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Biochemical Engineering Journal (v.49, #3)

BEJ Keywords (pp. ii).

Editorial Board (pp. co2).

Methanotrophs: Multifunctional bacteria with promising applications in environmental bioengineering by Hao Jiang; Yin Chen; Peixia Jiang; Chong Zhang; Thomas J. Smith; J. Colin Murrell; Xin-Hui Xing (pp. 277-288).

Methane is an important greenhouse gas which is produced from many natural and anthropogenic sources. It plays an important role in overall global warming. A significant amount of methane is removed through microbiological oxidation by methanotrophic bacteria, which are widespread in the environment, including many extreme environments. The key enzyme of these microorganisms, methane monooxygenase (MMO), especially the soluble MMO, is remarkable in its broad substrate specificity. This unique capability, i.e. catalyzing reactions of environmental importance, has attracted great attention for applied microbiologists and biochemical engineers. In this review, recent advances in the application of methanotrophs to environmental bioengineering are summarized, including biodiversity, catalytic properties, and cultivation, etc. We have focused on two aspects of the application and potential value of methanotrophs in environmental bioengineering, namely methane removal and biodegradation of toxic compounds. The removal of methane produced from landfills has been widely studied, and much of this work can be used as a source of reference for coal mine gas removal. Many bioreactors using methanotrophs in bioremediation have been developed in recent years. These reactors have two forms of configuration, single-stage and multi-stage. Current limitations which may affect the engineering applications of methanotrophs are discussed, such as the lack of suitable methanotrophic isolate, gas transfer limitation, competitive inhibition of MMO, regeneration of reducing equivalents for MMO and product toxicity.

Keywords: Methane; Methanotroph; Methane monooxygenase; Greenhouse gas; Biodegradation

Oxygen uptake rate in microbial processes: An overview by Felix Garcia-Ochoa; Emilio Gomez; Victoria E. Santos; Jose C. Merchuk (pp. 289-307).

In aerobic process oxygen must be continuously supplied in order to achieve acceptable productivities, Since the role of oxygen in microorganism growth and its metabolism is of vital importance, both the oxygen consumption by the cell and the oxygen transfer rate (OTR) into the system have to be understood.The main function of a properly designed bioreactor is to provide a controlled environment and a concentration of nutrients (dissolved oxygen, mainly) sufficient to achieve optimal growth and/or optimal product formation in a particular bioprocess. Dissolved oxygen in the broths is the result of a balance of its consumption rate in the cells, and the rate of oxygen transfer from the gas to the liquid phase. Monitoring dissolved oxygen in the broth is mandatory because often oxygen becomes the factor governing the metabolic pathways in microbial cells.In this work the oxygen uptake rate (OUR) in different fermentation broths is examined. Experimental techniques have been compiled from the literature and their applicability to microbial processes reviewed. The reciprocal influence of OUR and OTR is presented and an analysis of rate-limiting variables is carried out.Mathematical models are a fundamental tool in bioprocess design, optimisation, scale-up, operation and control at large-scale fermentation. Kinetic models describing aerobic bioprocesses have to include an oxygen balance taking into account OTR and OUR. Many different specific rate expressions for cell growth, substrate consumption, product formation and oxygen uptake have been developed and incorporated in the models, and simulations of different bioprocess have been carried out. Some of them are presented here.

Keywords: Oxygen uptake rate; Oxygen transfer rate; Bioprocesses description; Bioreactor design; Scale-up

Evaluation of amiodarone-induced phospholipidosis by in vitro system of 3D cultured rat hepatocytes in gel entrapment by Chong Shen; Guoliang Zhang; Qin Meng (pp. 308-316).

Various mechanisms are involved in drug hepatotoxicity including reactive intermediates, steatosis, phospholipidosis, mitochondrial dysfunction, etc. Although 3D cultured hepatocytes reflected more CYP 450 mediated reactive intermediates than traditional 2D cultured hepatocytes, the 3D model has not been evaluated for drug-induced phospholipidosis. This study applied 3D cultured hepatocytes in gel entrapment for drug-induced phospholipidosis, and amiodarone was used as a model drug. By amiodarone exposure for 48h, 3D cultured hepatocytes showed large number of lysosomal lamellar bodies (indicating phospholipidosis) and toxic response at a low dose of 2.5μM, equivalent to toxic serum concentration in rats. This sensitivity to amiodarone-induced phospholipidosis might relate to the more intracellular drug distribution in 3D cultured hepatocytes. Moreover, steatosis, mitochondrial injury and oxidative stress were all sensitively detected in 3D cultured hepatocytes, well reflecting the involvement of these mechanisms in amiodarone hepatotoxicity. In addition, pretreatment of 3D cultured hepatocytes by CYP 3A1/2 inhibitor (ketoconazole) significantly reduced the toxicity of amiodarone, indicating the positive mediation of CYP 3A1/2. By comparison, 2D cultured hepatocytes did not show significant phospholipidosis and involvement of CYP 3A1/2. In conclusion, hepatocytes in 3D culture well reflected amiodarone toxicity and thus could be a promising model for phospholipidosis study.

Keywords: Abbreviations; CYP; cytochrome; DTNB; 5,5-dithiobis(2-nitrobenzoic acid); GAPDH; glyceraldehyde-3-phosphate dehydrogenase; GSH; glutathione; LDH; lactate dehydrogenase; MDA; malondialdehyde; MTT; methyl thiazolyl tetrazolium; PBS; phosphate buffer solution; ROS; reactive oxygen species; RT-PCR; reverse transcription-polymerase chain reaction; TBA; thiobarbituric acidAmiodarone; Phospholipidosis; Hepatocytes; 3D culture; Gel entrapment; Drug accumulation

Adsorption of Reactive Red 4 by immobilized chitosan on glass plates: Towards the design of immobilized TiO₂–chitosan synergistic photocatalyst-adsorption bilayer system by M.A. Nawi; S. Sabar; A.H. Jawad; Sheilatina; W.S. Wan Ngah (pp. 317-325).

Immobilized chitosan adsorbent on glass plates (CS/glass) has been fabricated as a pre-step towards the design of bilayer immobilized TiO₂–chitosan synergistic photocatalyst-adsorbent system. The characterizations of the immobilized chitosan were studied by SEM, FTIR, BET, and strength test. Experiments were carried out as a function of adsorbent loading, pH, contact time, initial dye concentration and temperature using Reactive Red 4 anionic dye (RR4) as a model pollutant. The adsorption equilibrium data can be described well by the Langmuir and Freundlich isotherm models. The maximum adsorption capacity calculated from the Langmuir model was found to be 172.41mgg⁻¹. Experimental kinetic data were analyzed using pseudo-first and pseudo-second order rate models. Pseudo-second order model demonstrated to be the best kinetic model for the system suggesting that the rate-limiting step may be chemisorption. The negative value of free energy and enthalpy obtained indicates that the adsorption process is spontaneous and exothermic.

Keywords: Wastewater treatment; Adsorption; Separation; Immobilization; Chitosan; Reactive Red 4

The relationships between the metabolic fluxes and¹³C-labeled isotopomer distribution for the flux analysis of the main metabolic pathways by Yu Matsuoka; Kazuyuki Shimizu (pp. 326-336).

It has been known that¹³C-labeling technique is quite useful in estimating the metabolic fluxes. Although the program-based flux analysis is powerful, it is not easy to be confident with the result obtained without experiences and exhaustive trial and errors based on statistical analysis for the confidence intervals in practice. It is, therefore, quite important to grasp the relationship between the fluxes and the¹³C-labeled isotopomer distribution to get deeper insight into the metabolic flux analysis. In the present research, it was shown explicitly how the isotopomer distribution changes with respect to the fluxes in relation to the labeling patterns of the substrate, where either labeled glucose, acetate, or pyruvate was used as a carbon source. Some of the analytical expressions were derived based on the matrix representation, and they were utilized for analysis. It was shown that the isotopomer pattern does not necessarily change uniformly with respect to fluxes, but changes in a complicated way in particular for the case of using pyruvate as a carbon source where some isotopomers do not necessarily change monotonically. It was shown to be quite important to grasp how the isotopomer pattern changes with respect to fluxes and the labeling pattern of the substrate for flux determination and the experimental design. It was also shown that the mixture of [1-¹³C] acetate and [2-¹³C] acetate should not be used from the information index point of view. Some of the experimental data were evaluated from the present approach. It was also shown that the isotopomer distribution is less sensitive to the bidirectional fluxes in the reversible pathway.

Keywords: ¹³; C-labeling experiment; Isotopomer distribution; Metabolic flux analysis; Analytical expression

Effects of using various bioreactors on chitinolytic enzymes production by Paenibacillus taichungensis by Hua-Bing Chen; Po-Min Kao; Hung-Chang Huang; Chwen-Jen Shieh; Chih-I Chen; Yung-Chuan Liu (pp. 337-342).

In this study, various types of reactors were employed to investigate chitinolytic enzymes production via Paenibacillus taichungensis. It was found that the net-draft tube air-lift reactor (ALNR) gave the best performance among the reactors. Different aeration conditions of ALNR were carried out to evaluate cell growth and chitinolytic enzymes production. It was found that a 2vvm aeration produces an optimal chitinolytic enzymes activity level of 21.1mU/mL, which was 2.6 times greater than that obtained in a stirred-tank reactor. To explore the result, the shear rates and oxygen transfer rates of both reactors were evaluated and compared. Meanwhile, the morphologies of the strain for chitinolytic enzymes production were also investigated.

Keywords: Oxygen transfer rate; Shear rate; Chitinolytic enzyme production; Paenibacillus taichungensis; Net-draft tube air-lift reactor (ALNR)

Separation of recombinant β-glucuronidase from transgenic tobacco by aqueous two-phase extraction by Kristin C. Ross; Chenming Zhang (pp. 343-350).

Transgenic plants hold many promises as viable production hosts for therapeutic recombinant proteins. Many efforts have been devoted to increase the expression level of the proteins, but the efforts for developing economic processes to purify those proteins are lacking. In this report, aqueous two-phase extraction (ATPE) was investigated as an alternative for the separation of an acidic recombinant protein, β-glucuronidase (rGUS), from transgenic tobacco. Screening experiments by fractional factorial designs showed that PEG concentration and ionic strength of the system significantly affected the partitioning of native tobacco proteins and GUS. Response surface methodology was used to determine an optimized aqueous two-phase system for the purification of rGUS from transgenic tobacco. In a 13.4% (w/w) PEG 3400/18% (w/w) potassium phosphate system, 74% of the rGUS was recovered in the top PEG-rich phase while more than 90% of the native tobacco proteins were removed in the interphase and the bottom phase. A purification factor of about 20 was achieved in this process. The most important impurity from tobacco, Rubisco, was largely removed from the rGUS in the recovered phase.

Keywords: Transgenic tobacco; Recombinant protein; Downstream processing; Aqueous two-phase system; Aqueous two-phase extraction; Recombinant glucuronidase

Effect of surface contaminants on oxygen transfer in bubble column reactors by Maryam Asgharpour; Mohammad Reza Mehrnia; Navid Mostoufi (pp. 351-360).

Gas hold-up ( ɛ_g), sauter mean bubble diameter ( d₃₂) and oxygen transfer coefficient ( k_L a) were evaluated at four different alkane concentrations (0.05, 0.1, 0.3 and 0.5vol.%) in water over the range of superficial gas velocity ( u_g) of (1.18–23.52)×10⁻³m/s at 25°C in a laboratory-scale bubble column bioreactor. Immiscible hydrocarbons (n-decane, n-tridecane and n-hexadecane) were utilized in the experiments as impurity. A type of anionic surfactant was also employed in order to investigate the effect of addition of surfactant to organic-aqueous systems on sauter mean bubble diameter, gas hold-up and oxygen transfer coefficient. Influence of addition of alkanes on oxygen transfer coefficient and gas hold-up, was shown to be dependent on the superficial gas velocity. At superficial gas velocity below 0.5×10⁻³m/s, addition of alkane in air–water medium has low influence on oxygen transfer coefficient and also gas hold-up, whereas; at higher gas velocities slight addition of alkane increases oxygen transfer coefficient and also gas hold-up. Increase in concentration of alkane resulted in increase in oxygen transfer coefficient and gas hold-up and roughly decrease in sauter mean bubble diameter, which was attributed to an increase in the coalescence-inhibiting tendency in the presence of surface contaminant molecules. Bubbles tend to become smaller with decreasing surface tension of hydrocarbon, thus, oxygen transfer coefficient increases due to increasing of specific gas–liquid interfacial area ( a). Empirical correlations were proposed for evaluating gas hold-up as a function of sauter mean bubble diameter, superficial gas velocity and interfacial surface tension as well as evaluating Sherwood number as a function of Schmidt, Reynolds and Bond numbers.

Keywords: Bubble columns; Hydrodynamics; Mass transfer; Hydrocarbon contaminants; Surface tension; Surfactant

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification by Digantkumar Chapla; Jyoti Divecha; Datta Madamwar; Amita Shah (pp. 361-369).

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400U/g and validated xylanase activity was 8450U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.

Keywords: Xylanase; Lignocellulose degradation; Optimisation; Solid state; Chromatography; Saccharification

Tracking composition and dynamics of nitrification and denitrification microbial community in a biofilm reactor by PCR-DGGE and combining FISH with flow cytometry by Siqing Xia; Jixiang Li; Rongchang Wang; Junying Li; Zhiqiang Zhang (pp. 370-378).

A compact suspended carrier biofilm reactor (SCBR) was operated at three different C/N ratios (C/N=10:1, 5:1 and 3:1) with focus on reactor performance and microbial community composition of nitrifying and denitrifying bacteria. The process was capable of achieving over 90% removal of chemical oxygen demand (COD) and over 83.3% of simultaneous nitrification and denitrification (SND) efficiency. Denaturing gradient gel electrophoresis (DGGE) analysis showed that the diversity of ammonia-oxidizing bacteria (AOB) community decreased with inoculation sludge, C/N ratio 3:1, 10:1 and 5:1 in turn. Phylogenetics analysis indicated that there were three distinct groups of AOB in Betaproteobacteria subdivision, where Nitrosomonas were the dominant members in the biofilm. Fluorescence in situ hybridization-flow cytometry (FISH-FCM) results revealed that Alpha-, Beta- and Gamma-proteobacteria accounted for over 50% of all cells. Additionally, the clusters of Nitrosomonas, Nitrosospira, Nitrobacter, Nitrospira and AOB of Betaproteobacteria yielded a similar distribution pattern about 5–12% of all cells. Nested analysis of variance assay (ANOVA) demonstrated that the C/N ratio did not significantly affect the shift of populations in different groups of nitrifying and denitrifying bacteria detected by FISH-FCM.

Keywords: Suspended carrier biofilm reactor; Microbial community composition and dynamics; PCR-DGGE; Phylogenetic analysis; Fluorescence in situ hybridization; Flow cytometry

Effect of mechanical agitation on the production of cellulases by Trichoderma reesei RUT-C30 in a draft-tube airlift bioreactor by Aftab Ahamed; Patrick Vermette (pp. 379-387).

With the aim to produce cellulases and to study the effect of mechanical agitation, a 35L draft-tube airlift bioreactor equipped with a mechanical impeller was developed and validated to grow Trichoderma reesei RUT-C30 in a cellulose culture medium with lactose and lactobionic acid as fed batch. Cultures carried out without mechanical agitation resulted in higher volumetric enzyme productivity (200UL⁻¹h⁻¹), filter paper activity (17UmL⁻¹), carboxymethyl cellulase activity (11.8UmL⁻¹) and soluble proteins (3.2mgmL⁻¹) when compared to those with agitation. Stereo and polarized light microscopy analyses reveal that mechanical agitation resulted in shorter mycelial hyphae and larger numbers of tips.

Keywords: Airlift bioreactor; Cellulose; Lactose; Trichoderma reesei; Cellulase enzymes

Screening of agro-industrial wastes to produce ligninolytic enzymes by Phanerochaete chrysosporium by Fatma Gassara; Satinder K. Brar; R.D. Tyagi; M. Verma; R.Y. Surampalli (pp. 388-394).

Ligninolytic enzyme production by solid-state cultures of Phanerochaete chrysosporium BKM-F-1767 was investigated by employing different agro-industrial wastes, such as fishery residues, brewery waste, apple waste (pomace) and pulp and paper industry sludge. Different enzyme inducers, such as veratryl alcohol, Tween-80 and CuSO₄ at concentrations of 2mM, 0.5% (v/w) and 3mmole/kg, respectively were also tested. Use of veratryl alcohol and Tween-80 resulted in maximum manganese peroxidase (MnP) activity of 17.36±0.5, 540.2±5.1, 631.25±14, and 507.5±26.87U/gds (units/gram dry substrate), respectively, for different wastes. Maximum lignin peroxidase (LiP) activity of 141.38±3.39 and 14.1±0.5U/gds was attained with pomace and pulp and paper, respectively. Laccase activities were found to be insignificant for all wastes. Addition of Tween-80 and CuSO₄ resulted in highest values of MnP activity of 17.4±0.6, 291±2.8, 213.5±3, and 213.2±3.2U/gds for fishery waste, brewery waste, pomace and pulp and paper industry sludge, respectively. Addition of CuSO₄ to the culture medium enhanced laccase activity. Maximum laccase activities of 738.97±9.2, 719.97±14.6, 308.8±12.1, and 94.44±1.2U/gds were obtained for brewery waste, pomace, pulp and paper industry sludge and fishery waste, respectively. Brewery wastes and pomace served as excellent sources for the production of MnP, LiP and laccases.

Keywords: Abbreviations; ATCC; American type culture collection; cfu; colony forming unit; gds; gram dry substrate; LiP; lignin peroxidase; MnP; manganese peroxidase; MPN; most probable number; OD; optical density; PAP; pulp and paper industry sludge; PDA; potato–dextrose–agar; SSF; solid-state fermentation; U/gds; units per gram dry substrate; w/dw; weight per dry weight; w/v; weight per volumeLigninolytic enzymes; Agro-industrial wastes; Inducers; Phanerochaete chrysosporium

Medium optimization and production of secreted Renilla luciferase in Bacillus subtilis by fed-batch fermentation by Po Ting Chen; Chung-Jen Chiang; Yun-Peng Chao (pp. 395-400).

Luciferase of the soft coral Renilla reniformis (Rluc) has found a multitude of potential applications. However, its production in a secreted form has been solely performed in mammalian cells. In this work, a high production of secreted Rluc was approached in Bacillus subtilis by implementing the fermentation strategy. First, three critical medium components among others were selected by the Plackett–Burman design. Followed by the Box–Behnken design, the medium formulation was optimized and composed of 1.17% glucose, 2.27% yeast extract, and 0.55% glutamate. With the optimum medium, the shake-flask culture of the producer strain was carried out to secret Rluc with the yield of 55mg/L. Finally, the production scheme was scaled up using a 5-L jar fermenter operated in a fed-batch mode. By controlling the operational condition, the feeding solution was pumped in an impulse mode to the culture. As a consequence, the production of Rluc reaching 500mg/L was obtained after fermentation for 27h.

Keywords: Renilla; luciferase; Experimental design; Fed-batch fermentation; Bacillus subtilis

Comparing the efficiency of the laccase–NHA and laccase–HBT systems in eucalyptus pulp bleaching by Cristina Valls; José F. Colom; Carole Baffert; Isabelle Gimbert; M. Blanca Roncero; Jean-Claude Sigoillot (pp. 401-407).

Laccase–mediator systems have the disadvantage that the mediator is expensive and potentially toxic. In this work, we used N-hydroxyacetanilide (NHA) in combination with laccase for the first time to bleach eucalypt pulp and found it to be a very promising, advantageous alternative to 1-hydroxybenzotriazole (HBT) as mediator. Thus, NHA is efficiently oxidized by laccase to a radical that absorbs light at 350nm. Also, NHA is a better substrate for laccase than is HBT. An innovative result is that the enzyme is inactivated to a similar extent by both mediators under the typical treatment conditions of the bleaching step (L). This adverse effect, however, is strongly reduced in the presence of pulp. Moreover, the laccase–NHA system is as efficient as the laccase–HBT system in reducing the kappa number of eucalyptus pulp. Using a xylanase pretreatment or unbleached pulp boosts kappa number reduction and bleaching with the laccase–mediator system. Based on the results of cyclic voltammetry tests, NHA has a slightly lower redox potential than HBT, which further supports use of the former; also, unlike HBT, NHA is oxidized in a reversible, pH-dependent manner. Interestingly, the laccase–NHA system provides more efficient bleaching of eucalyptus pulp at pH 5 than it does at pH 4.

Keywords: Biobleaching; Eucalypt; Hydroxybenzotriazole (HBT); Laccase; N-hydroxyacetanilide (NHA); TCF

Stabilization ofd-amino acid oxidase from Rhodosporidium toruloides by encapsulation in polyallylamine-mediated biomimetic silica by I-Ching Kuan; Jian-Cheng Wu; Shiow-Ling Lee; Chen-Wei Tsai; Chiao-An Chuang; Chi-Yang Yu (pp. 408-413).

d-Amino acid oxidase from Rhodosporidium toruloides (RtDAO) and Fe₃O₄ magnetic nanoparticles were encapsulated simultaneously within biomimetic silica, as mediated by polyallylamine. The capacity for this enzyme reached 193mg/g of biomimetic silica when 15mg/ml RtDAO was used during encapsulation; the average encapsulation efficiency was approximately 74%. The T_m value (the temperature at which 50% of the initial activity was retained after 1h of incubation) was increased from 44.3°C of the free RtDAO to 57.7°C, clearly indicating the thermal stability was improved by encapsulation. In the presence of 50mM hydrogen peroxide, encapsulated RtDAO had a half-life of 148min, an approximately 2-fold increase in resistance to hydrogen peroxide as compared to 78-min half-life of the free form. The encapsulation process is simple and can be completed within minutes; besides, the resultant enzymes can be recovered easily under magnetic field. Such preparation of encapsulatedd-amino acid oxidase could be exploited for many potential applications.

Keywords: d; -Amino acid oxidase; Biomimetic silica; Enzyme technology; Immobilized enzymes; Protein recovery; Kinetic parameters

Immobilization of PLP-dependent enzymes with cofactor retention and enhanced stability by Carmen López; Sergio D. Ríos; Josep López-Santín; Gloria Caminal; Gregorio Álvaro (pp. 414-421).

Immobilization of PLP-dependent enzymes requires specific studies due to their special cofactor-enzyme bond. These enzymes were immobilized using different methods, and the recombinant serine hydroxymethyltransferase (SHMT) was used as a case study. The immobilization of SHMT on glyoxal-agarose resulted in a high retention yield (70%); however, the reduction step caused an enzymatic activity loss of 80%. The immobilization on Eupergit^® C was optimized by considering different ionic strengths, pH and temperatures. SHMT reached 53% retention on this support. Although the enzymatic activity of the derivative decreased by 36% during the treatment with methylamine and washing, it was totally recovered by incubation with the cofactor. SHMT immobilized on Eupergit^® C gained thermal stability with respect to the soluble enzyme. Finally, 6-His-tagged SHMT was adsorbed very rapidly on IMAC supports and reached a 98% immobilization yield and enzymatic retention. The capacity of Eupergit^® C beads to immobilize PLP-dependent enzymes was corroborated by the immobilization of alanine racemase and aspartate aminotransferase. The final immobilization yields were 85 and 74% respectively, and the derivatives were two- and nine-fold more stable than the soluble enzymes.

Keywords: PLP-dependent enzymes; Immobilization; Serine hydroxymethyltransferase; Alanine racemase; Aspartate aminotransferase

Kinetic analysis on the production of polyhydroxyalkanoates from volatile fatty acids by Cupriavidus necator with a consideration of substrate inhibition, cell growth, maintenance, and product formation by Jin Wang; Zheng-Bo Yue; Guo-Ping Sheng; Han-Qing Yu (pp. 422-428).

Volatile fatty acids (VFAs) could be utilized by Cupriavidus necator (previously named as Ralstonia eutropha) as its sole carbon and energy source for growth and polyhydroxyalkanoates (PHAs) synthesis. In such a biosynthesis, VFAs at a high concentration had an inhibition on cell growth and deteriorate metabolite production. In this work the kinetics of PHAs production and consumption of VFAs by C. necator with simultaneous considerations of substrate inhibition, cell growth, maintenance and product formation were explored. The growth of C. necator and production of PHAs were significantly affected by the initial VFAs concentration. The cell growth was inhibited under initial substrate-sufficient conditions, and the cell activities could be resumed with the consumption of VFAs in batch cultures. Experimental results verify that the model established in this work was able to appropriately describe the PHA production from VFAs by C. necator.

Keywords: Activated sludge; Biopolymers; Cupriavidus necator; Kinetics; Polyhydroxyalkanoates; Waste treatment

Sulfide assessment in bioreactors with gas replacement by Peng Hu; Lee T. Jacobsen; James G. Horton; Randy S. Lewis (pp. 429-434).

Many biological processes have utilized the addition of sulfide constituents, such as sodium sulfide or cysteine-sulfide, to affect the redox potential, remove residual oxygen, and/or provide a source of sulfur for metabolism. However, the effects of sulfide constituents and associated sulfide concentrations on growth and product formation of cellular systems have shown considerable variance. In this work, models were developed that explained sulfide loss in bottle studies (batch reactors) and continuously gas-purged reactors. Since sulfide in liquid can be converted to volatile hydrogen sulfide (H₂S), mass transfer plays a key role for sulfide loss in continuous reactors, whereas equilibrium is critical for sulfide loss in batch reactors. Models of sulfide can be used to understand the fate of sulfide during an experiment and to design experiments to maintain constant sulfide levels for providing greater clarity when interpreting experimental results. Cellular experiments for ethanol/acetic acid formation from syngas were carried out to demonstrate the maintenance of constant sulfide levels of 0–1.9mM throughout the experiment. Results showed that cell growth was slightly affected by the sulfide concentration, ethanol production was favored at higher sulfide concentrations, and acetic acid production was favored at lower sulfide concentrations.

Keywords: Sulfide; Bioreactor; Modeling; Syngas; Fermentation; Ethanol

Kaizen for improvement of rapid protein production for early reagent protein quantities by Beth Junker (pp. 435-444).

A kaizen improvement effort was undertaken focusing on the workflows of an in-house workgroup whose aim was the rapid production of small amounts of protein reagents. The kaizen's goal was to reduce process lead times to accommodate more deliveries per year and more complex protein types. Business requirements were established, and baseline performance was evaluated against projected needs. Then potential areas for change were identified, selected, and implemented. Improvements were quantified based on established metrics as well as customer input. Overall in-house group capacity was raised 11% from 1.1 to 1.2 deliveries per person per week at the same time that the percentage of non-platform (more difficult) requests was increased to nearly 50% from under 10%. In-house group lead times from request to shipping for platform (less difficult) purification deliveries were improved by 30% from 11.1 to 7.7 days.

Keywords: Kaizen; Improvement; Reagent protein; Transient transfection

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Biochemical Engineering Journal (v.49, #3)