Biochemical Engineering Journal (v.81, #C)

BEJ Keywords (IV).

α-Glucuronidase (EC 3.2.1.139) of family GH 115 from Scheffersomyces stipitis is a valuable enzyme for the modification of water-soluble xylan into insoluble biopolymers, due to its unique ability to act on polymeric xylans. The influence of growth rate on the production of α-glucuronidase by recombinant Saccharomyces cerevisiae MH1000pbk10D-glu in glucose-limited fed-batch culture was studied at 14 and 100 L scale. At and below the critical specific growth rate (μ crit ) of 0.12 h−1 at 14 L scale, the biomass yield coefficient (Y x/s ) remained constant at 0.4 g g−1 with no ethanol production, whereas ethanol yields relative to biomass (k eth/x ) of up to 0.54 g g−1 and a steady decrease in Y x/s were observed at μ  > 0.12 h−1. Production of α-glucuronidase was growth associated at a product yield (k α-glu/x ) of 0.45 mg g−1, with the highest biomass (37.35 g/L) and α-glucuronidase (14.03 mg/L) concentrations, were recorded during fed-batch culture at or near to μ crit . Scale-up with constant k L a from 14 to 100 L resulted in ethanol concentrations of up to 2.5 g/L at μ  = 0.12 h−1. At this scale, α-glucuronidase yield could be maximised at growth rates below μ crit , to prevent localised high glucose concentration pockets at the feed entry zone that would induce oxido-reductive metabolism. This is the first report where recombinant production of α-glucuronidase (EC 3.2.1.139) by S. cerevisiae was optimised for application at pilot scale.
Keywords: Saccharomyces cerevisiae; Fed-batch culture; Exponential feed; Critical specific growth rate; α-Glucuronidase; Scale-up;

Folding and aggregation of a multi-domain engineered immunotoxin by Ronald W. Maurer; Alan K. Hunter; Xiangyang Wang; William K. Wang; Anne S. Robinson; Christopher J. Roberts (8-14).
Engineered immunotoxins with specific targeting mechanisms have potential applications for the treatment of cancer and other diseases; however, their folding behavior is often poorly understood and this presents challenges during process development, manufacturing, and formulation. Folding thermodynamics of an antibody variable domain (VH/VL) genetically fused to a biological toxin payload were characterized at pH 6.0 and pH 8.0 in order to assess the relative domain stabilities, along with time scales on which they fold, and the competition between aggregation and folding. The toxin and VH/VL domains had considerably different unfolding free energies (ΔG UNF), leading to a thermodynamically-distinct intermediate species, with the toxin domain unfolded and the VH/VL folded. The intermediate is the majority species over a range of denaturant concentrations (∼4–6 M urea; ∼2–4 M guanidine HCl). Thermal unfolding resulted in reversible unfolding of the toxin domain at pH 8, but at pH 6 thermal unfolding was convoluted with aggregation due to irreversible unfolding and aggregation for the VH/VL domain. Chemical unfolding of both domains was more easily reversible, provided that the refold was done stepwise, allowing the antibody domain to fold first at intermediate denaturant concentration, as folding of the VH/VL domain played a key role in aggregation of this antibody fusion protein.
Keywords: Aggregation; Biophysical chemistry; Protein denaturation; Protein recovery;

Biodiesel production from soybean soapstock acid oil by hydrolysis in subcritical water followed by lipase-catalyzed esterification using a fermented solid in a packed-bed reactor by Diniara Soares; Andrei Ferreira Pinto; Alan Guilherme Gonçalves; David Alexander Mitchell; Nadia Krieger (15-23).
We investigated a new hydroesterification strategy for the production of biodiesel from low-value oil feedstocks: complete hydrolysis of the feedstock to fatty acids in subcritical water, followed by the use of a packed-bed reactor, containing a fermented solid with lipase activity, to convert the fatty acids to their ethyl esters. The fermented solids were produced by cultivating Burkholderia cepacia LTEB11 for 72 h on a 1:1 mixture, by mass, of sugarcane bagasse and sunflower seed meal. The esterification of fatty acids obtained from soybean soapstock acid oil was studied in the packed-bed bioreactor, in a solvent-free system, with the best results being a 92% conversion in 31 h, obtained at 50 °C. When the packed-bed reactor was reused in successive 48-h esterification reactions, conversions of over 84% of the fatty acids to esters were maintained for five cycles at 50 °C and for six cycles at 45 °C. Unlike previous hydroesterification processes that have used lipase-catalyzed hydrolysis followed by chemically-catalyzed esterification, our process does not expose the lipases to contaminants present in low quality feedstocks such as soapstocks. This advantage opens the possibility of operating the packed-bed esterification reactor in continuous mode.
Keywords: Biodiesel; Hydroesterification; Lipases; Burkholderia cepacia; Solid-state fermentation; Packed-bed reactor;

The influence of mixing on microorganism integrity and product formation is a critical design parameter for solid-state fermentation bioreactors. The effects of intermittent mechanical mixing on the solid-state fermentation of wet corn distillers grain with Trichoderma reesei NRRL 11460 for the production of cellulase were investigated. Experiments were conducted using the unbuffered media at mixing frequencies of 0, 1, 2, 3, and 6 d−1 at 27.5 °C with an initial moisture content of 50%. The results indicate that mixing caused about a tenfold increase in spore production compared to fermentations at static conditions. The cellulase enzyme activity produced was minimally affected by mixing with only a 5–10% decrease in filter paper activity for mechanically mixed fermentations compared to static fermentations. Mixing at lower frequencies of 1, 2, and 3 d−1 caused an increase in CO2 evolution compared to static conditions and higher mixing frequencies of 6 d−1. A correlation between substrate weight loss and cumulative CO2 evolution was established. The ability to intermittently mix a solid-state fermentation bioreactor with minimal detrimental effects increases the feasibility of onsite production of enzymes at biofuel facilities to lower the overall production costs of cellulosic biofuels.
Keywords: Mixing; Cellulase; Solid-state fermentation; Filamentous fungi; Spore production; Distillers grain;

This study evaluated the chronic impact of erythromycin, a macrolide antibiotic, on microbial activities, mainly focusing on changes in process kinetics induced on substrate biodegradation and all related biochemical processes of microbial metabolism. Experiments involved two fill/draw reactors sustained at steady state at two different sludge ages of 10 and 2.0 days, fed with peptone mixture and continuous erythromycin dosing of 50 mg/L. Oxygen uptake rate profiles were generated in a series of parallel batch reactors seeded with biomass from fill/draw systems at selected periods of steady-state operation. Experimental data were evaluated by model calibration reflecting inhibitory effect on process kinetics: continuous erythromycin dosing inhibited microbial growth, reduced the rate of hydrolysis, blocked substrate storage and accelerated endogenous respiration. Adverse impact was mainly due to changes inflicted on the composition of microbial community. Interruption of erythromycin feeding resulted in partial recovery of microbial response. Sludge age affected the nature of inhibition, indicating different process kinetics for faster growing microbial community. Kinetic evaluation additionally revealed the toxic effect of erythromycin, which inactivated a fraction of biomass. Mass balance using oxygen uptake rate data also identified a stoichiometric impact, where a fraction of available substrate, although completely removed, could not be utilized in metabolic activities.
Keywords: Biodegradation; Chronic inhibition; Erythromycin; Kinetic parameters; Modeling; Waste-water treatment;

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation by Cuong Mai Nguyen; Gyung Ja Choi; Yong Ho Choi; Kyoung Soo Jang; Jin-Cheol Kim (40-46).
The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L−1, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L−1  h−1 were obtained with lactic acid concentrations as high as 198.32 g L−1 for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L−1  h−1 with a maximum concentration of 186.40 g L−1 at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.
Keywords: Lactic acid; Fresh sweet potato; Starch; Fermentation; Enzymes; Simultaneous saccharification and fermentation;

Polyketides are important compounds with a staggering range of biological and medicinal activities. Previous studies have demonstrated that the addition of fatty acids can increase polyketides production. However, a detailed metabolic explanation of this phenomenon has not been established. The aim of this study was to explain the positive effect of exogenous fatty acids on polyketides production. Spinosyns are polyketide-derived macrolides. In our study, spinosyns were used, as an example, to study the positive effect of exogenous fatty acids on their production. In the presence of exogenous fatty acids, gene expression assays indicated that the transcription of de novo fatty acid biosynthesis was significantly decreased and the transcriptions of β-oxidation and spinosad biosynthesis were up-regulated. The decreased de novo fatty acid synthesis transcription and the increased β-oxidation transcription resulted in the increase of acetyl-CoA and malonyl-CoA. It is the up-regulated spinosad pathway coupling with the enhanced concentration of acetyl-CoA and malonyl-CoA that contributed to the increase of spinosad. Taken together, a metabolic link among de novo fatty acid synthesis, β-oxidation, and spinosad biosynthesis at the presence of exogenous fatty acids was established. The results presented here enable researchers to better understand why added fatty acids can increase polyketides production.
Keywords: Biosynthesis; Fermentation; Physiology; RNA; Spinosad; Saccharopolyspora spinosa;

Hydrolysis of lactose in whole milk catalyzed by β-galactosidase from Kluyveromyces fragilis immobilized on chitosan-based matrix by Danielle C. Vieira; Lionete N. Lima; Adriano A. Mendes; Wellington S. Adriano; Roberto C. Giordano; Raquel L.C. Giordano; Paulo W. Tardioli (54-64).
β-Galactosidase (β-gal) from Kluyveromyces fragilis was immobilized on chitosan-based matrices and agarose beads by different protocols to catalyze the hydrolysis of whole milk lactose in a batch system. The best derivative was obtained by immobilizing the enzyme on chitosan coagulated with KOH and activated with glutaraldehyde, both steps carried out at 50 °C. The immobilization yield and recovered activity were 100%. Maximum activity was achieved at pH 7.0 and 45 °C for both soluble and immobilized β-gal. The maximum β-gal load immobilized on chitosan-glutaraldehyde support was around 21 mg of enzyme per gram of support. Immobilized β-gal was 3–5-fold more stable than the soluble enzyme at 40 and 20 °C, respectively. The biocatalyst retained 80% of its initial activity after 3 months at 10 °C and pH 7.0 in the presence of the cofactors Mn2+ and Mg2+, twice more stable than soluble enzyme. The immobilized biocatalyst was capable to efficiently hydrolyze lactose into the whole milk at 25 °C (above 95% conversion), showing similar performance after four consecutive batches.
Keywords: Immobilized enzymes; Lactose; Whole milk; Batch processing; β-Galactosidase; Chitosan;

For predicting microbial metabolism in low energy yielding environments, various rate laws have been proposed to account for the effects of thermodynamic state (as a measure of product-inhibition) as well as maintenance requirements on energetics of mediated reactions. Explicit or implicit modeling of simplified ATP reactions allows distinction between energy and ATP producing (catabolic, treated as kinetic and reversible) and energy and ATP consuming (anabolic) processes including maintenance requirements. Here, we provide a comparison of several approaches for modeling microbial metabolism in anaerobic environments considering thermodynamic factors, and maintenance energy requirements. We develop a mathematical model for microbial metabolism in anaerobic systems, which couples catabolic and anabolic processes considering the limiting effects of intermediate concentrations on reaction rate through the reduction of chemical potential and reversibility, and that explicitly partitions energy (ATP) allocation between cell growth and maintenance. We include an approach where maintenance energy requirements are assumed to take precedence over ATP-consuming cell synthesis reactions. Also, substrate utilization terminates when the catabolic reactions reach thermodynamic equilibrium with respect to ATP formation, including maintenance energy. The comparison of the proposed model to other modeling approaches shows the benefits of incorporating product inhibition and maintenance requirements in situations which maintenance energy requirements are comparable in size to growth energy requirements. An example application is also presented, where the proposed model is applied to an experimental study of arsenate reduction by Bacillus arsenicoselenatis conducted by Blum et al. (Arch Microbiol. 171 (1998) 19-30), in which the rate of metabolism is controlled by thermodynamics.
Keywords: Thermodynamic equilibrium; Microbial growth kinetics; Maintenance energy; ATP synthesis; Product inhibition;

The entomogenous fungus Cordyceps taii, a traditional Chinese medicinal mushroom, exhibits potent important pharmacological effects and it has great potential for health foods and medicine. In this work, the effects of oxygen supply on production of biomass and bioactive helvolic acid were studied in shake-flask fermentation of C. taii mycelia. The value of initial volumetric oxygen transfer coefficient (K L a) within 10.1–33.8 h−1 affected the cell growth, helvolic acid production and expression levels of biosynthetic genes. The highest cell concentration of 17.2 g/L was obtained at 14.3 h−1 of initial K L a. The highest helvolic acid production was 9.6 mg/L at 10.1 h−1 of initial K L a. The expression levels of three genes encoding hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and squalene synthase were down-regulated on day 2 and day 8 but up-regulated on day 14 at an initial K L a value of 10.1 h−1 vs. 33.8 h−1, which well corresponded to the helvolic acid biosynthesis in those conditions. The information obtained would be helpful for improving the biomass and helvolic acid production in large-scale fermentation of C. taii.
Keywords: Fermentation; Oxygen transfer; Biosynthesis; Filamentous fungi; Biosynthetic gene expression; Medicinal mushroom;

The effect of various feeding strategies with the use of glycerol and the mixture of glycerol and lactose on lovastatin biosynthesis in the continuous fed-batch stirred tank bioreactor by Aspergillus terreus ATCC20542 was studied. Within the first 24 h of feeding the increase in lovastatin titre, even by 100%, compared with batch runs, was obtained. In this work, it was proved that apart from carbon substrate feeding other factors played a crucial role in lovastatin formation. These were redox potential level, air flow rate and carbonate carbon assimilation rate. Thus, their impact was also thoroughly examined. Under the conditions of the elevated redox potential, both initial and feeding carbon substrates were faster utilised, which led to the enhancement of lovastatin production. In the process with the highest redox potential levels maximum lovastatin concentration was equal to 83.8 mgLOV  l−1, while at the lowest redox level it did not reach 67 mgLOV  l−1. In turn, carbonate carbon as well as organic carbon were considerably better assimilated at the higher aeration rates, which also positively influenced lovastatin formation. All in all, there were several factors that had to occur simultaneously to achieve the satisfactory lovastatin titres.
Keywords: Lovastatin; Biosynthesis; Fed-batch culture; Bioreactor; Aeration; Redox level;

Escherichia coli BA002, the ldhA and pflB deletion strain, cannot utilize glucose anaerobically due to the inability to regenerate NAD+. To regulate NAD(H) pool size and NADH/NAD+ ratio, overexpression of the enzymes in the NAD(H) biosynthetic pathways in BA002 was investigated. The results clearly demonstrate that the increased NAD(H) pool size and the decreased NADH/NAD+ ratio improved the glucose consumption and cell growth, which improved succinic acid production. When the pncB and the nadD genes were co-overexpressed in CA102, the ratio of NADH/NAD+ was decreased from 0.60 to 0.12, and the concentration of NAD(H) was the highest among that of all the strains. Moreover, the dry cell weight (DCW), glucose consumption, and the concentration of succinic acid in CA102 were also the highest. Based on the sufficient NAD+ supply after gene modification in the NAD(H) biosynthetic pathways, reductive carbon sources with different amounts of NADH can further change the distribution of metabolites. When sorbitol was used as a carbon source in CA102, the byproducts were lower than those of glucose fermentation, and the yield of succinic acid was increased.
Keywords: Anaerobic processes; Bioconversion; Enzymes; Microbial growth; NAD(H) pool size; NADH/NAD+;

Developing a cyclin blueprint as a tool for mapping the cell cycle in GS-NS0 by D.G. García Münzer; M. Kostoglou; M.C. Georgiadis; E.N. Pistikopoulos; A. Mantalaris (97-107).
The cell cycle is at the center of growth, productivity, and death of mammalian cell cultures. There exists a need to identify and quantify major landmarks in the cell cycle of industrially relevant mammalian cell lines and its association with productivity; central for designing productivity optimization strategies. Herein, we studied the expression of three cyclins, under both perturbed and unperturbed growth, by flow cytometry in batch cultures of GS-NS0. The perturbed systems involved two different DNA synthesis inhibitors, thymidine and dimethyl sulfoxide (DMSO). This approach enables the establishment of characteristic cyclin profiles, timings, and thresholds. In particular, two G1 class cyclins (D1 and E1), and one G2 cyclin (B1) were investigated. Cyclin B1 showed a clear cell cycle phase-specific expression increasing during G2 phase where it was approximately 40% higher when compared to G1 phase. Similarly, cyclin E1 showed a clear pattern being expressed approximately 10% higher in G1 compared to G2 phase and decreased through S phase. Cyclin D1 expression was fairly invariable throughout the cell cycle phases. The observed patterns provide a blueprint of the cell line's cell cycle, which can be used for the development of biologically accurate and experimentally validated distributed cell cycle models.
Keywords: Cell cycle; Cyclin; Hybridoma cultures; Growth kinetics; Monoclonal antibodies; Modeling; Bioprocess monitoring;

The performance of a mixed-culture on the removal of caffeine (CFN), sulfamethoxazole (SMX), ranitidine (RNT), carbamazepine (CZP) and ibuprofen (IBP) in a suspended growth reactor has been studied. The sorption and biodegradation of these compounds were examined when they were individually or simultaneously tested. The sorption of individual compounds was significantly low except from RNT (K d  = 0.42 L/g). In contrast, the sorption of SMX and CFN increased in detriment of RNT when all the pharmaceutical compounds were simultaneously present. The biodegradation removal also exhibited significant differences. Thus, the simultaneous treatment showed higher biodegradation rates (K b up to 97.55 × 10−6  L/mg h) than the individual treatment (K b up to 8.13 × 10−6  L/mg h) of the pharmaceuticals. In general, the simultaneous treatment leads to increased sorption distribution coefficients and biodegradation rates. Results seem to reveal that the enhanced biomass efficiency on the simultaneous elimination process was due to the synergistic effects of pharmaceutical compounds onto mixed-culture. During the simultaneous removal, CFN, SMX and CZP were removed consistently (5.3 ± 4.4%, 73.2 ± 21.3% and 4.2 ± 2.3%, respectively), whereas RNT and IBP showed an unsteady removal over time. Finally, a kinetic model capable of describing the influence of biomass growth and nutrients utilization on the sorption and biodegradation of the pollutants was successfully demonstrated.
Keywords: Bioremediation; Modeling; Waste-water treatment; Carbon assimilation; PPCPs removal; Synergistic interactions;

Cultivation of Chlorella vulgaris in tubular photobioreactors: A lipid source for biodiesel production by Davide Frumento; Alessandro Alberto Casazza; Saleh Al Arni; Attilio Converti (120-125).
Chlorella vulgaris was cultivated in two different 2.0 L-helicoidal and horizontal photobioreactors at 5 klux using the bicarbonate contained in the medium and ambient air as the main CO2 sources. The influence of bicarbonate concentration on biomass growth as well as lipid content and profile was first investigated in shake flasks, where the stationary phase was achieved in about one half the time required by the control. The best NaHCO3 concentration (0.2 g L−1) was then used in both photobioreactors. While the fed-batch run performed in the helicoidal photobioreactor provided the best result in terms of biomass productivity, which was (84.8 mg L−1  d−1) about 2.5-fold that of the batch run, the horizontal configuration ensured the highest lipid productivity (10.3 mg L−1  d−1) because of a higher lipid content of biomass (22.8%). These preliminary results suggest that the photobioreactor configuration is a key factor either for the growth or the composition of this microalga. The lipid quality of C. vulgaris biomass grown in both photobioreactors is expected to meet the standards for biodiesel, especially in the case of the helicoidal configuration, provided that further efforts will be made to optimize the conditions for its production as a biodiesel source.
Keywords: Chlorella vulgaris; Microalgae; Fed-batch culture; Bioreactors; Growth kinetics; Biodiesel;

Development of a fed-batch cultivation for antibody-producing cells based on combined feeding strategy of glucose and galactose by Ya-ting Sun; Liang Zhao; Zhaoyang Ye; Li Fan; Xu-ping Liu; Wen-Song Tan (126-135).
In order to achieve enhanced cell mass and productivity with less lactate accumulation, a fed-batch culture based on a combined feeding strategy of glucose and galactose was developed. Cell performance was first examined with feeding of galactose alone. While cell growth was improved compared with glucose-feeding culture, cell maintenance was inefficient with rapid lactate depletion and considerable ammonium accumulation. Subsequently, to improve cell maintenance, a combined feeding strategy of glucose and galactose was proposed focusing on optimizing the ratio of glucose to galactose and feeding time. In addition, the compositions of amino acids and vitamins in feeding medium were refined for balanced supply of nutrients. With the combined feeding strategy, the metabolic shift of lactate from production to consumption occurred, but not accompanied by rapid lactate depletion and ammonium production. Furthermore, energy metabolism was more efficient and better utilization of carbon sources was achieved. Compared with the glucose-feeding culture in bioreactor, maximum lactate concentration was reduced by 55%; IVCC and the specific production rate of antibody were increased by 45% and 143%, respectively.
Keywords: CHO cells; Fed-batch culture; Antibody production; Lactate metabolism; Galactose; Process development;

Robust adaptive controller for continuous bioreactors by Ming-Feng Jang; Yuh-Jong Chern; Yi-Shyong Chou (136-145).
Bioreactor control has become very important in recent years due to the difficulty in controlling highly non-linear behavior, model mismatch and parameter variation. Adaptive back-stepping control is a recursive design method that employs the Lyapunov stability theory in its design procedures. Adaptive back-stepping control is applied to the problem, which drives a continuous bioreactor to its optimal productivity point. However the designed control law may not work at the optimal productivity point, since controlling the optimal productivity point frequently causes the control system to fall into a singular point. Thus in this study, we apply the back-stepping design to a bioreactor system by introducing a simple parameter into the design techniques to prevent the control system from operating at a singular point. The parameter introduced in the control law can be understood intuitively and the proposed control scheme is endowed with strong robustness properties. The resulting controllers are compared to the feedback linearization based proportional-integral controller and internal model control, as well as nonlinear model predictive control and observer-based linearizing control by theoretical simulations. It is shown that the proposed control strategy is versatile and effective in implementing robust control for regulating productivity in the presence of model mismatch, model uncertainty and parameter variation. The proposed control scheme successfully drives the productivity of the controlled continuous bioreactor to its optimal point.
Keywords: Bioreactors; Control; Fermentation; Dynamic simulation; Back-stepping control; Nonlinear control;

Current developments in solid-state fermentation by Leya Thomas; Christian Larroche; Ashok Pandey (146-161).
Solid-state fermentation (SSF) is a three-phase heterogeneous process, comprising solid, liquid and gaseous phases, which offers potential benefits for the microbial cultivation for bioprocesses and products development. Over the last two decades, SSF has gained significant attention for the development of industrial bioprocesses, particularly due to lower energy requirement associated with higher product yields and less wastewater production with lesser risk of bacterial contamination. In addition, it is eco-friendly, as mostly utilizes solid agro-industrial wastes (resides) as the substrate (source of carbon). This article aims to present and analyze the current development on SSF taken place mainly during the last five years, linking the developments with earlier two papers published in this journal in 2003 (Pandey, 2003 [1]) and in 2009 (Singhania et al., 2009 [2]). The article reviews the current state-of-art scenario and perspectives on the development of bioprocesses and products in SSF and also discusses microbes employed in these processes, the types of bioreactors used for these and also presents the modeling and kinetics aspects.
Keywords: Solid-state fermentation; Bioprocess design; Enzyme production; Modeling production kinetics; Agro-industrial residues; Other industrial products;

Production of yeast hybrids for improvement of cider by protoplast electrofusion by Mengqi Ye; Tianli Yue; Yahong Yuan; Linsong Wang (162-169).
This study aimed to construct new yeast hybrid strains for introducing flavor and aroma diversity to ciders. The inactivated protoplasts of Saccharomyces cerevisiae and Candida krusei were electric-induced fused under the optimized electric condition of pulse field density 2200 V/cm, pulse time 20 μs, pulse number 2 times and pulse interval 1 s, and 69 fusants were initially obtained. By performing Durham's fermentation for ten generations, 9 stable hybrid strains were screened. The chemical analysis showed that the alcoholic degree of ciders fermented by R2, R4, R5, R6 and R8 achieved about 12% (v/v), which was statistically the same level as the one fermented by parental strain WF1. The GC–MS results showed different strain generated totally different aroma profiles. R4 produced significant higher concentration of 2-methyl-butanoic acid ethyl ester, 2-methyl-1-propanol, 3-methyl-butanol acetate, 1-butanol, acetic acid hexyl ester, 1-hexanol and 1-octanol. The 9 hybrid yeast strains and parental strains were further compared through fuzzy comprehensive evaluation combining sensory score and aroma components content. The results showed that the hybrid R4 scored highest and displayed desirable properties of both parents.
Keywords: Yeast; Hybridoma cultures; Protoplasts electrofusion; Brewing; Apple wine; Fuzzy logic;

Converting microalgae oil into biodiesel is considered a promising route in the field of biofuel production. However, the cost of microalgae-based biodiesel is still too high to be economically feasible. The high cost of microalgae-based biodiesel is mainly a result of downstream processing, in particular from the extraction of oil out of the microalgal biomass. This study proposes a bacterial (enzymatic) destruction pretreatment of the microalgae cell wall to render the microalgal oil extraction more efficient. It has been found that the cell wall of microalgae can be modified if the microalgal biomass is co-cultured with an indigenous bacterial isolate Flammeovirga yaeyamensis in a salt concentration of 3% and a pH of 8.0. Following this treatment, the activities of some hydrolytic enzymes (i.e., amylase, cellulases, and xylanase) have been detected in the co-culture of F. yaeyamensis and the oil-rich microalga (Chlorella vulgaris ESP-1). The SEM micrographs clearly show specific damage to the microalgae cell wall caused by the bacterial treatment. We found that when the microalgae is pretreated with a concentrated co-culture supernatant (containing the hydrolytic enzymes), a nearly 100% increase in lipid extraction efficiency is obtained. The proposed bacterial disruption method seems to be an effective and environmentally friendly way of improving the efficiency of oil extraction and biofuel production from a microalgal biomass.
Keywords: Microalgae; Cell disruption; Enzyme activity; Amylase; Batch processing;

Design of polymeric microparticles for pH-responsive and time-sustained drug release by Wenjie Liu; Cordelia Selomulya; Xiao Dong Chen (177-186).
We described the design of uniform microencapsulates with almost 100% encapsulation efficiency, synthesized without organic solvents, via microfluidic spray drying of water-based dispersions of pH-responsive methacrylic acid polymers (Eudragit® L 30D-55). The effects of incorporating water-based network-forming materials in the formulations on pH-responsiveness and controlled release patterns of enteric microparticles were observed. Acid hydrolysed tetraethoxysilane (TEOS) was used to form an interpenetrating, rigid framework of silica, whereas Eudragit® NE (a copolymer based on ethyl acrylate and methyl methacrylate) was added to produce a more flexible polymeric network. The spray-dried microparticles generally displayed crumbled or buckled morphologies dependent on drying temperatures, due to large hydrodynamic sizes of solutes in feed dispersions. The drug release kinetics of microparticles were sensitive to the type and the added amount of network-forming materials, due to different colloidal interactions between Eudragit® L and either silica or the copolymer. This study demonstrated a strategy to design enteric microparticles with different microstructural properties and drug release behaviours through understanding of colloidal interactions between constituents of matrix materials.
Keywords: Uniform microencapsulates; Microfluidic-jet spray drying; pH-responsive release; Time-sustained release; Colloidal interactions; Microstructural design;