therapy requires a suitable bioreactor for production and delivery of the gene products into a target tissue, and keratinocyte is suitable model in this regard because of its potential for systemic release of proteins. To establish a keratinocyte-specific expression system, a mammalian-based expression plasmid equipped with a 2,240-bp fragment from the human keratin 14 (k14) gene enhancer/promoter region was constructed and used for the insertion of the human coagulation factor IX (hFIX)-cDNA downstream the K14-derived regulatory elements. The human epidermal keratinocytes isolated from neonatal foreskin were cultivated in keratinocyte serum-free media and transfected with the recombinant plasmid. The K14-promoter-driven expression of recombinant hFIX (rhFIX) was evaluated by performing coagulation test as well as enzyme-linked immunosorbent assay on the cultured media collected from the transfected cells at various stages. The rhFIX corresponding transcript and protein were confirmed by performing reverse transcription PCR as well as immunoblotting experiments, respectively. Based on the coagulation activities obtained from the conditioned media of nine isolated clones, the hFIX expression levels vary from 5% to 39% of normal human plasma. Expression levels of the hFIX obtained in this study are comparable to those reported for viral systems. The obtained data supported the potential of keratinocyte for the expression and secretion of biologically active rhFIX and underscore the importance of the examined cis sequences for enhancing gene expression in a mammalian expression system. Besides, it has provided means for further bioengineering strategies to improve the expression efficiency of the hFIX in keratinocytes and other mammalian host cells.
Keywords: Human Factor IX; Keratinocyte; Hemophilia B; Keratin 14 gene promoter; Ex vivo gene therapy

Use of Mesophilic Fungal Amylases Produced by Solid-state Fermentation in the Cold Hydrolysis of Raw Babassu Cake Starch by Aline Machado de Castro; Thiago Vetter de Andréa; Leda dos Reis Castilho; Denise Maria Guimarães Freire (1612-1625).
Amylases constitute one of the most important groups of industrial enzymes, presenting several applications, such as in the food, textile, and ethanol manufacturing. In this work, a starchy residue from the Brazilian agroindustry, namely babassu cake, was used for the production of amylases by solid-state fermentation and for obtaining sugar hydrolysates, which can be used as building blocks for future bioconversions. Eight filamentous fungi from the genera Aspergillus and Penicillium were screened. Regarding amylase production, A. awamori strains showed well-balanced endoamylase and exoamylase activities, while A. wentii produced an amylolytic complex much richer in the endo-acting enzymes. Simultaneous liquefaction and saccharification using the crude enzyme extracts from the four most promising fungal strains was then investigated applying DOE techniques. The highest total reducing sugar content (24.70 g L−1) was obtained by the crude extract from A. awamori IOC-3914, corresponding to a hydrolysis yield of 52% of total starch in the cake, while A. awamori IOC-3915 produced the most appropriate extract in terms of glucose release (maximum of 5.52 g L−1). Multivariate analysis of the DOE studies indicated that these extracts showed their best performance at 50–57 °C under acid conditions (pH 3.6–4.5), but were also able to act satisfactorily under milder conditions (36 °C and pH 5.0), when TRS and glucose released were about 65% of the maximum values obtained. These data confirm the high potential of the enzyme extracts under study for cold hydrolysis of starch.
Keywords: Mesophilic amylases; Babassu cake; Aspergillus ; Filamentous fungi; Raw starch hydrolysis; Desirability function; Cold hydrolysis; Simultaneous liquefaction and saccharification

Isolation and Identification of Two New Fungal Strains for Xylanase Production by Yasser Bakri; Magali Masson; Philippe Thonart (1626-1634).
Fungi are well known for their ability to excrete enzymes into the environment. The aim of this work was to evaluate xylanase production by fungi isolated from soil. One hundred and thirty-six fungal isolates were screened for xylanase production. Two xylanase producing isolates, FSS117 and FSS129, were identified on the basis of analyses of 5,8S gene sequencing. The closest phylogenetic neighbors according to 5,8S gene sequence data for the two isolates were Aspergillus tubingensis and Aspergillus terreus, respectively. When birchwood xylan or corn cob hulls was used as a substrate for 5 days under submerged culture cultivation, xylanase production from A. terreus FSS129 was 113 and 174 IU ml−1, respectively. The pH and temperature for optimum xylanase activity were 8 and 65 ºC.
Keywords: Aspergillus terreus ; A. tubingensis ; Xylanase; Submerged culture

A newly isolated thermophilic fungal strain from Tunisian soil samples was identified as Talaromyces thermophilus and was selected for its ability to produce extracellular hemicellulases when grown on various lignocellulosic substrates. Following the optimization of carbon source, nitrogen source, and initial pH of the growth medium in submerged liquid cultures, yields as high as 10.00 ± 0.15 and 0.21 ± 0.02 U/ml were obtained for xylanase and β-xylosidase, respectively. In fact, wheat bran was found to be a good inducer of hemicellulase enzymes, mainly β-xylosidase. The optimal temperature and pH of the xylanase activity were 75°C and 8.0, respectively. This enzyme exhibited a remarkable stability and retained 100% of its original activity at 50°C for 7 days at pH 7.0–8.0. The half-lives of the enzyme were 4 h at 80°C, 2 h at 90°C, and 1 h at 100°C. T. thermophilus could therefore be considered as a satisfactory and promising producer of thermostable xylanases. Crude enzyme of T. thermophilus rich in xylanase and β-xylosidase was established for the hydrolysis of lignocellulosic materials as wheat bran.
Keywords: Xylanase; β-xylosidase; Talaromyces thermophilus ; Wheat bran; Xylose

Potential of Biocellulose Nanofibers Production from Agricultural Renewable Resources: Preliminary Study by Yaser Dahman; Kithsiri E. Jayasuriya; Magdalina Kalis (1647-1659).
In the present preliminary study, we report results for the biocellulose nanofibres production by Gluconacetobacter xylinus. Production was examined by utilizing different feedstocks of single sugars and sugar mixtures with compositions similar to the acid hydrolyzates of different agriculture residues. Profiles for cell proliferation, sugar consumption, and the subsequent pH changes were thoroughly analyzed. Highest biocellulose production of 5.65 g/L was achieved in fructose medium with total sugar consumption of 95.57%. Moreover, the highest production using sugar mixtures was 5.2 g/L, which was achieved in feedstock with composition identical to the acid hydrolyzate of wheat straws. This represented the highest biocellulose yield of 17.72 g/g sugars compared with 14.77 g/g fructose. The lowest production of 1.1 and 1.75 g/L were obtained in xylose and glucose media, respectively, while sucrose and arabinose media achieved relatively higher production of 4.7 and 4.1 g/L, respectively. Deviation in pH of the fermentation broths from the optimum value of 4–5 generally had marked effect on biocellulose production with single sugars in feedstock. However, the final pH values recorded in the different sugar mixtures were ∼3.3–3.4, which had lower effect on production hindrance. Analyzing profiles for sugars' concentrations and cell growth showed that large amount of the metabolized sugars were mainly utilized for bacterial cell growth and maintenance, rather than biocellulose production. This was clearly observed with single sugars of low production, while sugar consumption was rather utilized for biocellulose production with sugar mixtures. Results reported in this study demonstrate that agriculture residues might be used as potential feedstocks for the biocellulose nanofibres production. Not only this represents a renewable source of feedstock, but also might lead to major improvements in production if proper supplements and control were utilized in the fermentation process.
Keywords: Bacterial cellulose; Nanofibers; Feedstock; Agriculture residues; Wheat straw; Corn fibre; DDGS

Sweetgum (Liquidambar styraciflua L.): Extraction of Shikimic Acid Coupled to Dilute Acid Pretreatment by Elizabeth Martin; Julie Duke; Mathew Pelkki; Edgar C. Clausen; Danielle Julie Carrier (1660-1668).
Liquidambar styraciflua L., also known as sweetgum, is an understory hardwood species that has widespread distribution in the southeast USA, especially in pine plantations. In addition to being a possible biorefinery feedstock, sweetgum contains shikimic acid, which is a precursor for the drug Tamiflu®. Sweetgum bark was extracted with 65 °C water and yielded 1.7 mg/g of shikimic acid, while sweetgum de-barked wood yielded 0.2 mg/g of shikimic acid. Because shikimic acid can be extracted with water, the coupling of the phytochemical hot water extraction with dilute acid pretreatment was examined. The addition of a 65 °C shikimic acid extraction step coupled to pretreatment with 0.98% H2SO4 at 130 °C for 50 min resulted in 21% and 17% increases in xylose percent recovery from bark and de-barked wood, respectively. These results indicate that, in addition to recovering a high value product, the 65 °C wash step also increases xylose recovery.
Keywords: Extraction; Pretreatment; Shikimic acid; Water; Dilute acid; Sweetgum

Enzymatic Oxidation and Separation of Various Saccharides with Immobilized Glucose Oxidase by Danica Mislovičová; Vladimír Pätoprstý; Alica Vikartovská (1669-1677).
Glucose oxidase from Aspergillus niger, the specific enzyme for β-d-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of d-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for d-glucose and other saccharides examined. As a result, k cat/K M ratio for d-glucose was determined to be 42 times higher than d-mannose, 61.6 times higher than d-galactose, 279 times higher than d-xylose, and 254 times higher than for d-fructose and d-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove d-glucose from d-cellobiose, d-glucose from d-xylose, and d-xylose from d-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of d-glucose and d-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease d-glucose or d-xylose content to 0–0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides d-xylose, d-cellobiose, and d-lyxose, respectively, was observed.
Keywords: Glucose oxidase; Catalase; Saccharides; Oxidation kinetics; Immobilized enzymes

β-d-Galactosidase from Enterobacter cloacae: Production and Some Physicochemical Properties by Anamika Ghatak; Arun Kumar Guha; Lalitagauri Ray (1678-1688).
A bacterial strain isolated from soil and identified as Enterobacter cloacae had been found to be capable of producing both intra and extracellular β-d-galactosidase.The intracellular enzyme was thermostable and its optimum temperature, pH and time for enzyme—substrate reaction were found to be 50 °C, 9.0 and 5 min respectively, using ONPG as substrate. The maximum β-galactosidase production in shake flask was achieved at 30 °C, pH 7.0, incubation time 72 h using 50 ml medium in 250 ml Erlenmeyer flask. Only Mg2+ stimulated the activity of enzyme. Cetyl trimethyl ammonium bromide showed stimulatory effect on catalytic activity of the enzyme whereas EDTA inhibited enzyme activity. The enzyme retained its activity upto 55 °C after incubating at that temperature for 1 h.The maximum activity of crude intracellular enzyme was 14.35 IU/mg of protein. The K m and V max values of β-galactosidase using ONPG as substrate at 50 °C were 2.805 mM and 37.45 × 10−3 mM/min/mg, respectively.
Keywords: β-Galactosidase; Enterobacter cloacae ; Intracellular; Production; Thermostable

Aerobic Biodegradation of a Sulfonated Phenylazonaphthol Dye by a Bacterial Community Immobilized in a Multistage Packed-Bed BAC Reactor by Alfredo Ruiz-Arias; Cleotilde Juárez-Ramírez; Daniel de los Cobos-Vasconcelos; Nora Ruiz-Ordaz; Angélica Salmerón-Alcocer; Deifilia Ahuatzi-Chacón; Juvencio Galíndez-Mayer (1689-1707).
A microbial community able to aerobically degrade the azo dye Acid Orange 7 was selected from riparian or lacustrine sediments collected at sites receiving textile wastewaters. Three bacterial strains, pertaining to the genera Pseudomonas, Arthrobacter, and Rhizobium, constitute the selected community. The biodegradation of AO7 was carried out in batch-suspended cell culture and in a continuously operated multistage packed-bed BAC reactor. The rapid decolorization observed in batch culture, joined to a delay of about 24 h in COD removal and cell growth, suggests that enzymes involved in biodegradation of the aromatic amines generated after AO7 azo-bond cleavage (1-amino-2-naphthol [1-A2N] and 4-aminobenzenesulfonic acid [4-ABS]), are inducible in this microbial consortium. After this presumptive induction period, the accumulated byproducts, measured through COD, were partially metabolized and transformed in cell mass. At all azo dye loading rates used, complete removal of AO7 and 1-A2N was obtained in the multistage packed-bed BAC reactor (PBR).; however, the overall COD (η COD ) and 4-ABS (η ABS ) removal efficiencies obtained in steady state continuous culture were about 90%. Considering the toxicity of 1-A2N, its complete removal has particular relevance. In the first stages of the packed-bed BAC reactor (Fig. 4a–c), major removal was observed. In the last stage, only a slight removal of COD and 4-ABS was obtained. Comparing to several reported studies, the continuously operated multistage packed-bed BAC reactor showed similar or superior results. In addition, the operation of large-packed-bed BAC reactors could be improved by using several shallow BAC bed stages, because the pressure drop caused by bed compaction of a support material constituted by small and fragile particles can be reduced.
Keywords: Acid Orange 7; Sulfanilic acid; 4-aminobenzenesulfonic acid; 1-amino-2-naphthol; Packed-bed reactor; Biofilm; Biodegradation; Arthrobacter ; Rhizobium

AnSBBR Applied to the Treatment of Metalworking Fluid Wastewater: Effect of Organic and Shock Load by Pedro P. Carvalhinha; Anderson Flôres; José A. D. Rodrigues; Suzana M. Ratusznei; Marcelo Zaiat; Eugenio Foresti (1708-1724).
An investigation was performed regarding the application of a mechanically stirred anaerobic sequencing batch biofilm reactor containing immobilized biomass on inert polyurethane foam (AnSBBR) to the treatment of soluble metalworking fluids to remove organic matter and produce methane. The effect of increasing organic matter and reactor fill time, as well as shock load, on reactor stability and efficiency have been analyzed. The 5-L AnSBBR was operated at 30 °C in 8-h cycles, agitation of 400 rpm, and treated 2.0 L effluent per cycle. Organic matter was increased by increasing the influent concentration (500, 1,000, 2,000, and 3,000 mg chemical oxygen demand (COD)/L). Fill times investigated were in the batch mode (fill time 10 min) and fed-batch followed by batch (fill time 4 h). In the batch mode, organic matter removal efficiencies were 87%, 86%, and 80% for influent concentrations of 500, 1,000, and 2,000 mgCOD/L (1.50, 3.12, and 6.08 gCOD/L.d), respectively. At 3,000 mgCOD/L (9.38 gCOD/L.d), operational stability could not be achieved. The reactor managed to maintain stability when a shock load twice as high the feed concentration was applied, evidencing the robustness of the reactor to potential concentration variations in the wastewater being treated. Increasing the fill time to 4 h did not improve removal efficiency, which was 72% for 2,000 mgCOD/L. Thus, gradual feeding did not improve organic matter removal. The concentration of methane formed at 6.08 gCOD/L was 5.20 mmolCH4, which corresponded to 78% of the biogas composition. The behavior of the reactor during batch and fed-batch feeding could be explained by a kinetic model that considers organic matter consumption, production, and consumption of total volatile acids and methane production.
Keywords: AnSBBR; Metalworking fluid wastewater; Organic load; Fill time; Shock load

Biodiesel Preparation from Jatropha curcas Oil Catalyzed by Hydrotalcite Loaded With K2CO3 by Guangyuan Teng; Lijing Gao; Guomin Xiao; Hu Liu; Jianhua Lv (1725-1736).
This paper discusses the synthesis of biodiesel catalyzed by solid base of K2CO3/HT using Jatropha curcas oil as feedstock. Mg–Al hydrotalcite was prepared using co-precipitation methods, in which the molar ratio of Mg to Al was 3:1. After calcined at 600 °C for 3 h, the Mg–Al hydrotalcite and K2CO3 were grinded and mixed according to certain mass ratios, in which some water was added. The mixture was dried at 65 °C, and after that it was calcined at 600 °C for 3 h. Then, this Mg–Al hydrotalcite loaded with potassium carbonate was obtained and used as catalyst in the experiments. Analyses of XRD and SEM characterizations for catalyst showed the metal oxides formed in the process of calcination brought about excellent catalysis effect. In order to achieve the optimal technical reaction condition, five impact factors were also investigated in the experiments, which were mass ratio, molar ratio, reaction temperature, catalyst amount and reaction time. Under the best condition, the biodiesel yield could reach up to 96%.
Keywords: Biodiesel; Transesterification; Hydrotalcite; Solid base; Jatropha curcas oil

Deficiency of Cellulase Activity Measurements for Enzyme Evaluation by Scott W. Pryor; Nurun Nahar (1737-1750).
Switchgrass was used as a model feedstock to determine the influence of pretreatment conditions and biomass quality on enzymatic hydrolysis using different enzyme products. Dilute sulfuric acid and soaking in aqueous ammonia pretreatments were used to produce biomass with varied levels of hemicellulose and lignin sheathing. Pretreated switchgrass solids were tested with simple enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) with three commercial enzyme products: Accellerase 1000 (Genencor), Spezyme CP (Genencor)/Novozyme 188 (Novozymes), and Celluclast/Novozyme 188 (Novozymes). Enzymes were loaded on a common activity basis (FPU/g cellulose and CBU/g cellulose). Despite identical enzyme loadings, glucose yields were significantly different for both acid and alkaline pretreatments but differences diminished as hydrolysis progressed for acid-pretreated biomass. Cellobiose concentrations in Accellerase treatments indicated an initial β-glucosidase limitation that became less significant over time. SSF experiments showed that differences in glucose and ethanol yields could not be attributed to enzyme product inhibition. Yield discrepancies of glucose or ethanol in acid pretreatment, alkaline pretreatment, and acid pretreatment/SSF were as much as 15%, 19%, and 5%. These results indicate that standardized protocols for measuring enzyme activity may not be adequate for assessing activity using pretreated biomass substrates.
Keywords: Cellulase activity; Enzymatic hydrolysis; Pretreatment; Cellobiose; β-glucosidase

Metabolic Effects of the Initial Glucose Concentration on Microbial Production of Hyaluronic Acid by Aline Mara Barbosa Pires; Maria Helena A. Santana (1751-1761).
The objective of the present work was to evaluate the metabolic effects induced by the initial glucose concentration (IGC) on the cultivation of Streptococcus zooepidemicus for the production of hyaluronic acid (HA). These effects were monitored along non-controlled pH cultivations, carried out in 250-mL Erlenmeyer flasks (natural aeration) and in a 3-L bioreactor (forced aeration) as well. Effects of the IGC were observed with focus on the main metabolites, cell growth, production, and average molecular weight of HA. The absence of glucose resulted in a mixed acid metabolism independent of the oxygen supply, while, for IGCs ranging from 5 to 90 g L-1, the homolactic metabolism was prevalent. The IGC had no influence on the amounts of either biomass or HA produced in the cultivations carried out in flasks; however, cultivations in 3-L bioreactor were found to be strongly dependent on it. The highest concentration of HA (1.21 g L-1) was obtained from 25 g L-1 IGC, the only cultivation where the conversion of glucose to HA was higher than the one of glucose to biomass. Average molecular weight of HA increased concomitant with the IGC, independently of aeration; nevertheless, it decreased along cultivation under forced aeration, due to the shear imparted by stirring.
Keywords: Hyaluronic acid; Initial glucose concentration; Metabolites; Streptococcus zooepidemicus

In this study, a glucose feedback controller had been designed. Under the controller, the glucose concentration in l-lactic acid culture could be automatically controlled at 15 ± 1 g/L. Furthermore, l-lactic acid production by Lactobacillus rhamnosus LA-04-1 in fed-batch culture was significantly increased to 170 g/L l-lactic acid using the controller compared with 130 g/L l-lactic acid in pulse fed-batch and 135 g/L in l-lactic acid constant feed rate fed-batch cultures. The results showed that the controller was effective at glucose control in l-lactic acid culture. The control strategy could also be used in other fermentation processes.
Keywords: l-Lactic acid; Feedback; Controller; Glucose; Lactobacillus rhamnosus

Evaluation of High Solids Alkaline Pretreatment of Rice Straw by Yu-Shen Cheng; Yi Zheng; Chao Wei Yu; Todd M. Dooley; Bryan M. Jenkins; Jean S. VanderGheynst (1768-1784).
Fresh-harvested, air-dried rice straw was pretreated at a water content of 5 g H2O/g straw using sodium hydroxide (NaOH) and compared to pretreatment at 10 g H2O/g straw by hydrated lime (Ca(OH)2). Full factorial experiments including parallel wash-only treatments were completed with both sources of alkali. The experiments were designed to measure the effects of alkaline loading and pretreatment time on delignification and sugar yield upon enzymatic hydrolysis. Reaction temperature was held constant at 95°C for lime pretreatment and 55°C for NaOH pretreatment. The range of delignification was 13.1% to 27.0% for lime pretreatments and was 8.6% to 23.1% for NaOH pretreatments. Both alkaline loading and reaction time had significant positive effects (p < 0.001) on delignification under the design conditions, but only alkaline loading had a significant positive effect on enzymatic hydrolysis. Treatment at higher temperature also improved delignification; delignification with water alone ranged from 9.9% to 14.5% for pretreatment at 95°C, but there was little effect observed at 55°C. Post-pretreatment washing of biomass was not necessary for subsequent enzymatic hydrolysis. Maximum glucose yields were 176.3 mg/g dried biomass (48.5% conversion efficiency of total glucose) in lime-pretreated and unwashed biomass and were 142.3 mg/g dried biomass (39.2% conversion efficiency of total glucose) in NaOH-pretreated and unwashed biomass.
Keywords: Lime; Calcium hydroxide; Sodium hydroxide; Alkaline pretreatment; Biomass; Rice straw; Delignification; Enzymatic hydrolysis

Research on biomass pretreatment to enhance enzymatic digestibility has been done for more than decades, but a viable continuous pretreatment method needs to be developed. Extrusion has the potential to be a viable continuous pretreatment method. This study investigated the effect of compression ratio (2:1 and 3:1), screw speed (50, 100, and 150 rpm), and barrel temperature (50, 100, and 150 °C) on the sugar recovery from switchgrass (SG) and prairie cord grass (PCG) over a range of moisture contents (15, 25, 35, and 45% wb).The pretreated samples were subjected to enzymatic hydrolysis for sugar recovery measurement. Statistical analyses revealed that a 3:1 screw compression ratio (compared to 2:1) increased glucose recovery by 12% and 8% and combined sugar recovery by 37% and 40% for SG and PCG, respectively. For SG, the highest sugar recovery (45.2%) was obtained at the lowest screw speed (50 rpm) and the highest temperature (150 °C) with moisture content of 15%. The highest glucose, xylose, and combined sugar recovery of 61.4%, 84.3%, and 65.8% were recorded for PCG extruded at a screw speed of 50 rpm and a temperature of 50 °C with a moisture content of 25%. Glycerol and acetic acid were byproducts found in low concentration (0.02–0.18 g/L) for both biomass.
Keywords: Biomass; Pretreatment; Switchgrass; Prairie cord grass; Screw speed; Temperature; Extruder

Streptomyces sp. 7b showed highest xylanase activity among 41 bacterial isolates screened under submerged fermentation. The organism grew over broad pH (5–11) and temperatures range (25–55 °C) and displayed maximum xylanase production on wheat bran (1230 U/g) under solid-state fermentation. Xylanase production was enhanced substantially (76%–77%) by inclusion of trypton (2180 U/g) or beef extract (2170 U/g) and moderately (36%–46%) by yeast extract (1800 U/g) or soybean meal (1670 U/g). Inclusion of readily utilizable sugars such as glucose, maltose, fructose, lactose or xylose in the substrate repressed the xylanase production. The optimum initial pH of the medium for maximum enzyme production was 7 to 8; however, appreciable level of activity was obtained at pH 6 (1,680 U/g) and 9 (1,900 U/g). Most appropriate solid to liquid ratio for maximum xylanase production in solid-state fermentation was found to be 1:2.5. The organism produced a single xylanase of molecular weight of approximately 30 kDa as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis after purification with ammonium sulfate precipitation, and carboxy methyl sephadex chromatography. The enzyme was purified to the extent of 5.68-fold by salt precipitation and ion-exchange chromatography. Optimum temperature and pH for maximum xylanase activity were 50 °C and 6, respectively.
Keywords: Xylanase; Solid-state fermentation; Streptomyces sp. 7b; Wheat bran; Optimization; Purification