Applied Biochemistry and Biotechnology (v.162, #8)

The Influence of Mineral Ions on the Microbial Production and Molecular Weight of Hyaluronic Acid by Aline Mara B. Pires; Silvia Y. Eguchi; Maria Helena Andrade Santana (2125-2135).
This study aimed to evaluate the influence of the culture medium supplementation with mineral ions, focusing on the growth of Streptococcus zooepidemicus as well as on the production and average molecular weight (MW) of hyaluronic acid (HA). The ions were investigated in terms of individual absence from the totally supplemented medium (C+) or individual presence in the non-supplemented medium (C−), where C+ and C− were used as controls. Differences between the effects were analyzed using the Tukey's test at p < 0.05. The adopted criteria considered required the ions, whose individual absence attained at 80% or less of the C+ and their individual presence was 20% or more than the C−. The supplementation was either inhibitory or acted in synergy with other ions, when the individual absence or presence was 20% higher than C+ or 20% lower than C−, respectively. Results showed that the effects of C+ or C− were equal for both the production of HA and its yield from glucose. However, C+ showed to be beneficial to cell growth while the individual absence of Na+ was beneficial to the production of HA. The highest MW of HA (7.4 × 107 Da) was observed in the individual presence of Na+ in spite of the lowest HA concentration (0.65 g.L−1). These results suggest that the quality of HA can be modulated through the mineral ion supplementation.
Keywords: Hyaluronic acid; Ion supplementation; Streptococcus zooepidemicus

A β-glucosidase gene designated gluc3m was cloned through construction of a genomic library of Martelella mediterranea 2928. The gluc3m consisted of 2,496 bp and encoded a peptide of 832 amino acids that shared the greatest amino acid similarity (59%) with a β-glucosidase of family 3 glycoside hydrolase from Agrobacterium radiobacter K84. The optimum reaction temperature and pH of Gluc3M were 45 °C and 8.0, respectively. The K m and V max for p-nitrophenyl-β-d-glucopyranoside were 0.18 mg/ml and 196.08 µmol/min/mg enzyme, respectively. Gluc3M was found to be highly alkali stable, retaining 80% of its maximum enzymatic activity after treatment with pH 11.0 buffers for 24 h. Furthermore, the activity of Gluc3M improved remarkably in the presence of univalent metal ions, whereas it was inhibited in the presence of divalent ions. Gluc3M also exhibited significant activities toward various substrates including pNPGlu, pNPGal, salicin, and konjac powder. It is important to note that Gluc3M is a cold-active enzyme that showed over 50% of the maximum enzymatic activity at 4 °C. SWISS-MODEL revealed that the amino acids near the conserved domain SDW of Gluc3M contributed to the cold-active ability. Based on these characteristics, Gluc3M has the potential for use in additional studies and for industrial applications.
Keywords: β-Glucosidase; Martelella mediterranea ; Glycosyl hydrolase family 3; Cold active; Alkaline stability

Substrate concentration in lactic acid fermentation broth could not be controlled well by traditional feeding methods, including constant, intermittent, and exponential feeding methods, in fed-batch experiments. A simple feedback feeding method based on pH was proposed to control pH and substrate concentration synchronously to enhance lactic acid production in fed-batch culture. As the linear relationship between the consumption amounts of alkali and that of substrate was concluded during lactic acid fermentation, the alkali and substrate in the feeding broth were mixed together proportionally. Thus, the concentration of substrate could be controlled through the adjustment of pH automatically. In the fed-batch lactic acid fermentation with Lactobacillus lactis-11 by this method, the residual glucose concentration in fermentation broth was controlled between 4.1 and 4.9 g L−1, and the highest concentration of lactic acid, maximum cell dry weight, volumetric productivity of lactic acid, and yield were 96.3 g L−1, 4.7 g L−1, 1.9 g L−1 h−1, and 0.99 g lactic acid per gram of glucose, respectively, compared to 82.7 g L−1, 3.31 g L−1, 1.7 g L−1 h−1, and 0.92 g lactic acid per gram of glucose in batch culture. This feeding method was simple and easily operated and could be feasible for industrial lactic acid production in the future.
Keywords: Lactic acid; Fed-batch; Lactobacillus lactis-11; Ammonium hydroxide; pH feedback

High Level Expression of an Acid-Stable Phytase from Citrobacter freundii in Pichia pastoris by Wei Zhao; Aisheng Xiong; Xiaoyan Fu; Feng Gao; Yongsheng Tian; Rihe Peng (2157-2165).
To obtain a high level expression of phytase with favorable characteristics, a codon-optimized phytase gene from Citrobacter freundii was synthesized and transferred into Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. After purified by Ni2+–NTA agarose affinity column, the characterizations of the recombinant phytase were determined. The recombinant phytase (r-phyC) had two distinct pH optima at 2.5 and 4.5 and an optimal temperature at 50 °C. It retained more than 80% activity after being incubated under various buffer (pH 1.5–8.0) at 37 °C for 1 h. The specific activity, Km, and Vmax values of r-phyC for sodium phytate were 2,072 ± 18 U mg−1, 0.52 ± 0.04 mM, and 2,380 ± 84 U mg−1 min−1, respectively. The enzyme activity was significantly improved by 1 mM of K+, Ca2+, and Mg2+. These characteristics contribute to its potential application in feed industry.
Keywords: Citrobacter freundii ; Codon optimization; Pichia pastoris ; pH-stable; Phytase

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Rhodotorula glutinis CCMI 145 cells grown in shake flasks. Changes in the side light scatter and forward light scatter were detected during the yeast batch growth, which were attributed to the different yeast growth phases. A progressive increase in the proportion of cells stained with PI (cells with permeabilized cytoplasmic membrane) was observed during the yeast growth, attaining 79% at the end of the fermentation. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional gravimetric lipid analysis was found for this yeast, making this method a suitable and quick technique for the screening of yeast strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of the yeast oil production is now in progress.
Keywords: Lipids; Rhodotorula glutinis ; Flow cytometry; Nile red; Light scatter

Fucoidan-Degrading Fungal Strains: Screening, Morphometric Evaluation, and Influence of Medium Composition by Rosa M. Rodríguez-Jasso; Solange Inês Mussatto; Lorenzo Pastrana; Cristóbal N. Aguilar; José A. Teixeira (2177-2188).
Ten different fungal strains from the genus Aspergillus, Penicillium, and Mucor were screened for fucoidan hydrolyzing ability aiming to find microorganisms able to produce sulfated fucan-degrading enzymes. Screening was carried out by measuring the strains kinetic and morphometric behavior over plate assays using Laminaria japonica fucoidan as only carbon source, testing three nitrogen sources (urea, peptone, and sodium nitrate). The selected fungal strains were subsequently used in submerged fermentations, which were performed for (1) selection of the strains able to growth over fucoidan medium and (2) media selection, testing the synergy of fucoidan with other sugars for inducing high enzyme titles. Radial expansion and hyphae parameters were observed for Aspergillus niger PSH, Mucor sp. 3P, and Penicillium purpurogenum GH2 grown only over fucoidan-urea medium. A. niger PSH showed the maximum enzymatic activity values, which were significantly different (p < 0.05) from those achieved by the other selected fungi. Sucrose addition to fucoidan media proportioned the highest fucoidanase activity values for this fungal strain. This research allowed establishing optimal conditions for metabolites synthesis by fungal stains able to act toward fucoidan ramified matrix.
Keywords: Fucoidan; Fucoidanases; Fungal strains; Screening; Fermentation

Enzyme biosensor based on the immobilization of HRP on SiO2/BSA/Au composite nanoparticles by Shirong Yuan; Ruo Yuan; Yaqin Chai; Ying Zhuo; Xia Yang; Yali Yuan (2189-2196).
In this work, an enzyme biosensor based on the immobilization of horseradish peroxidase (HRP) on SiO2/BSA/Au/thionine/nafion-modified gold electrode was fabricated successfully. Firstly, nafion was dropped on the surface of the gold electrode to form a nafion film followed by chemisorption of thionine (Thi) as an electron mediator via the ion-exchange interaction between the Thi and nafion. Subsequently, the SiO2/BSA/Au composite nanoparticles were assembled onto Thi film through the covalent bounding with the amino groups of Thi. Finally, HRP was immobilized on the SiO2/BSA/Au composite nanoparticles due to the covalent conjugation to construct an enzyme biosensor. The surface topographies of the SiO2/BSA/Au composite nanoparticles were investigated by using scanning electronic microscopy. The stepwise self-assemble procedure of the biosensor was further characterized by means of cyclic voltammetry and chronoamperometry. The enzyme biosensor showed high sensitivity, good stability and selectivity, a wide linear response to hydrogen peroxide (H2O2) in the range of 8.0 × 10-6 ∼ 3.72 × 10-3 mol/L, with a detection limit of 2.0 × 10-6 mol/L. The Michaelies-Menten constant $$ K_M^{app} $$ value was estimated to be 2.3 mM.
Keywords: SiO2/BSA/Au composite nanoparticles; Nafion (Nf); Thionine (Thi); HRP; Hydrogen peroxide (H2O2)

Catalytic properties of two glucoamylases, AmyC and AmyD, without starch binding domains from Rhizopus oryzae strain 99-880 are determined using heterologously expressed enzyme purified to homogeneity. AmyC and AmyD demonstrate pH optima of 5.5 and 6.0, respectively, nearly one unit higher than the Rhizopus AmyA glucoamylase enzyme. Optimal initial activities are at 60 and 50 °C for AmyC and AmyD, respectively. Inactivation of both enzymes occurs at 50 °C following 30 min pre-incubation. The two enzymes demonstrate substantially slower catalytic rates toward soluble starch relative to AmyA. AmyC has similar k cat and K m for oligosaccharides to other Rhizopus and Aspergillus glucoamylases; however, the enzyme has a 2-fold lower K m maltose . AmyD has a 3-fold higher K m and lower k cat for maltooligosaccharides than AmyC and other glucoamylases. AmyC (but not AmyD) exhibits substrate inhibition. K i for substrate inhibition decreases with increasing length of the oligosaccharides. Data from pre-steady-state binding of AmyC to maltose and maltotriose and pre-steady-state to steady-state catalytic turnover experiments of AmyC acting on maltotriose were used to interrogate models of substrate inhibition. In the preferred model, AmyC accumulates an enzyme-maltose-maltotriose dead-end complex in the steady state.
Keywords: Glucoamylase; Glycoside hydrolase; Substrate inhibition; Enzyme mechanism; Pre-steady-state kinetics; Rhizopus oryzae ; Pichia pastoris

Ethanol Production Using Immobilized Saccharomyces cerevisiae in Lyophilized Cellulose Gel by Eleonora Winkelhausen; Elena Velickova; Samuel A. Amartey; Slobodanka Kuzmanova (2214-2220).
A new lyophilization technique was used for immobilization of Saccharomyces cerevisiae cells in hydroxyethylcellulose (HEC) gels. The suitability of the lyophilized HEC gels to serve as immobilization matrices for the yeast cells was assessed by calculating the immobilization efficiency and the cell retention in three consecutive batches, each in duration of 72 h. Throughout the repeated batch fermentation, the immobilization efficiency was almost constant with an average value of 0.92 (12–216 h). The maximum value of cell retention was 0.24 g immobilized cells/g gel. Both parameters indicated that lyophilized gels are stable and capable of retaining the immobilized yeast cells. Showing the yeast cells propagation within the polymeric matrix, the scanning electron microscope images also confirmed that the lyophilization technique for immobilization of S. cerevisiae cells in the HEC gels was successful. The activity of the immobilized yeast cells was demonstrated by their capacity to convert glucose to ethanol. Ethanol yield of 0.40, 0.43 and 0.30 g ethanol/g glucose corresponding to 79%, 84% and 60% of the theoretical yield was attained in the first, second and third batches, respectively. The cell leakage was less than 10% of the average concentration of the immobilized cells.
Keywords: Lyophilized gels; Hydroxyethylcellulose; Immobilization; Saccharomyces cerevisiae ; Ethanol

Microbial Oxidation of (-)-α-pinene to Verbenol Production by Newly Isolated Strains by Ieda Rottava; Priscila F. Cortina; Camila A. Zanella; Rogerio L. Cansian; Geciane Toniazzo; Helen Treichel; Octavio A. C. Antunes; Enrique G. Oestreicher; Debora de Oliveira (2221-2231).
Verbenol is a bicyclicbicycle secondary allylic alcohol, with pronounced camphor and mint flavor notes, mainly used as food flavoring. This compound is also used to control harmful insects, and hence has potential for using in agriculture, and is an intermediate in the synthesis of valuable perfume and medicinal substances. This work is focused on the microbial oxidation of (-)-α-pinene to verbenol production. To carry out the present study, 405 microorganisms were tested for their ability to bioconvert the substrate. From the isolated microorganisms, 193 were selected in the pre-screening using mineral medium for limonene degradation. At the screening step, 31 strains were able to convert (-)-α-pinene in verbenol. The highest concentration in verbenol from (-)-α-pinene was about 125.6 mg/L for yeast isolated from orange juice industrial residue.
Keywords: Screening; Biotransformation; (-)-α-Pinene; Verbenol; Monoterpenes

Application of Supermacroporous Monolithic Hydrophobic Cryogel in Capturing of Albumin by Nesibe Avcibaşi; Murat Uygun; M. Emin Çorman; Sinan Akgöl; Adil Denizli (2232-2243).
Supermacroporous poly{2-hydroxyethyl methacrylate-co-[N,N-bis(2,6-diisopropylphenyl)-perylene-3,4,9,10-tetracarboxylic diimide]} [poly(HEMA-co-DIPPER)] monolithic cryogel column was prepared by radical cryocopolymerization of HEMA with DIPPER as functional comonomer and N,N′-methylene-bisacrylamide (MBAAm) as crosslinker directly in a plastic syringe for adsorption of albumin. The monolithic cryogel contained a continuous polymeric matrix having interconnected pores of 10–50 μm size. Poly(HEMA-co-DIPPER) cryogel was characterized by swelling studies, FTIR, scanning electron microscopy, and elemental analysis. The equilibrium swelling degree of the poly(HEMA-co-DIPPER) cryogel was 14.7 g H2O/g dry cryogel. Poly(HEMA-co-DIPPER) cryogel was used in the adsorption/desorption of albumin from aqueous solutions. The nonspecific adsorption of albumin onto plain poly(HEMA) cryogel was very low (3.36 g/g polymer). The maximum amount of albumin adsorption from aqueous solution in acetate buffer was 40.9 mg/g polymer at pH 5.0. It was observed that albumin could be repeatedly adsorbed and desorbed with the poly(HEMA-co-DIPPER) cryogel without significant loss of adsorption capacity.
Keywords: Cryogel; Affinity separation; Albumin; HEMA

In this study, we evaluated the feasibility of solid-state fermentation (SSF) on polyurethane foam (PUF) for xanthan production. The effects of air pressure pulsation (APP) on biomass accumulation and final xanthan concentration were also studied. Under suitable conditions (15% inoculum, 0.5-cm (side length) PUF cubes, 15 mL medium per gram cubes and 4.5 cm bed depth), the broth was dispersed on the PUF as a film. When the initial glucose concentration in the media was low (20 and 40 g L−1), there was no significant difference between the final xanthan concentration in static SSF and submerged fermentation (SMF). When high initial glucose concentrations (60 and 80 g L−1) were used, the final gum concentrations in SSF were much higher than those in SMF. When the APP technique was applied in xanthan production with a medium containing a high glucose concentration (80 g L−1), the oxygen consumption rate of Xanthomonas campestris was significantly enhanced at the later stages of fermentation, and both the biomass and xanthan concentration were improved. The results indicated that SSF on PUF is suitable for xanthan preparation, especially when the initial glucose concentration ranged from 60 to 80 g L−1. Those results also demonstrated that APP technology can be used to enhance xanthan yields.
Keywords: Xanthan gum; Solid-state fermentation; Inert support; Air pressure pulsation; Xanthomonas campestris

Industrial Sustainability of Competing Wood Energy Options in Canada by Emmanuel K. Ackom; Warren E. Mabee; John N. Saddler (2259-2272).
The amount of sawmill residue available in Canada to support the emerging cellulosic ethanol industry was examined. A material flow analysis technique was employed to determine the amount of sawmill residue that could possibly be available to the ethanol industry per annum. A combination of two key trends—improved efficiency of lumber recovery and increased uptake of sawmill residues for self-generation and for wood pellet production—have contributed to a declining trend of sawmill residue availability. Approximately 2.3 × 106 bone-dry tonnes per year of sawmill residue was estimated to be potentially available to the cellulosic ethanol industry in Canada, yielding 350 million liters per year of cellulosic ethanol using best practices. An additional 2.7 billion liters of cellulosic ethanol might be generated from sawmill residue that is currently used for competing wood energy purposes, including wood pellet generation. Continued competition between bioenergy options will reduce the industrial sustainability of the forest industry. Recommendations for policy reforms towards improved industrial sustainability practices are provided.
Keywords: Cellulosic ethanol; Wood pellets; Industrial sustainability; Material flow analysis; Sawmill residues

Thiazolidinediones as a Novel Class of Algicides Against Red Tide Harmful Algal Species by Yeon-Mi Kim; Ying Wu; Thi Uyen Duong; Gajanan S. Ghodake; Si Wouk Kim; EonSeon Jin; Hoon Cho (2273-2283).
This paper reports the synthesis of 28 thiazolidinedione derivatives along with their algicidal activity against microalgae causing harmful algal blooming. Among the 28 compounds tested, most showed effective algicidal activity against Heterosigma akashiwo, Chattonella marina, and Cochlodinium polykrikoides, while non-harmful algae were relatively tolerant to these thiazolidinedione derivatives. Compounds 6, 13, and 22 were the most potent against C. polykrikoides with IC50 values <0.5 µM. Among the thiazolidinedione derivatives tested, compounds 7, 13, 27, and 28 were extremely competent and selective to C. polykrikoides with IC50 values ranging from 0.1 to 2 µM, while C. marina and H. akashiwo showed an IC50 value ranging from 30 to 130 µM. These results show that some thiazolidinedione derivatives can act as potent algicides against harmful algal blooms.
Keywords: Harmful algal blooms; Toxicity; Thiazolidinediones; Algicides; Red tide

The random bacterial genomic DNA microarray chip (RBGDMC), which was fabricated using random genomic DNA fragments obtained from the fragmentation of bacterial genome by using four different pairs of restriction enzymes, was found to discriminate bacterial species in the same genus and resulted in the determination of dominant bacteria in enriched cultures. The identification of a dominant bacterial species was successfully conducted in the co-culture of three different bacteria using the RBGDMC. In addition, the analysis of the chip data could confirm if any of the selected bacteria is the most abundant or if some bacteria were enriched and became the dominant species within the consortium after the samples were prepared from the repeated cultures of real sludge in a complex medium. This study shows the successful implementation of the RBGDMC for the identification and monitoring of dominant bacteria in complex environmental bacterial communities simply without any PCR amplification of the target nucleic acids.
Keywords: Random fragmentation; Genomic DNA; DNA microarray chip; Bacteria identification; Complex culture

Insights into pH-Induced Conformational Transition of β-Galactosidase from Pisum sativum Leading to its Multimerization by Alka Dwevedi; Vikash Kumar Dubey; Medicherla V. Jagannadham; Arvind M. Kayastha (2294-2312).
Although β-galactosidases are physiologically a very important enzyme and have may therapeutics applications, very little is known about the stability and the folding aspects of the enzyme. We have used β-galactosidase from Pisum sativum (PsBGAL) as model system to investigate stability, folding, and function relationship of β-galactosidases. PsBGAL is a vacuolar protein which has a tendency to multimerize at acidic pH with protein concentration ≥100 μg mL−1 and dissociates into its subunits above neutral pH. It exhibits maximum activity as well as stability under acidic conditions. Further, it has different conformational orientations and core secondary structures at different pH. Substantial predominance of β-content and interfacial interactions through Trp residues play crucial role in pH-dependent multimerization of enzyme. Equilibrium unfolding of PsBGAL at acidic pH follows four-state model when monitored by changes in the secondary structure with two intermediates: one resembling to molten globule-like state while unfolding seen from activity and tertiary structure of PsBGAL fits to two-state model. Unfolding of PsBGAL at higher pH always follows two-state model. Furthermore, unfolding of PsBGAL reveals that it has at least two domains: α/β barrel containing catalytic site and the other is rich in β-content responsible for enzyme multimerization.
Keywords: PsBGAL; Secondary structure; Intrinsic fluorescence; Hydrophobic domains

Formic Acid as a Potential Pretreatment Agent for the Conversion of Sugarcane Bagasse to Bioethanol by Raveendran Sindhu; Parameswaran Binod; Karri Satyanagalakshmi; Kanakambaran Usha Janu; Kuttavan Valappil Sajna; Noble Kurien; Rajeev Kumar Sukumaran; Ashok Pandey (2313-2323).
In recent years, growing attention has been focused on the use of lignocellulosic biomass as a feedstock for the production of ethanol, a possible renewable alternative to fossil fuels. Several pretreatment processes have been developed for decreasing the biomass recalcitrance, but only a few of them seem to be promising. In this study, effect of various organic solvents and organic acids on the pretreatment of sugarcane bagasse was studied. Among the different organic acids and organic solvents tested, formic acid was found to be effective. Optimization of process parameters for formic acid pretreatment was carried out. The structural changes before and after pretreatment was investigated by scanning electron microscopy, X-ray diffraction (XRD), and Fourier transform infrared (FTIR) analysis. The X-ray diffraction profile showed that the degree of crystallinity was more for pretreated biomass than that of untreated. The FTIR spectra shown at the stretching of hydrogen bonds of pretreated sugarcane bagasse arose at higher number. It also revealed that the cellulose content in the solid residue increased because the hemicelluloses fraction in raw materials was released by acid hydrolytic reaction.
Keywords: Sugarcane bagasse; Bioethanol; Formic acid; Pretreatment; Lignocellulosic biomass

Semi-continuous Cultivation of Chlorella vulgaris for Treating Undigested and Digested Dairy Manures by Liang Wang; Yingkuan Wang; Paul Chen; Roger Ruan (2324-2332).
The present study, based on a previous batch-wise experiment, investigated a lab-scale semi-continuous cultivation of green microalgae Chlorella vulgaris (UTEX 2714), as a useful means for nutrient reduction as well as production of algal biomass which can be used as potential feedstock for the production of biofuel and other commodities, on 20× diluted dairy manures. Both undigested and digested samples were applied in parallel experiments for comparison regarding the requirements of hydraulic retention times (HRTs), removal efficiencies of nitrogen, phosphorus, and chemical oxygen demand (COD), biomass productivities, and CO2 sequestration abilities. It was demonstrated that algae grown in undigested dairy manure achieved removal rates of 99.7%, 89.5%, 92.0%, and 75.5% for NH 4 + –N, TN, TP, and COD, respectively, under a 5-day HRT, while the HRT had to extend to 20 days in order to achieve 100.0% removal of NH 4 + –N in digested one with simultaneous removals of 93.6% of TN, 89.2% of TP, and 55.4% of COD. The higher organic carbon contained in undigested dairy manure helped boost the growth of mixotrophic Chlorella, thus resulting in a much shorter HRT needed for complete removal of NH 4 + –N. Moreover, algae grown in digested dairy manure provided more penitential than those grown in undigested one in CO2 sequestration per milligram of harvested dried biomass (1.68 mg CO2/mg dry weight (DW) vs 0.99 mg CO2/mg DW), but did not surpass in total the amount of CO2 sequestered on a 15-day period basis because of the better productivity gained in undigested dairy manure.
Keywords: Dairy manure; Nutrients removal; CO2 sequestration; Chlorella

Strain-Dependent Carotenoid Productions in Metabolically Engineered Escherichia coli by Han Seung Chae; Kong-Hwan Kim; Sun Chang Kim; Pyung Cheon Lee (2333-2344).
Seven Escherichia coli strains, which were metabolically engineered with carotenoid biosynthetic pathways, were systematically compared in order to investigate the strain-specific formation of carotenoids of structural diversity. C30 acyclic carotenoids, diaponeurosporene and diapolycopene were well produced in all E. coli strains tested. However, the C30 monocyclic diapotorulene formation was strongly strain dependent. Reduced diapotorulene formation was observed in the E. coli strain Top10, MG1655, and MDS42 while better formation was observed in the E. coli strain JM109, SURE, DH5a, and XL1-Blue. Interestingly, C40 carotenoids, which have longer backbones than C30 carotenoids, also showed strain dependency as C30 diapotorulene did. Quantitative analysis showed that the SURE strain was the best producer for C40 acyclic lycopene, C40 dicyclic β-carotene, and C30 monocyclic diapotorulene. Of the seven strains examined, the highest volumetric productivity for most of the carotenoids structures was observed in the recombinant SURE strain. In conclusion, we showed that recombinant hosts and carotenoid structures influenced carotenoid productions significantly, and this information can serve as the basis for the subsequent development of microorganisms for carotenoids of interest.
Keywords: Carotenoids; E. coli ; Strain dependency; Metabolic engineering; Pathway engineering

Using immobilized cells of a novel strain of Microbacterium hydrocarbonoxydans L29-9 in polymers of polyvinyl alcohol (PVA)–alginate–boric acid, enantioselective resolution of racemic γ-lactam to produce (−)γ-lactam was successfully carried out. A 6:1 ratio of PVA:sodium alginate not only prevented agglomeration of the matrix but also produced beads with high gel strength. The optimum biotransformation conditions were 1 g/L substrate, pH 7.0, reaction temperature of 30 °C, and reaction time of 3 h. After every two cycles, the immobilized cell beads were separated and immersed in 0.5 mM KCl solution at 4 °C for preservation. At optimum conditions, the enantiomeric excess and the yield of (−)γ-lactam were >99% and 34%, respectively. The beads showed a slight decrease in the enantiomeric excess when re-used up to 14 cycles (the enantioselectivity of the immobilized cells decreased slightly after 14 cycles of usage).
Keywords: Abacavir; Enantioselective resolution; γ-Lactam; Immobilized Microbacterium hydrocarbonoxydans cells; Polyvinyl alcohol

Functional Expression of phaCAB Genes from Cupriavidus taiwanensis Strain 184 in Escherichia coli for Polyhydroxybutyrate Production by Chih-Ching Chien; Chian-Chiao Hong; Po-Chi Soo; Yu-Hong Wei; Shan-Yu Chen; Mei-Ling Cheng; Yi-Ming Sun (2355-2364).
Polyhydroxyalkanoates are polyesters synthesized by numerous microorganisms. These polyesters are biodegradable and have similar properties to those of conventional plastics. Cupriavidus taiwanensis strain 184 is phylogenetically related to the well-known polyhydroxybutyrate (PHB) producer Ralstonia eutropha (Cupriavidus necator) and is also shown to be able to accumulate significant amounts of PHB. In this study, we cloned the PHB synthesis genes (phaCAB) from C. taiwanensis 184 into Escherichia coli for biosynthesis of PHB. The recombinant E. coli strains were able to synthesize significant amounts of PHB. The PHB amounted to about 66∼70% of total cell material of these recombinant strains.
Keywords: Polyhydroxyalkanoates; PHA; Cupriavidus taiwanensis ; PHB; Polyhydroxybutyrate

ASBR Applied to the Treatment of Biodiesel Production Effluent: Effect of Organic Load and Fill Time on Performance and Methane Production by Vivian C. Selma; Luís H. B. Cotrim; José A. D. Rodrigues; Suzana M. Ratusznei; Marcelo Zaiat; Eugenio Foresti (2365-2380).
The effect of organic matter and fill time on anaerobic sequencing batch reactor (5 L, 30°C, 8-h cycles, 50 rpm) efficiency has been analyzed. Organic matter was increased by the influent concentration. Fill times investigated were in the batch mode and fed-batch followed by batch. In the batch mode organic matter removal were 93%, 81%, and 66% for influent concentration of 500, 1,000, and 2,000 mgCOD/L (0.6, 1.29, and 2.44 gCOD/L.d), respectively. At 3,000 mgCOD/L (3.82 gCOD/L.d) operational stability could not be achieved. Removal efficiency was improved by increasing the fill time, and was 85% for the 1,000 mgCOD/L condition and fill times of 2 and 4 h, and 80 and 77% for the 2,000 mgCOD/L condition and fill times of 2 and 4 h, respectively. Hence, gradual feeding seemed to improve and to smooth the profiles of organic matter and volatile acids along the cycle with 78 to 96 NmLCH4/gCOD.
Keywords: ASBR; Biodiesel effluent; Organic load; Fill time; Methane

The extractive acetone–butanol–ethanol (ABE) fermentations of Clostridium acetobutylicum were evaluated using biodiesel as the in situ extractant. The biodiesel preferentially extracted butanol, minimized product inhibition, and increased production of butanol (from 11.6 to 16.5 gL−1) and total solvents (from 20.0 to 29.9 gL−1) by 42% and 50%, respectively. The fuel properties of the ABE-enriched biodiesel obtained from the extractive fermentations were analyzed. The key quality indicators of diesel fuel, such as the cetane number (increased from 48 to 54) and the cold filter plugging point (decreased from 5.8 to 0.2 °C), were significantly improved for the ABE-enriched biodiesel. Thus, the application of biodiesel as the extractant for ABE fermentation would increase ABE production, bypass the energy intensive butanol recovery process, and result in an ABE-enriched biodiesel with improved fuel properties.
Keywords: Biobutanol; Biodiesel; Butanol inhibition; Clostridium ; Extractant; Low cold filter plugging point (CFPP); Solvent extractive fermentation

A lipase gene from Serratia marcescens ECU1010 was cloned into expression vector pET28a, sequenced, and overexpressed as an N terminus His-tag fusion protein in Escherichia coli. Through the optimization of culture conditions in shake flask, the lipase activity was improved up to 1.09 × 105 U/l, which is a great improvement compared to our previous reports. It was purified to homogeneity by Ni-NTA affinity chromatography with an overall yield of 59.4% and a purification factor of 2.4-fold. This recombinant lipase displayed excellent stability below 30 °C and within the pH range of 5.0−6.8, giving temperature and pH optima at 40 °C and pH 9.0, respectively. The lipase activity was found to increase in the presence of metal ions such as Ca2+, Cu2+, and some nonionic surfactants such as PEG series. In addition, among p-nitrophenyl esters of fatty acids with varied chain length, the recombinant lipase showed the maximum activity on p-nitrophenyl laurate (C12). Using racemic trans-3-(4′-methoxy-phenyl)-glycidyl methyl ester [(±)-MPGM] as substrate, which is a key chiral synthon for production of diltiazem, a 50% conversion yield was achieved after 4 h in toluene–water (100 mM KPB phosphate buffer, pH 7.5) biphasic system (5:5 ml) at 30 °C under shaking condition (160 rpm), affording (−)-MPGM in nearly 100% ee. The K m and V max values of the lipase for (±)-MPGM were 222 mM and 1.24 mmol min−1 mg−1, respectively. The above-mentioned features make the highly enantioselective lipase from Serratia marcescens ECU1010 a robust biocatalyst for practical use in large-scale production of diltiazem intermediate.
Keywords: Serratia marcescens ECU1010; Lipase; Soluble expression; Purification; Characterization; Enantioselectivity; Biocatalytic resolution

The influence of 12 biochemical stimulants, namely 2-phenylacetic acid (PAA; 30 ppm), indole-3 butyric acid (IBA; 10 ppm), 1-naphthaleneacetic acid (NAA; 2.5, 5 and 10 ppm ), gibberellic acid (GA3, 10 ppm), zeatin (ZT; 0.002 ppm), thidiazuron (0.22 ppm), humic acid (20 ppm), kelp extract (250 ppm), methanol (500 ppm), ferric chloride (3.2 ppm ), putrescine (0.09 ppm), spermidine (1.5 ppm) were prescreened for their impact on growth and chlorophyll for the green alga—Chlorella sorokiniana. C. sorokiniana responded best to phytohormones in the auxin family, particularly NAA. Thereafter, two studies were conducted on combinations of phytohormones to compare blends from within the auxin family as well as against other families. These treatments were NAA5 ppm+PAA30 ppm, NAA2.5 ppm+PAA15 ppm, NAA5 ppm+IBA10 ppm, NAA5 ppm+GA310 ppm, NAA5 ppm+ZT1 ppm, and NAA5 ppm+GA310 ppm+ZT1 ppm. Combinations of NAA with other auxins did not have synergistic or antagonistic effects on the growth. However, combinations of compounds from different phytohormone families, such as NAA5 ppm+GA310 ppm+ZT1 ppm, dramatically increased the biomass productivity by 170% over the control followed by the treatments: NAA 5 ppm+GA310 ppm (138%), NAA 5 ppm+ZT1 ppm (136%), and NAA 5 ppm ( 133%). The effect of biochemical stimulants were also measured on metabolites such as chlorophyll, protein, and lipids in C. sorokiniana. Renewed interest in microalgae for biotechnology and biofuel applications may warrant the use of biochemical stimulants for cost reduction in large-scale cultivation through increased biomass productivity.
Keywords: Auxins; Bioenergy; Biofuels; Biomass; Biostimulants; Microalgae; Phytohormones