Biochemical Engineering Journal (v.91, #C)

IFC (IFC).

BEJ Keywords (IV).

Simulation of gas-inducing reactor couples gas–liquid mass transfer and biochemical reaction by Hou-Sheng Hong; Zi-Jin Cai; Jun-Qing Li; Dong-Sheng Shi; Wen-Qi Wan; Li Li (1-9).
In the present study Euler–Euler's two fluid model coupled with mass transfer and biochemical reaction and experiment were applied to investigate hydrodynamic characteristic and bio-reaction parameters in self-induced bioreactor. Biochemistry parameters, like biomass, residual sugar concentration and dissolved oxygen concentration were measured. Higbie's penetration theory was applied to gas–liquid mass transfer and Monod's equation was used to build a growth model for yeast in self-induced bioreactor. The computational model was mapped on ANSYS CFX 10.0. The biomass, residual sugar and dissolved oxygen concentration predicted by numerical simulation were validated with results from experiments. The distribution of local biomass, residual sugar concentration and dissolved oxygen were successfully predicted by simulation, which reflect the effect of geometry of reactor and physical-chemistry conditions.
Keywords: Gas-inducing reactor; Yeast; Gas–liquid mass transfer; Biokinetics; Scale-up;

Cofermentation of Bacillus licheniformis and Gluconobacter oxydans for chitin extraction from shrimp waste by Pei Liu; Shanshan Liu; Na Guo; Xiangzhao Mao; Hong Lin; Changhu Xue; Dongzhi Wei (10-15).
Shrimp processing and consumption generate each year thousand tons of wastes, which are the sources of raw material for the production of chitin. Successive cofermentation was applied to biologically extract chitin from shrimp head waste in combination with a protease-producing bacterium, Bacillus licheniformis 21886 (B. 21886), and an acid-producing bacterium, Gluconobacter oxydans DSM-2003 (G. 2003). When they were cultivated individually, B. 21886 removed 83.1% of the protein in the absence of glucose and G. 2003 95% of minerals from shrimp heads. When the waste was first cofermented with B. 21886 followed by G. 2003, the deproteinization (DP) and demineralization (DM) efficiencies were 87 and 93.5% respectively at a chitin content of 90.8%. In the cofermentation broth eight organic acids with a total amount of 16 g/l were found. Major acids were lactic, formic and acetic acid, followed by gluconic, succinic, pyroglutaminc and propionic acid. In the cofermenation, three times more organic acids were released than in the monofermentation of G. 2003. The fermentation time of 96 h was almost half of the cultivated cycle reported.
Keywords: Bacillus licheniformis; Gluconobacter oxydans; Waste treatment; Fermentation; Biodegradation; Proteolysis;

Improved capsular polysaccharide production by Streptococcus pneumoniae serotype 14 using continuous cultivation by Verônica Maria Rodege Gogola-Kolling; Rafaela Taís Zanardo; Talita Souza Carmo; Natália Dalfré Zampoli; Douglas Borges Figueiredo; Viviane Maimoni Gonçalves (16-22).
The capsular polysaccharide (PS) is the most important pneumococcal virulence factor and is currently used as antigen in all pneumococcal vaccines. Despite its physiological and epidemiological importance, meager studies have been devoted to improve PS production and understand its relationship with pneumococcal central metabolism. In this study, kinetics of growth and production of PS by Streptococcus pneumoniae serotype 14 (PS14) in batch and continuous cultivation were investigated. Strong cell lysis was observed in batch cultivation, while accumulation of organic acids and autolysis was avoided in continuous cultivation. In the continuous cultivation was possible to achieve higher concentration of biomass and PS14. Calculation of kinetic parameters demonstrated that PS14 is a cell-associated product. The coefficients for growth-associated stoichiometric true yield and maintenance were determined as 0.25 gglucose  gbiomass −1 and 1.24 gglucose  (gbiomass  h)−1, respectively. The maximum productivity of PS14 released in the supernatant (PS14R) and cell-bound PS14 (PS14C) were obtained at a dilution rate of 0.8 h−1, respectively, 85 and 122 mg  (gbiomass  h)−1. Compared to batch fermentation, both PS14R and PS14C productivities were increased by about 300% in the continuous process. These findings demonstrate that continuous cultivation is a promising strategy for PS production to be used in pneumococcal vaccines.
Keywords: Pneumococcus; Continuous culture; Batch processing; Production kinetics; Microbial growth; Kinetic parameters;

Microbial production of enzymes: Nonlinear state and kinetic reaction rates estimation by Dan Selişteanu; Sihem Tebbani; Monica Roman; Emil Petre; Vlad Georgeanu (23-36).
The nonlinearity of the biotechnological processes and the absence of cheap and reliable instrumentation require an enhanced modelling effort and estimation strategies for the state and the kinetic parameters. This work approaches nonlinear estimation strategies for microbial production of enzymes, exemplified by using a process of lipase production from olive oil by Candida rugosa. First, by using a dynamical mathematical model of this process, an asymptotic observer which reconstructs the unavailable state variables is proposed. The design of this kind of observers is based on mass and energy balances without the knowledge of kinetics being necessary; only minimal information concerning the measured concentrations is used. Second, a nonlinear high-gain observer is designed for the estimation of imprecisely known kinetics of the bioprocess. An important advantage of this high-gain estimator is that the tuning is reduced to the calibration of a single parameter. Numerical simulations in various scenarios are provided in order to test the behaviour and performances of the proposed nonlinear estimation strategies.
Keywords: Enzymes; Lipase; Modelling; Nonlinear estimation; Observers; Dynamic simulation;

Increased CHO cell fed-batch monoclonal antibody production using the autophagy inhibitor 3-MA or gradually increasing osmolality by S. Soroush Nasseri; Navid Ghaffari; Katrin Braasch; Mario A. Jardon; Michael Butler; Malcolm Kennard; Bhushan Gopaluni; James M. Piret (37-45).
Modulating autophagy provides a new method to increase CHO cell protein production. A fed-batch protocol using the autophagy inhibitor 3-methyl adenine (3-MA), developed for a tissue-plasminogen activator (t-PA) expressing DHFR based CHO cell line, was successfully adapted to a monoclonal antibody (MAb) expressing CHOK1-SV based CHO cell line. By optimizing the timing and dose of 3-MA treatment, the cell-specific productivity was increased 4-fold, resulting in 2-fold increased total MAb production. The positive effect of the 3-MA treatment appeared to be reduced when the amino acid feed concentration was increased 5-fold. Further investigation revealed that by slowly increasing osmolality up to ∼450 mOsm/kg, both the cell-specific productivity and the total MAb almost doubled. This effect was replicated with a DUXB-based CHO cell line expressing a human–llama chimeric antibody. The positive effect of gradually increasing osmolality was then combined with the positive effects of the 3-MA treatment, however their combined effect were not additive. Thus, either increased osmolality or 3-MA treatment were equally effective in increasing MAb-CHO cell fed-batch production on the cell lines tested. Analysis of protein glycosylation showed that both of these fed-batch modifications did not substantially influence the overall glycan profiles of the MAb product.
Keywords: Autophagy; 3-MA; Osmolality; Glycosylation; Fed-Batch; Recombinant protein production;

Octamethylcyclotetrasiloxane removal using an isolated bacterial strain in the biotrickling filter by Jiajia Wang; Weijiang Zhang; Jiao Xu; Yunhui Li; Xuetao Xu (46-52).
Octamethylcyclotetrasiloxane (D4) is a volatile organosilicon compound present in biogas derived from widely using industrial and consumer products. Due to being harmful to many organisms at very low concentrations and forming SiO2 deposits during biogas combustion, the degradation of D4 was investigated. The D4-degrading culture was taken from the effluent of an organic silicon manufacturer. The isolation and the purification were performed. The isolated bacterial strain was inoculated in a biotrickling filter. The effects of the pH, the D4 inlet concentration, the empty bed residence time (EBRT), and the liquid flowrate on the removal performance of D4 were investigated. It is shown that the optimum pH for D4 degradation was 4.0–6.0. The max removal efficiency (RE) of D4 of 60.2% and the max elimination capacity (EC) of 165.4 mg m−3  h−1 were obtained. The main metabolic products consist of HO―SiMe2 ―OH, HO―SiMe2 ―O―SiMe2 ―OH, Me3SiO(CO)2OSiMe3, ▪, and Si(OH)4. The degradation pathways and mechanism for D4 degradation by Phyllobacterium myrsinacearum were proposed.
Keywords: Octamethylcyclotetrasiloxane (D4); Biodegradation; Biofilms; Separation; Purification; Identification.;

Although various methods have been developed for microalgae harvesting, problems such as chemical toxicity, low efficacy, low flexibility, and high cost, still remain with the current methods. In the present study, an effective biological separation method has been developed to harvest microalgae via immobilization on exogenous fungal mycelium. Fungal mycelium was added into microalgae culture and air mixed for the immobilization. The immobilized microalgae with fungal mycelium were naturally precipitated within 10 min after stopping the mixing. Both marine (e.g. Nannochloropsis sp.) and freshwater (e.g. Chlorella vulguris) microalgae were almost completely (94–97%) precipitated using the mycelium of Aspergillus nomius CCK-PDA 7#6. The precipitated mixed biomass was separated by simple filtration using sieve. A 70% initial nutrients supplement to de-algated culture medium gave high growth yield (0.43 g/l) in subsequent microalgae cultivation. The mycelium can be obtained free or at low-cost as waste from a fungal fermentation process of producing valuable products. The developed method is, therefore, very promising for economical harvesting of microalgae.
Keywords: Microalgae; Immobilization; Separation; Optimization; Screening fungus; Mycelium;

Tubular structures made of alginate and similar hydrogels have been extensively investigated for drug delivery, tissue engineering and other biomedical applications. Alginate fibers are usually fabricated using complicated techniques such as coaxial flow extrusion or electro-spinning. This paper discusses the fabrication of hollow and solid alginate fibers using a simple membrane based molding technique. A hollow-fiber microfiltration membrane served both as the mold as well as reservoir for the cross-linking agent. The pores of the membrane were first filled with the cross-linker (i.e. calcium chloride) solution, after which the lumen was filled with sodium alginate solution. The calcium ions diffused from the membrane pores into the lumen, cross-linking the alginate in a radially inward direction. Solid alginate fibers were obtained by cross-linking all the alginate within the lumen, while hollow fibers were fabricated by pushing out un-cross-linked alginate from the central core using calcium chloride solution. The alginate fibers fabricated as described above were expelled from the membrane mold using water under pressure. These were then characterized by optical, fluorescence and scanning electron microscopy. The most attractive features of this fabrication method are its simplicity and flexibility with regards to alginate concentration. The fibers obtained were straight with excellent definition and uniform thickness, and could be fabricated in a highly reproducible manner. Other complicated solid and hollow 3-dimensional structures suitable for biomedical, and indeed other applications could also potentially be fabricated using similar membrane-based molding techniques.
Keywords: Alginate; Molding; Biomedical; Tissue Cell Culture; Hollow Fibres; Microporous Membranes;

Promoting the growth of Rubrivivax gelatinosus in sewage purification by the addition of magnesium ions by Pan Wu; Jian-zheng Li; Yan-ling Wang; Xian-shu Liu; Cong Du; Qing-yue Tong; Ning Li (66-71).
This work investigated the improvement in biomass yield and production and organics reduction of Rubrivivax gelatinosus (R. gelatinosus) by adding magnesium (Mg2+) for sewage purification under natural light micro-oxygen conditions. The results showed that with the optimal Mg2+ dosage (10 mg/L), biomass production (4125.6 mg/L) was enhanced by 59%. The biomass yield was improved by 43.3%. COD and protein removal increased more than 90%. Hydraulic retention time was decreased by 25%. Moreover, 5–10 mg/L Mg2+ improved R. gelatinosus yield in wastewater to medium levels. Analyses revealed that different Mg2+ concentrations had different mechanisms for impacting biomass production and COD removal. This was because there was an upper limit (15 mg/L) and an optimal value (6 mg/L) of intracellular Mg2+ concentrations for R. gelatinosus. Appropriate Mg2+ concentrations enhanced ATP production, which improved the secretion and activity of protease, indicating that more protein and COD were removed and more biomass accumulated. However, excessive Mg2+ was harmful for biomass accumulation and COD removal because excess Mg2+ decreased ATP production.
Keywords: R. gelatinosus; Mg2+; Soybean processing wastewater treatment; Biomass resources recycling; ATP production;

Efficient production of ethyl (R)-2-hydroxy-4-phenylbutyrate using a cost-effective reductase expressed in Pichia pastoris by Xiao-Long Qian; Jiang Pan; Nai-Dong Shen; Xin Ju; Jie Zhang; Jian-He Xu (72-77).
Recombinant strains of Pichia pastoris expressing carbonyl reductase CgKR2 from Candida glabrata were constructed for stereoselective reduction of ethyl-2-oxo-4-phenylbutyrate (OPBE) to ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE], an important building block for synthesis of angiotensin-converting enzyme (ACE) inhibitors. An intracellular expression level of 6.67 g L−1 and a secretion expression level of 3.0 g L−1 were obtained, respectively, by high cell density fermentation. By using whole cells of KM71/CgKR2 in aqueous system, the CgKR2-mediated bioreduction was performed, achieving a complete conversion of 1.0 M OPBE at 0.5 L scale, with a final yield of 77.9% and an enantiomeric excess (ee) of 97.3%. The secreted enzyme CgKR2 appeared to be a better catalyst with respect to the perfect ee of the product (R)-HPBE (>99%) when comparing with the recombinant cells of KM71/CgKR2. The cost of the resultant (R)-HPBE was reduced by 1/4 using the secreted CgKR2.
Keywords: Biocatalysis; (R)-HPBE; Enzyme production; Recombinant Pichia pastoris; Fermentation; Cost-effective reductase; Fed-batch culture;

Glucose oxidase enzyme immobilized porous silica for improved performance of a glucose biosensor by Anees Y. Khan; Santosh B. Noronha; Rajdip Bandyopadhyaya (78-85).
High activity of glucose oxidase (GOD) enzyme (immobilized in porous silica particles) is desirable for a better glucose biosensor. In this work, effect of pore diameter of two porous hosts on enzyme immobilization, activity and glucose sensing was compared. The hosts were amine functionalized: (i) microporous silica (NH2-MS) and (ii) mesoporous silica (NH2-SBA-15). Based on whether the dimension of GOD is either larger or smaller than the pore diameter, GOD was immobilized on either external or internal surface of NH2-MS and NH2-SBA-15, with loadings of 512.5 and 634 mg/g, respectively. However, GOD in NH2-SBA-15 gave a higher normalized absolute activity (NAA), which led to an amperometric sensor with a larger linear range of 0.4–13.0 mM glucose. In comparison, GOD in NH2-MS had a lower NAA and a smaller linear range of 0.4–3.1 mM. In fact, the present GOD-NH2-SBA-15 electrode based sensor was better than other MS and SBA-15 based electrodes reported in literature. Thus, achieving only a high GOD loading (as in NH2-MS) does not necessarily give a good sensor performance. Instead, a host with a relatively larger pore than enzyme, together with optimized electrode composition ensures the sensor to be functional in both hyper- and hypoglycemic range.
Keywords: Biocatalysis; Biosensors; Enzyme activity; Immobilized enzymes; Mesoporous silica; Glucose oxidase;

Recycling biodiesel-derived glycerol by the oleaginous yeast Rhodosporidium toruloides Y4 through the two-stage lipid production process by Xiaobing Yang; Guojie Jin; Zhiwei Gong; Hongwei Shen; Fengwu Bai; Zongbao Kent Zhao (86-91).
Direct utilization of crude glycerol, a major byproduct in biodiesel industry, becomes imperative, because its production has outpaced the demand recently. We demonstrated that the oleaginous yeast Rhodosporidium toruloides Y4 had a great capacity to convert glycerol into lipids with high yield using the two-stage production process. Significantly higher cell mass and lipid yield were observed when the media were made with synthetic crude glycerol than pure glycerol. The process achieved a lipid yield of 0.22 g g−1 glycerol, which was comparable with the lipid yield using glucose as the substrate. Lipid samples showed similar fatty acid compositional profiles to those of vegetable oils, suggesting that such microbial lipids were potential feedstock for biodiesel production. Our data provided an attractive route to integrate biodiesel production with microbial lipid technology for better resource efficiency and economical viability.
Keywords: Bioconversion; Biodiesel; Glycerol; Microbial growth; Optimization; Oleaginous yeast;

Fermentation process for continuous production of succinic acid in a fibrous bed bioreactor by Qiang Yan; Pu Zheng; Sheng-Tao Tao; Jin-Jun Dong (92-98).
Succinic acid (SA) was produced from Actinobacillus succinogenes with high cell density by continuous fermentation using fibrous bed bioreactor (FBB). The effects of feeding glucose concentration, dilution rate, and pH on continuous production of SA were examined to achieve an efficient and economical bioprocess. The optimum feeding glucose concentration, dilution rate, and pH were 80 g/L, 0.05 1/h, and 6.0–6.5, respectively. A SA concentration of 55.3 ± 0.8 g/L, productivity of 2.77 ± 0.04 g/L/h, and yield of 0.8 ± 0.02 g/g were obtained, and the continuous fermentation exhibited long-term stability for as long as 18 days (440 h) with no obvious fluctuations in both SA and biomass levels. The Jerusalimsky equation for the specific rate of SA production presented the inhibition phenomenon of the product, demonstrating that 60 g/L SA might be a critical concentration in this continuous FBB system. The results obtained could be beneficial for future fermentor designs and improvements in SA production.
Keywords: Actinobacillus succinogenes; Succinic acid; Bioprocess design; Bioreactors; Fermentation; Immobilized cells;

Although there have been remarkable progresses in hepatocytes cultures in terms of mimicking microenvironments of in vivo liver, oxygen supply is still a critical issue. In this study, we investigated the effect of direct oxygenation through oxygen-permeable membranes on functionalities of hepatocytes in two widely accepted advanced culture models, sandwich culture and 3D culture. Rat hepatocytes were cultured on the polydimethylsiloxane (PDMS) membranes for 14 days in monolayer culture, sandwich culture with Matrigel and 3D culture with microporous expanded polytetrafluoroethylene (ePTFE) membranes in the presence and absence of direct oxygenation from the other side of the membranes. The present results showed remarkable enhancement of hepatocytes duration and their functions by oxygen transfer through PDMS membranes in all these three cultures. The hepatocytes cultured in sandwich with oxygen exhibited extended survival and highest maintenance of metabolic activities, such as albumin productivity and Cyp1a1/2 activity. Additionally, the expression levels of various drug-metabolism genes, as examined by PCR arrays, were also closest to those of freshly isolated hepatocytes. As the cellular maintenance has been greatly improved by microporous ePTFE membranes, the hepatocytes in 3D culture performed increased functions that comparable to those in sandwich culture. This study clearly illustrates that oxygenation is a critical factor to be considered in optimization of the microenvironments of hepatocytes cultures.
Keywords: Tissue cell culture; Oxygen transfer; Microporous membrane; Sandwich culture; 3D culture; Optimization;

Bio-ethanol production through simultaneous saccharification and fermentation using an encapsulated reconstituted cell-free enzyme system by Muhammad Wajid Ullah; Waleed Ahmad Khattak; Mazhar Ul-Islam; Shaukat Khan; Joong Kon Park (110-119).
An encapsulated reconstituted cell-free enzyme system was developed through liquid-droplet forming method by using endogenous glycolytic and fermentation enzymes from yeast cells and exogenously added saccharification enzymes, cofactors, and ATPase. It was evaluated for bio-ethanol production through simultaneous saccharification and fermentation (SSF) at various temperatures, pH, and cell-free enzyme and substrate concentrations. Using 1% starch as substrate, encapsulated system illustrated maximum efficiency at 45 °C and pH 7.0. SSF with encapsulated and bare reconstituted cell-free enzyme systems produced 3.47 g/L and 2.98 g/L bio-ethanol corresponding to 62% and 53% of maximum theoretical yield, respectively. It was explicable that encapsulated system provided better substrate utilization and product formation at elevated temperatures than bare system. Kinetic profile of SSF process of both systems was affected differently by variations in pH, temperature, and substrate and cell-free enzyme concentrations. Under appropriate conditions, system retained 90%, 64%, and 40% of initial enzyme concentration and produced 3.21, 2.24, and 0.83 g/L bio-ethanol after 5, 10, and 15 consecutive batches, respectively. The current system offered several advantages and was superior compared to previously reported SSF systems. This system can effectively overcome the major barriers associated with successful development of SSF processes for bio-ethanol production on an industrial scale.
Keywords: Encapsulation; Reconstituted cell-free system; Ethanol; Enzyme technology; Yeast; Batch processing;

Human transferrin (hTF), an α/β protein, transforms from its native soluble form to proto-fibrils and amyloid fibrils at 20% TFE after prolonged incubation. This type of amyloid fibrils is observed in a number of pathological disorders. Existence of dry molten-globule state, at 5% TFE, was characterized by native-like secondary structure, Trp fluorescence and negligible ANS binding, indicating its dry interior. At 15% TFE, decrease in Trp and increase in ANS fluorescence was observed with native-like secondary structure, indicating exposure to water molecule and hence, this was referred to as Wet MG state. AFM revealed protofibrils as smaller in size howbeit amyloid fibrils were long and stiffer in morphology. Amyloid fibrils were found to possess cross-linked β-sheet, lack of tertiary contacts, as revealed by CD and ATR-FTIR, enhanced Thioflavin T fluorescence and shift in Congo red absorbance. These results showed that formation of amyloid fibrils becomes favorable when protein is destabilized in suitable conditions and non-covalent interactions, particularly intermolecular hydrogen bonding becomes prominent. Protofibrils were genotoxic in nature albeit amyloid fibrils lack this effect.
Keywords: Aggregation; Biophysical chemistry; Fibrils; Kinetic parameters; Transferrin;

Mathematical modeling of immobilized enzymes under different kinetics mechanism viz. simple Michaelis–Menten, uncompetitive substrate inhibition, total competitive product inhibition, total non-competitive product inhibition and reversible Michaelis–Menten reaction are discussed. These five kinetic models are based on reaction diffusion equations containing non-linear terms related to Michaelis–Menten kinetics of the enzymatic reaction. Modified Adomian decomposition method is employed to derive the general analytical expressions of substrate and product concentration for all these five mechanisms for all possible values of the parameters Φ S (Thiele modulus for substrate), Φ P (Thiele modulus for product) and α (dimensionless inhibition degree). Also we have presented the general analytical expressions for the mean integrated effectiveness factor for all values of parameters. Analytical results are compared with the numerical results and also with the limiting case results, which are found to be good in agreement.
Keywords: Mathematical modeling; Immobilized enzymes; Non-linear reaction-diffusion equations; Modified Adomian decomposition method;

Evaluating fermentation effects on cell growth and crude extract metabolic activity for improved yeast cell-free protein synthesis by Alaksh Choudhury; C. Eric Hodgman; Mark J. Anderson; Michael C. Jewett (140-148).
Saccharomyces cerevisiae is a promising source organism for the development of a practical, eukaryotic crude extract based cell-free protein synthesis (CFPS) system. Crude extract CFPS systems represent a snapshot of the active metabolism in vivo, in response to the growth environment at the time of harvest. Therefore, fermentation plays a central role in determining metabolic activity in vitro. Here, we developed a fermentation protocol using chemically defined media to maximize extract performance for S. cerevisiae-based CFPS. Using this new protocol, we obtained a 4-fold increase in protein synthesis yields with extracts derived from wild-type S288c as compared to a previously developed protocol that uses complex growth media. The final luciferase yield in our new method was 8.86 ± 0.28 μg mL−1 in a 4 h batch reaction. For each of the extracts processed under different fermentation conditions, synthesized protein, precursor monomers (amino acids), and energy substrates (nucleotides) were evaluated to analyze the effect of the changes in the growth environment on cell-free metabolism. This study underscores the critical role fermentation plays in preparing crude extract for CFPS reactions and offers a simple strategy to regulate desired metabolic activity for cell-free synthetic biology applications based on crude cell extracts.
Keywords: Cell-free protein synthesis; Defined media; Enzyme biocatalysis; Fermentation; Growth kinetics; Modelling Yeast;

Utilization of nano-gold tracing technique: Study the adsorption and transmission of laccase in mediator-involved enzymatic degradation of lignin during solid-state fermentation by Chen Zhang; Liang Liu; Guang-Ming Zeng; Dan-Lian Huang; Cui Lai; Chao Huang; Zhen Wei; Ning-Jie Li; Piao Xu; Min Cheng; Fang-Ling Li; Xiao-xiao He; Ming-yong Lai; Yi-bin He (149-156).
The degradation of lignin waste by ligninolytic enzymes and the effect of mediators on lignin degradation were studied. Moreover, nano-gold tracing technique was innovatively used to study the adsorption and transmission mechanism of ligninolytic enzymes in the process of SSF. The results showed that the effects of three common mediators on the degradation of lignin were in descending order: veratryl alcohol > ABTS > Mn2+, and the optimum dosages were: Mn2+ (20 μM g−1), ABTS (0.2 μM g−1), veratryl alcohol (4 μM g−1). A significantly high degradation ratio of lignin (31.8%) was achieved at the optimum dosages of ligninolytic enzymes and mediators. In addition, laccase, as a typical ligninolytic enzyme, was selected to conjugate to nano-gold for the study of adsorption and transmission mechanism. An obvious decrease of nano-gold-laccase conjugates was observed after 10 days of enzymatic hydrolysis and there were few laccase residues on the surface of straw fibers after 30 days of enzymatic hydrolysis. The present findings will advance the understanding of mediator-involved enzymatic degradation of lignin, as well as the adsorption and transmission mechanism of ligninolytic enzymes in lignin degradation, which could provide useful references for developing waste biotreatment technology.
Keywords: Enzymes; Lignin; Mediators; Solid-state fermentation; Nano-gold tracing technique; Adsorption;

Enabling the biosynthesis of Antroquinonol in submerged fermentation of Antrodia camphorata by Yong-Dan Hu; Huan Zhang; Rui-Qiu Lu; Xiang-Ru Liao; Bo-Bo Zhang; Gan-Rong Xu (157-162).
In recent years, Antroquinonol has been considered as one of the most potent bioactive components in the medicinal mushroom Antrodia camphorata. However, Antroquinonol could not be produced via conventional submerged fermentation of A. camphorata. In this study, the biosynthesis of Antroquinonol was successfully enabled and stimulated in the submerged fermentation by the addition of its precursors including p-hydroxybenzoic acid, geraniol and coenzyme Q0. Referred to their structures, they involve in the biosynthesis of the quinonoid nucleus and polyprenyl side chain of Antroquinonol, respectively. Moreover, the appropriate concentration and addition time of these precursors were systematically studied. The maximum production of Antroquinonol could be achieved at 82.22 ± 0.49 mg/L with the multiple additions of coenzyme Q0 and p-hydroxybenzoic acid. This study demonstrated the bioprocess regulation of Antroquinonol production, which will be of great significance for the scientific study of A. camphorata.
Keywords: Antrodia camphorata; Antroquinonol; Bioprocess design; Biosynthesis; Fermentation; Optimisation;

Concentration of docosahexaenoic and eicosapentaenoic acids by enzymatic alcoholysis with different acyl-acceptors by Lorena Martín Valverde; Pedro A. González Moreno; Luis Esteban Cerdán; Elvira Navarro López; Beatriz Castillo López; Alfonso Robles Medina (163-173).
The aim of this work was to produce docosahexaenoic (DHA) and eicosapentaenoic acid (EPA) enriched acylglycerols by alcoholysis of tuna and sardine oils, respectively, using isobutanol and 1-butanol as acyl-acceptors. The alcoholysis reactions were catalyzed by lipases Lipozyme® TL IM from Thermomyces lanuginosus and lipase QLG® from Alcaligenes sp., because these lipases have shown selectivity towards DHA and EPA, respectively. Studies were made to determine the influence of reaction time, alcohol/oil molar ratio, lipase amount and temperature. In the optimized conditions for the alcoholysis of tuna and sardine oils catalyzed by Lipozyme TL IM and lipase QLG, respectively, the DHA and EPA contents were trebled (from 22 to 69% for DHA, and from 19 to 61% for EPA). The stability of both lipases was also determined. Although Lipozyme TL IM is much more stable in isobutanol than in ethanol, with the former the conversion attained after four reaction cycles was about 40% of the initial conversion. In similar conditions, the conversion obtained with lipase QLG was about 88% of the initial conversion. In addition, the separation of DHA enriched acylglycerols and isobutyl esters from an alcoholysis reaction was studied by liquid–liquid fractionation using the ethanol–water–hexane biphasic system. The DHA enriched acylglycerols obtained were 97.6% pure (64.4% DHA).
Keywords: Docosahexaenoic acid (DHA); Eicosapentaenoic acid (EPA); Lipase; Biokinetic; Enzyme deactivation; Liquid–liquid extraction;

Characterization of dewatering process of activated sludge assisted by cationic surfactants by Long-Fei Wang; Dong-Qin He; Zhong-Hua Tong; Wen-Wei Li; Han-Qing Yu (174-178).
In this work, the dewatering of activated sludge assisted by cationic surfactants was investigated. Dose of dodecyl trimethyl ammonium bromide (DTAB) and cetyl trimethyl ammonium bromide (CTAB) resulted in the release of extracellular polymeric substances (EPS) from sludge and decrease in sludge negative charge. The surfactants significantly promoted sludge dewaterability, as reflected by decreased specific resistance of filtration (SRF) and water content in sludge cakes. The treated sludge were analyzed by combined use of differential scanning calorimetry (DSC), thermo gravimetric analysis (TGA) and Brunauer–Emmett–Teller (BET) analysis. CTAB was found more effective in releasing bound water from sludge than DTAB, due to its superior surface activity and strong adsorption/bridge capacities with sludge. The specific surface area and pore size of sludge cakes declined after surfactant treatment, indicating an enhanced compressibility. With these results, the surfactant-assisted dewatering mechanism could be attributed to the integrated effects of electrostatic neutralization, enhanced compaction and release of EPS and bound water. Our study clearly characterizes the accelerated dewatering process assisted by cationic surfactants, and demonstrates that cationic surfactants could be used as a potential agent for sludge dewatering.
Keywords: Cationic surfactant; Dewatering; Adsorption; Filtration; Separation; Waste-water treatment;

Effect of arsanilic acid on anaerobic methanogenic process: Kinetics, inhibition and biotransformation analysis by Hai-Long Wang; Zhen-Hu Hu; Zi-Lin Tong; Qiao Xu; Wei Wang; Shoujun Yuan (179-185).
Arsanilic acid (4-aminophenylarsonic acid) is widely used in the poultry and animal industries as a feed additive in the diets. Nearly all the added arsanilic acid is excreted unchanged in manure resulting in the risk of arsenic contamination. In this study, the effects of arsanilic acid on the kinetics, inhibition of methanogenic process and its biotransformation were investigated. The methane yield was not affected by arsanilic acid loading at concentration <0.46 mM, while the methane production was completely inhibited at concentration of 0.92 mM. The IC50 of arsanilic acid in this study was 0.47 mM. After 115 days of incubation, 37–59% of the added arsanilic acid was degraded. The species analysis indicated that at lower initial arsanilic acid concentration, the soluble inorganic arsenic mainly existed in the species of arsenate (As(V)), while at higher initial arsanilic acid concentration (>0.460 mM), the soluble inorganic arsenic mainly existed in the species of arsenite (As(III)), which explains why higher arsanilic acid concentration has severe inhibition to methanogens.
Keywords: Methane production; Arsanilic acid; Biotransformation; Organoarsenic; Modelling; Wastewater treatment;

The combination of cell morphology investigation and metabolomics analysis were carried out to improve rapamycin production rationally in Streptomyces hygroscopicus. First, the correlation between morphology and rapamycin production by S. hygroscopicus was investigated with statistical experiments, and the longer filaments and higher branching frequency in hyphal growth in the periphery of the pellets were conducive to high production of rapamycin. Then, to explore the potential mechanism influencing morphology of S. hygroscopicus, metabolomics-based approach was employed to reveal intracellular metabolic change under different morphology characteristics conditions and identify the potential key metabolites in response to different morphology characteristics in submerged fermentation process. Furthermore, 19 potential key metabolites related pellet morphology and rapamycin production were revealed. Based on the analysis of key metabolites change combined with related metabolic pathway, as well as the change in pellet morphology characteristics, the dissolved oxygen supplied strategies were proposed, and rapamycin production was ultimately increased by 63.9%, reaching 536 mg/L under optimal oxygen supplied strategy condition. The strategy developed here could be extended to titer improvement and process optimization of other products.
Keywords: Filamentous Bacteria; Fermentation; Biosynthesis; Morphology; Metabolomics analysis; Dissolved oxygen;

Mutations introduced to wild-type proteins naturally, or intentionally via protein engineering, often lead to protein aggregation. In particular, protein aggregation within mammalian cells has significant implications in the disease pathology and biologics production; making protein aggregation modulation within mammalian cells a very important engineering topic. Previously, we showed that the semi-rational design approach can be used to reduce the intracellular aggregation of a protein by recovering the conformational stability that was lowered by the mutation. However, this approach has limited utility when no rational design approach to enhance conformational stability is readily available. In order to overcome this limitation, we investigated whether the modification of residues significantly displaced upon the original mutation is an effective way to reduce protein aggregation in mammalian cells. As a model system, human copper, zinc superoxide dismutase mutant containing glycine to alanine mutation at position 93 (SOD1G93A) was used. A panel of mutations was introduced into residues substantially displaced upon the G93A mutation. By using cell-based aggregation assays, we identified several novel variants of SOD1G93A with reduced aggregation propensity within mammalian cells. Our findings successfully demonstrate that the aggregation of a mutant protein can be suppressed by mutating the residues significantly displaced upon the original mutation.
Keywords: Protein aggregation; Mammalian cells; Mutation; Protein engineering; Superoxide dismutase;

An efficient preparation of l-Tle was developed through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant E. coli coexpressing leucine dehydrogenase and NAD+-dependent formate dehydrogenase.Enantiopure l-tert-leucine (l-Tle) was synthesized through reductive amination of trimethylpyruvate catalyzed by cell-free extracts of recombinant Escherichia coli coexpressing leucine dehydrogenase (LeuDH) and formate dehydrogenase (FDH). The leudh gene from Lysinibacillus sphaericus CGMCC 1.1677 encoding LeuDH was cloned and coexpressed with NAD+-dependent FDH from Candida boidinii for NADH regeneration. The batch reaction conditions for the synthesis of l-Tle were systematically optimized. Two substrate feeding modes (intermittent and continuous) were addressed to alleviate substrate inhibition and thus improve the space-time yield. The continuous feeding process was conveniently performed in water at an overall substrate concentration up to 1.5 M, with both conversion and ee of >99% and space-time yield of 786 g L−1  d−1, respectively. Furthermore, the preparation was successfully scaled up to a 1 L scale, demonstrating the developed procedure showed a great industrial potential for the production of enantiopure l-Tle.
Keywords: l-tert-Leucine; Reductive amination; Biocatalysis; Biotransformation; Bioprocess design; Optimization;

Quantitative modeling of inducer transport in fed-batch cultures of Escherichia coli by Daniel Calleja; Alfred Fernández-Castañé; Martina Pasini; Carles de Mas; Josep López-Santín (210-219).
An unsteady, unstructured, unsegregated and based on first principles mathematical model has been proposed to describe IPTG (isopropyl-β-d-tiogalactopiranoside) transport in induced fed-batch cultures of E. coli M15 ΔglyA [pQEαβrham] [pREP4] producing rhamnulose 1-phosphate aldolase (RhuA). The model predicts extracellular and intracellular IPTG concentration. Experimental extracellular IPTG concentrations under different operational conditions were obtained by HPLC–MS analysis. These experimental data were used to fit the parameters of the model. The model was also able to predict the experimental behavior of two different E. coli strains producing fuculose 1-phosphate aldolase (FucA). IPTG transport to cells was the contribution of three processes: a diffusion process, and two active processes (one non-specific and another specific).
Keywords: E. coli; Mathematical model; Inducer transport; IPTG; Lac permeases;

Our recent work showed that Chlorella vulgaris JSC-6 can accumulate over 50% carbohydrates per dry weight, which mainly contain glucose and xylose as the monosaccharides. In this study, synthetic mixed sugars simulating the hydrolyzed biomass of C. vulgaris JSC-6 (primarily containing glucose and xylose at a ratio of 5:1–6.5:1) was used as the carbon source for bio-butanol production with Clostridium acetobutylicum ATCC824. The growth and product formation kinetics based on glucose, xylose, and mixtures of the two sugars were determined using Monod-type and Michaelis–Menten models, respectively. C. acetobutylicum ATCC824 can grow faster and produce butanol more efficiently on glucose, while the performance was markedly poorer when using xylose. The butanol production kinetics were quite similar when using glucose alone or using mixed sugars (glucose to xylose ratio = 5:1 or 6.5:1), resulting in a maximum butanol production rate of 0.89–0.93 g/h/L. This work demonstrated the feasibility of using the microalgae-based carbohydrates as the feedstock for biobutanol production with C. acetobutylicum ATCC824.
Keywords: Butanol; Fermentation; Carbohydrates; Growth kinetics; Production kinetics; Microalgae;

It is known that trehalose and sodium chloride (NaCl) can both effectively inhibit acid-induced protein denaturation, but the thermodynamic and kinetic behaviors of acid-induced protein unfolding synergistically inhibited by trehalose and NaCl are unclear. In this study, the synergistic inhibition effects of trehalose and NaCl on the acid-induced unfolding of ferricytochrome c were studied at pH 2.0. Thermodynamic parameters were firstly derived based on fluorescence spectroscopic data. Then, kinetic behaviors were studied using stopped-flow fluorescence spectroscopy. It was found that the kinetics of the acid-induced protein unfolding transformed from a triphasic process (i.e., fast, intermediate and slow phases) into a biphasic one (i.e., intermediate and slow phases) and then a single slow phase process with increasing either trehalose or NaCl concentration in the mixture. The rate constants for all the unfolding phases change slightly, while the amplitudes for the fast and intermediate phases diminish greatly with increasing the concentration of trehalose or NaCl. This clearly indicates that the mixture of trehalose and NaCl could synergistically inhibit acid-induced protein unfolding by reducing the extent of protein conformational changes, thus inducing a stable molten-globule state at higher concentrations of the agents.
Keywords: Protein; Biophysical chemistry; Protein denaturation; Kinetic parameters; Trehalose; Ferricytochrome c;

Succinic acid production from sucrose and molasses by metabolically engineered E. coli using a cell surface display system by Jiangfeng Ma; Feng Li; Rongming Liu; Liya Liang; Yaliang Ji; Ce Wei; Min Jiang; Honghua Jia; Pingkai Ouyang (240-249).
To achieve sucrose-metabolizing capability, different sucrose utilization operons have been introduced into E. coli that cannot utilize sucrose. However, these engineered strains still suffer from low growth rates and low sucrose uptake rates. In this study, cell surface display system was adopted in engineered E. coli AFP111 for succinic acid production from sucrose and molasses directly. Invertase (CscA) from E. coli W was successfully anchored to outer membrane by fusion with OmpC anchoring motif, and the displayed CscA showed high extracellular activity. Compared with the sucrose permease system, the cell surface display system consumed less ATP during sucrose metabolism. When less ATP was consumed by AFP111/pTrcC-cscA, the succinic acid productivity from sucrose was 23% higher than that by AFP111/pCR2.1-cscBKA that having the sucrose permease system. As a result, 41 g L−1 and 36.3 g L−1 succinic acid were produced by AFP111/pTrcC-cscA from sucrose and sugarcane molasses respectively at 34 h in 3-L fermentor during dual-phase fermentation. In addition, 79 g L−1 succinic acid was accumulated with recovered AFP111/pTrcC-cscA cells at the end of dual-phase fermentation in 3-L fermentor, and the overall yield was 1.19 mol mol−1 hexose.
Keywords: Sucrose; Cell surface display; CscA; Bioconversion; Fermentation; Fed-batch culture;

Multiobjective evolutionary optimization in antibody purification process design by Richard Allmendinger; Ana S. Simaria; Suzanne S. Farid (250-264).
To contribute towards designing more cost-efficient, robust and flexible downstream processes for the manufacture of monoclonal antibodies (mAbs), a framework consisting of an evolutionary multiobjective optimization algorithm (EMOA) linked to a biomanufacturing process economics model is presented. The EMOA is tuned to discover sequences of chromatographic purification steps and column sizing strategies that provide the best trade-off with respect to multiple objectives including cost of goods per gram (COG/g), robustness in COG/g, and impurity removal capabilities. Additional complexities accounted for by the framework include uncertainties and constraints. The framework is validated on industrially relevant case studies varying in upstream and downstream processing train ratios, annual demands, and impurity loads. Results obtained by the framework are presented using a range of visualization tools, and indicate that the performance impact of uncertainty is a function of both the level of uncertainty and the objective being optimized, and that uncertainty can cause otherwise optimal processes to become suboptimal. The optimal purification processes discovered outperform the industrial standard with, e.g. savings in COG/g of up to 10%. Guidelines are provided for choosing an optimal purification process as a function of the objectives being optimized and impurity levels present.
Keywords: Biopharmaceutical manufacture; Monoclonal antibodies; Downstream processing; Modeling; Optimisation; Evolutionary multiobjective optimisation;

Quasi-continuous fermentation in a reverse-flow diafiltration bioreactor by Kristina Meier; Frederike Carstensen; Matthias Wessling; Lars Regestein; Jochen Büchs (265-275).
Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse-flow diafiltration utilizes an internal membrane device, but pulsed feeding causes spatial heterogeneities. In this study, the influence of conventional reverse-flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non-feeding at a dilution rate of 0.2 h−1 results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short-term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi-continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h−1 and 0.5 h−1, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h−1 to 0.5 h−1. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time.
Keywords: In situ product recovery; Hansenula polymorpha; Hollow fibers; Membrane bioreactors; Dissolved Oxygen; Glucose pulse;

Lipases catalyze the hydrolysis of carboxylic acid esters and owing to their vast substrate specificity, they have many industrial applications. Due to the demand of thermostable lipases in industrial applications, we have enhanced the thermostability of lipase from Bacillus licheniformis RSP-09. The thermostable mutant lipases of Bacillus licheniformis RSP-09 were isolated following two rounds of directed evolution using error-prone PCR. The best mutant lipases obtained after first and second round of error-prone PCR were purified and characterized. The mutant lipases showed increased thermostability and retained catalytic function. The best mutant lipase (eP-231-51) showed 13.5-fold increase in percentage thermal stability (% remaining activity after incubation of purified enzyme at 60 °C for 1 h) than wild-type lipase. Also, this mutant lipase (ep-231-51) showed 30% improved catalytic efficiency compared with the wild-type which is due to significant decrease in K m and marginal increase in k cat. In addition, the thermostable mutant lipases have shown resistance to hydrophobic organic solvents. The role of mutations in the best mutant lipases of second round i.e. eP-231-51 (Asp72Gly, Asp61Gly, Tyr129His, and Thr101Pro) and eP-231-137 (Leu49Arg, Thr101Pro, Asp72Gly), that led to thermostability have been postulated after the comparison of molecular models of wild-type and mutated enzymes.
Keywords: Lipase; Biocatalysis; Enzymes; Protein; Directed evolution; Thermostability;

Acute impact of tetracycline and erythromycin on the storage mechanism of polyhydroxyalkanoates by Tugce Katipoglu-Yazan; Ilke Pala-Ozkok; Emine Ubay-Cokgor; Derin Orhon (283-289).
The study investigated acute impact of tetracycline and erythromycin on substrate storage under aerobic conditions. A fill and draw reactor fed with peptone mixture was maintained at steady-state at a sludge age of 10 days; the acclimated biomass was used in a series of batch runs. The first run served as control reactor with organic substrate alone and the others were started with antibiotic doses of 50 mg/L and 200 mg/L for assessing intracellular storage. Parallel batch reactors were also conducted for recording oxygen uptake rate profiles. Both antibiotics enhanced substrate storage, leading to higher levels of polyhydroxyalkanoates incorporated into biomass, but they impaired its internal utilization for microbial growth. The observed decrease in oxygen consumption under the acute effect of antibiotics could partially be related to substrate storage – except for 50 mg/L of erythromycin dosing – suggesting an additional substrate binding mechanism by antibiotics, leading to residual biodegradable substrate.
Keywords: Substrate storage; Polyhydroxyalkanoates; Kinetic parameters; Dissolved oxygen; Peptone; Antibiotic;