Biochemical Engineering Journal (v.68, #C)

BEJ Keywords (IV).

► The ribosome binding site originating from pET30a was used in bicistronic constructs. ► A pET30a-derived BioBrick base vector was constructed for dual-promoter constructs. ► The effect of gene location was investigated in the work. ► Co-expression constructs offered high level expression of two recombinant enzymes.The enzymes used for biomass decomposition comprise a major cost in the production of biofuels from lignocellulosic feedstocks. Engineering of key enzymes de novo in heterologous hosts provides one strategy for the rational improvement of enzyme cocktails. Until recently, Escherichia coli has remained the most commonly used host for recombinant protein expression. Nevertheless, to our knowledge, there are few reports describing the co-expression of biomass degrading enzymes in E. coli. In this study, bicistronic and dual-promoter constructs based on pET30a were built for the co-expression of an endo-β-xylanase gene (xyn) and a β-glucosidase gene (bgl) from Trichoderma reesei QM 9414 in E. coli. The internal ribosome binding site used in the bicistronic constructs was originally found in pET30a. In the dual-promoter constructs described here, a pET30a-derived BioBrick base vector was built for the standard assembly of two targeted genes. Compared with monocistronic constructs, the crude enzyme expressed from a bicistronic construct (xyn located upstream of bgl) and a dual-promoter construct (xyn located upstream of bgl) offered the comparable activity of two recombinant proteins. Our results indicated that the common commercial vectors, such as pET30a, could be modified and optimized for a particular co-expression strategy in E. coli.
Keywords: Co-expression; BioBrick vector; Cellulase; Enzyme production; Biodegradation; Enzyme activity;

Continuous production of biodiesel using whole-cell biocatalysts: Sequential conversion of an aqueous oil emulsion into anhydrous product by Ayumi Yoshida; Shinji Hama; Naoki Tamadani; Hideo Noda; Hideki Fukuda; Akihiko Kondo (7-11).
► A continuous production of biodiesel using recombinant fungal whole-cells. ► Packed-bed reactors were applied to methanolysis of an aqueous plant oil emulsion. ► Significant difference in dependence of methanolysis activity on water content. ► An aqueous oil emulsion was sequentially converted into the anhydrous product.A continuous production of biodiesel from an aqueous plant oil emulsion was attempted using immobilized fungal whole cells. Six packed-bed reactors were connected in series and operated with stepwise methanol addition. In the first column, more than 3% water was necessary for methanolysis to proceed. Despite the low initial water content of 0.36%, the methyl ester content in the effluent from the second column increased similarly in a wide range of contents of water (0–20%) added, which shows the water-content-independent reaction in this column in contrast to the reaction in the first column. Further investigations using reaction mixture models suggested a contribution of the composition of the reaction mixture to the phenomenon. On the basis of these findings, the sequential methanolysis through six columns was attempted, where the upper layer of the effluent from each column was supplied into the next column without further addition of water. Consequently, an aqueous plant oil emulsion with 3% water was converted into the final product with 96.1% methyl ester and 0.15% water. Therefore, the system developed is useful for producing biodiesel enzymatically from water-containing feedstocks.
Keywords: Biodiesel feedstocks; Immobilized cells; Lipase; Methanolysis; Packed-bed reactor;

Cell disruption enhanced the pure EGFP recovery from an EGFP-intein-surface protein production system in recombinant E. coli by Jiun-Yan Wu; Chih-I Chen; Chi-Ming Chen; Chia-Chi Lin; Shu-Chen Kan; Chwen-Jen Shieh; Yung-Chuan Liu (12-18).
► Approach to obtain highly pure EGFP by INP-INT system was developed. ► Osmotic shock method gave the highest EGFP yield. ► Cell disruption method gave the highest EGFP purity by only centrifugations. ► An EGFP yield of 63 mg/L with 97% purity was obtained via cell disruption method. ► This approach is useful for pure recombinant protein production in large scale.In this study, three approaches for protein production were devised to enhance the efficiencies in yielding high purity protein via surface display system. A plasmid carrying enhanced green fluorescent protein (EGFP), an intein (INT) and ice nucleation protein (INP) was constructed to produce EGFP via surface display in Escherichia coli. To obtain high purity of the produced EGFP, several procedures, including osmotic shock, surfactant addition and cell disruption were employed. Among these approaches, the cell disruption method gave the highest EGFP purity by simply conducting several centrifugations. An EGFP yield of 63 mg/L with 97% purity was obtained. The result demonstrated that pure EGFP can be harvested only through centrifugation; no complicated processes or expensive equipment are required. This approach shows potential for the production of pure recombinant proteins in scale-up processes for biotechnological, academic and industrial uses.
Keywords: Protein; Purification; Surface display; Intein; Centrifugation; Recombinant DNA;

► A new process for isolating lignin of pre-hydrolysis liquor was proposed. ► Pre-hydrolysis liquor was treated with surfactant and acid/lime under different sequences/conditions. ► The maximum lignin removal was obtained via overliming and surfactant treatments. ► The isolated lignocelluloses were evaluated by FTIR, TGA, and particle size analyser.Hemicelluloses present in the pre-hydrolysis liquor (PHL) of the kraft-based dissolving pulp production process can potentially be converted into value-added products such as ethanol and xylitol. However, the lignin present in the PHL is an inhibitor of fermentation processes, and thus should be isolated from the PHL prior to fermentation. In this study, a new process was proposed for isolating the lignin of PHL by overliming and surfactant treatment. The fundamentals associated with the interactions of lignin, surfactants and calcium hydroxide were discussed. In the experimental part of this work, three different cationic surfactants were applied to industrially produced PHL, and the most effective one, dodecyltrimethyl ammonium chloride (DTAC), was selected for further analysis. The optimum dosage of 0.3% DTAC/PHL was identified via turbidity and particle size analyses. Additionally, overliming via CaO treatment resulted in removing 36.1% lignin and 23% hemicelluloses. Overliming followed by the DTAC treatment was more effective than the DTAC treatment followed by overliming in removing lignin. In the former, the maximum removals of 43.6% lignin and 27% hemicelluloses were obtained. The formation of lignocelluloses/surfactant complexes was confirmed by means of a Fourier transform infrared spectroscopy (FTIR) and thermo gravimetric analyzer (TGA).
Keywords: Bioseparation; Surfactant; Fuel; Fermentation; Biochemical processes; Lignin;

Decoupling of oxygen transfer and power dissipation for the study of the production of pristinamycins by Streptomyces pristinaespiralis in shaking flasks by N. Mehmood; E. Olmos; J.-L. Goergen; F. Blanchard; P. Marchal; W. Klöckner; J. Büchs; S. Delaunay (25-33).
► Decoupling of k L a and power dissipation in shaking flasks. ► Respective influence of OTR and power dissipation on antibiotic production. ► The release of pristinamycins is related to the oxygen transfer. ► The final concentration of pristinamycins is impacted by the power dissipation. Streptomyces pristinaespiralis is a filamentous bacterium used in the pharmaceutical industry for the production of pristinamycins. In previous works, it was shown that the occurrence of production and the antibiotics concentration could be related to gas–liquid transfer and power dissipation in shaking flasks. Nevertheless, in standard cultures, both mechanisms are coupled. It is then a difficult task to assign a precise physiological response to either oxygen transfer or power dissipation. The aim of the present study was to decouple the oxygen transfer coefficient (k L a) and the power dissipation per unit of volume (P/V) to study their respective impact on pristinamycin production. During cultures in flasks, the rotation diameter of the shaking table was changed to modify the k L a but not the power dissipation P/V. The influence of operating conditions with P/V ranging from 0.55 to 10.3 kW m−3 and k L a ranging from 30 to 490 h−1 have been determined on the microbial kinetics and also on the pellet diameters. The final biomass concentration and the release of antibiotics were then related to k L a, whereas the final pristinamycins concentrations, as well as the bacterial pellet diameter were mainly correlated to P/V independently of the k L a. The change in the pellet diameter could be the crucial parameter for the pristinamycins production as it might influence the nutriment transfer inside the pellets and the ratio of active cells in each pellet.
Keywords: Streptomyces; Pristinamycins; Power dissipation; Gas–liquid mass transfer coefficient; Shaking flask;

► Application of mathematical modelling for optimization of 1,3-PD production has been attempted. ► Batch mathematical model for 1,3-PD production has been proposed. ► Model parameters identified by non-linear regression technique using original batch experimental data. ► Proposed model extrapolated to fed-batch for enhanced 1,3-PD production. ► Highest 1,3-PD concentration ever reported in literature achieved using model-based fed-batch cultivation.1,3-Propanediol (1,3-PD) is an organic compound of immense importance particularly for polycondensation reactions to synthesise polyesters, polyethers and polyurethanes. Batch cultivation of obligate anaerobe Clostridum diolis DSM15410 for 1,3-PD production using statistically optimised medium was attempted in the present investigation. A mathematical model for the description of batch 1,3-PD production kinetics was then proposed which also incorporated substrate/product inhibition data. The kinetic parameters of the model were identified by non-linear regression technique using batch experimental data. The developed model adequately described the experimental data to the extent of 99% accuracy as tested by statistical validity test. The batch model was then extrapolated to fed-batch cultivation primarily to identify fresh nutrient feeding strategies (off-line on the computer) for enhanced production of 1,3-PD. Experimental implementation of one such fed-batch fermentation strategy involving constant feed rate and subsequent comparison of the observed kinetics with model simulation further established the accuracy of the developed model. A 1,3-PD concentration of 61.2 g/L with a productivity of 1.02 g/L/h of 1,3-PD was obtained in this mathematical-model guided fed-batch fermentation which is the highest 1,3-PD concentration ever reported in literature using this strain.
Keywords: 1,3-Propanediol; Clostridium diolis; Modelling; Glycerol; Fed-batch culture; Kinetic parameters;

► The solutions for rate of phenol degradation which yet to be reported. ► Established the efficiency of the HAM for handling non-linear structures. ► Our results are in good agreement with the limiting cases. ► A validation of the procedure shows good results in fluidized bed bioreactor.A theoretical model for fluidized bioreactor (FBR) is presented. The model is based on system of reaction-diffusion equation containing a non-linear term related to specific growth rate of biomass. Homotopy analysis method (HAM) is successfully employed in the development of non-linear steady state phenol degradation. In this paper simple and approximate polynomial expression of concentration of phenol and oxygen and rate of phenol degradation are derived for all possible values of parameters ϕ O (dimensionless modulus for oxygen), ϕ S (dimensionless modulus for phenol), K i * (dimensionless inhibition constant for phenol) and K O * , K S * (dimensionless monod constant for oxygen and phenol). The limiting case results are compared with our common results and are found to be in good agreement.
Keywords: Biodegradation; Homotopy analysis method; Bioreactors; Diffusion; Mathematical modelling; Fluidization;

► We coupled ClO2 and ultrasonic for sludge disruption and studied the optimal parameters of the ClO2-ultrasonic disruption. ► We revealed that lysis-cryptic growth using ClO2-ultrasonic disintegration result in considerable sludge reduction. ► We studied the effect of lysis-cryptic growth on effluent and sludge activity. ► We did economical evaluation for lysis-cryptic growth using ClO2-ultrasonic disruption.A lysis-cryptic growth system, which combined chlorine dioxide and ultrasonication for sludge disruption (ClO2-ultrasonication disruption), was proposed to reduce the excess sludge in an activated sludge process. The optimal parameters of the ClO2-ultrasonication disruption were determined by sequencing batch jar experiment and orthogonal jar experiments. Subsequently, a ClO2-ultrasonication disruption system was combined with a 1.8 m3/d pilot SBR for lysis-cryptic growth. The results indicated that most of the disintegrated sludge was inactivated and turned into biodegradable material at the optimal parameters of a ClO2 dosage of 4 mg/g dry sludge, a ClO2 treatment time of 60 min, an ultrasonic intensity of 0.5 W/mL and an ultrasonication time of 6 min. The enzymatic activity of the disrupted sludge decreased by 96.5% and the value of BOD/COD reached 0.4. The operating data from the pilot SBRs showed that the discharged excess sludge was reduced by 55% by returning 70% of the excess sludge pretreated by the ClO2-ultrasonication disruption. Although lysis-cryptic growth resulted in an increase of 0.11 mg/L TP and 1.1 mg/L TN in the effluent, the impacts on the COD, ammonium and SS present in the effluent, as well as the sludge activity, were minimal.
Keywords: ClO2-ultrasonication; Sludge reduction; Cell disruption; Waste treatment; Process integration; Aerobic process;

► Production and characterization of chitosan–TPP nanoparticles. ► Optimization of chitosan nanoparticle characteristics by response surface modeling. ► Controlled release of rHu-Erythropoietin from chitosan nanoparticles.In this work we report the production and characterization of nanoparticles using the ionotropic gelation method between the biopolymer chitosan (CS) and tripolyphosphate (TPP) ion with simultaneous encapsulation of the glycoprotein Recombinant human erythropoietin (rHu-EPO). Chitosan concentration, chitosan to TPP mass ratio, and solution pH were optimized using response surface modeling to produce nanoparticles containing rHu-EPO. Nanoparticles were produced with diameters ranging from 100 nm to 400 nm, particle polydispersity from 0.2 to 0.4 and nanoparticle surface charge (zeta potential) ranging between 30 and 50 mV. Controlled release of rHu-EPO from CS-TPP nanoparticles was studied and showed 30% release of the original encapsulated rHu-EPO in 24 h and 68% release in two weeks. These rHu-EPO release results can be used as a basis for more research on the application of nanoparticles within animal models.
Keywords: Chitosan; Controlled release; Encapsulation; Erythropoietin; Ionotropic gelation; Nanoparticles; Response surface modeling;

A critical evaluation of CO2 supplementation to algal systems by direct injection by N.M. Langley; S.T.L. Harrison; R.P. van Hille (70-75).
Chlorella vulgaris was cultivated in 3.2 l internal loop airlift photobioreactors. ► Overall gas mass transfer coefficient calculated at 0.0094 s−1. ► P CO 2 in excess of 0.0012 atm (1200 ppm) did not improve productivity further. ► CO2 sequestration efficiency at 0.0012 atm was 26%. ► Direct injection may not be effective for CO2 sequestration from flue gas.Microalgae are currently cultivated on a relatively small scale for nutritional products, supplements and aquaculture feed. In recent years there has been renewed interest in algal bioenergy, which would require cultivation at far greater scales. A key component of large-scale production systems is the delivery of CO2 to the algal cells, which is often a limiting factor in ponds and air sparged systems. Although many methods of CO2 supplementation to algal reactors have been investigated, the most commonly suggested method is still the direct injection of CO2 enriched gas into the growth medium. A sound understanding of CO2 gas–liquid mass transfer is critical to efficient operation of cultivation systems as mixing and gas compression may represent significant operational expenses. For carbon capture or sequestration through algal culture, CO2 recovery is equally important, particularly where carbon trading is involved. Chlorella vulgaris was grown in internal loop airlift reactors under varied CO2 partial pressures in the inlet gas. In these reactors, with an overall mass transfer coefficient of 0.0094 s−1, an inlet CO2 partial pressure of 0.0012 atm (1200 ppm CO2 by volume) was sufficient to overcome any mass transfer limitations. Under these operating conditions, a CO2 recovery of 26% resulted. Increasing the partial pressure of CO2 in the inlet gas above 0.0012 atm did not increase the algal productivity and caused significant decreases in CO2 recovery to 9.7% and 2.1% at inlet CO2 partial pressures of 0.00325 atm and 0.0145 atm respectively. Much research into algal growth is done without analysis of CO2 gas–liquid mass transfer, using influent CO2 partial pressures well in excess of the minimum value required to overcome CO2 gas–liquid mass transfer limitations, even in poorly mixed systems. This could result in algal growth being optimised under conditions that are not industrially practical or desirable.
Keywords: Microalgae; Photobioreactors; Gas–liquid mass transfer; Carbon sequestration; Algal productivity; Chlorella vulgaris;

► Amperometric biosensor for detection of phenolic compounds based on Lac/NiNPs/cMWCNT/PANI/Au. ► Linear range and detection limit were 0.1–00 μM and 0.05 μM respectively. ► Biosensor measured total phenolic content in fruit juices. ► Enzyme electrode was used 100 times over a period of four months, when stored at 4 °C.A method is described for construction of an enzyme electrode for detection of phenolic compounds based on covalent immobilization of laccase onto nickel nanoparticles (NiNPs) decorated carboxylated multiwalled carbon nanotubes (cMWCNTs)/polyaniline (PANI) composite electrodeposited onto gold (Au) electrode. The modified electrode was characterized at different stages of its construction by scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, cyclic voltammograms and electrochemical impedance spectroscopy (EIS). An amperometric biosensor for phenolic compounds was fabricated by connecting enzyme electrode (Lac/NiNPs/cMWCNTs/PANI/AuE) as working electrode, with Ag/AgCl as reference electrode and Pt wire as auxiliary electrode through potentiostat. The biosensor showed optimum response at pH 5.5 (0.1 M acetate buffer) and 35 °C, when operated at a scan rate of 20 mV s−1. Linear range, response time, detection limit and sensitivity of biosensor were 0.1–10 μM (lower concentration range) and 10–500 μM (higher concentration range), 8 s, 0.05 μM and 0.694 μA μM−1  cm−2 respectively. The biosensor measured total phenolic content in fruit juices. The enzyme electrode was used 200 times over a period of four months, when stored at 4 °C.
Keywords: Biosensors; Enzymes; Immobilisation; Optimisation; NiNPs; cMWCNT;

► The mass transfer mechanism of dyes biosorption by Spirulina platensis nanoparticles was studied. ► The external mass transfer model and the homogeneous solid diffusion model were applied. ► The external mass transfer coefficients ranged from 1.67 × 10−6 to 11.40 × 10−6  cm s−1. ► The intraparticle diffusion coefficients ranged from 0.70 × 10−14 to 4.30 × 10−14  cm2  s−1. ► Biosorption was controlled by the external mass transfer and intraparticle diffusion mechanisms.In this research, the mass transfer kinetics for the biosorption of synthetic dyes (acid blue 9 and FD&C red no. 40) by Spirulina platensis nanoparticles was analyzed under different experimental conditions. The external mass transfer model (EMTM) and the homogeneous solid diffusion model (HSDM) were employed to study the mass transfer kinetics and also to estimate the values of external mass transfer coefficient (k f ) and intraparticle diffusion coefficient (D int). The Biot number (Bi) was used to verify the importance of external mass transfer in relation to intraparticle diffusion. The values of external mass transfer coefficient (k f ) ranged from 1.67 × 10−6 to 11.40 × 10−6  cm s−1 and the intraparticle diffusion coefficient (D int) ranged from 0.70 × 10−14 to 4.30 × 10−14  cm2  s−1. The Biot numbers (0.53 ≤ Bi ≤ 10.33) showed that, for both dyes, the biosorption onto S. platensis nanoparticles was controlled simultaneously by external mass transfer and intraparticle diffusion.
Keywords: Biot number; Biosorption; Diffusion; Dyes; Mass transfer; Microalgae;

► A comparison among different start-up strategies is presented. ► Both experimental and model results were employed. ► An innovative calibration protocol was employed for the calibration of the integrated MBR model. ► The fouling rate was lower in the case of the start-up with inoculum. ► The start-up strategy defines the features and the physiology of biomass.The performance of a membrane bioreactor (MBR), and mechanisms of fouling formation, may differ due to the start-up. Therefore, the start-up can constitute an aspect that critically influences MBR performance during its lifespan. Indeed, the start-up can influence the mechanisms of membrane fouling, which is of paramount importance in an MBR. In order to gain insights on the effects of the start-up, both experimental and mathematical modelling studies were carried out on an MBR pilot plant. The MBR pilot plant constituted of a hollow fibre membrane module, in a submerged configuration, was fed by real wastewater. Two experimental periods were carried out, lasting 65 days each, characterised by two different MBR start-ups: without inoculum of activated sludge, and with inoculum of activated sludge. An integrated MBR mathematical model was applied to the two periods in order to combine the insights gained from the experimental survey. The MBR model simulates both biological and physical processes simultaneously, taking into account the interactions between them. Two MBR model calibrations were carried out for the two different experimental start-ups. Model calibration was carried out by means of an innovative calibration protocol, based on the global sensitivity analysis, and the Generalised Likelihood Uncertainty Estimation. Thus, two sets of model parameters were assessed and compared. Both experimental and mathematical model results were analysed to quantify the effects of the two start-ups on membrane fouling. The results showed only slight differences in terms of total pollutant removal efficiency. However, relevant differences appeared with respect to the fouling phenomenon between the two start-ups. The results demonstrated that the best choice, in terms of the start-up of MBR systems, comes from a compromise between minimisation of the fouling issues and high system performance.
Keywords: Experimental pilot plant; ASM1-SMP; Calibration protocol; Fouling; MBR modelling; Start-up;

Single-chamber bioelectrochemical hydrogen fermentation from garbage slurry by Kengo Sasaki; Masahiko Morita; Daisuke Sasaki; Norio Matsumoto; Naoya Ohmura; Yasuo Igarashi (104-108).
► Single-chamber bioelectrochemical system was operated because design is simple. ► Hydrogen fermentation from artificial garbage slurry was easily constructed. ► Hydrogen production rate of 2196 mL L−1  day−1 was attained. ► Acetate and butyrate were the main products in volatile fatty acids. ► High potential on the anodic electrode and low pH was important.Bioelectrochemical systems (BESs) can control electric flow and resultant microbial activity. We designed a single chamber BES to investigate hydrogen production from artificial garbage slurry. A 3-electrode system was applied and the potential of the working electrode was regulated to −1.0 V (vs. Ag/AgCl). Thus, the potential on the counter electrode was 1.38 V (vs. Ag/AgCl). Hydrogen fermentation in the BES was initiated by methanogenic seed sludge. The BES achieved a hydrogen production rate of 2196 mL L−1  day−1 at an organic loading rate of 58.7 g dichromate chemical oxygen demand L−1  day−1. Acetate and butyrate were the main products, indicating that favorable hydrogen fermentation occurred in the system. Combination of high potential on the counter electrode and relatively low pH condition (5.5–6.4) was effective for constructing hydrogen fermentation even in single chamber system by inhibiting methanogenesis. However, it may be necessary to increase the space between the working and counter electrodes so as to decrease the electrical input. The single-chamber BES could be scaled-up for efficient hydrogen fermentation from garbage slurry.
Keywords: Bioprocess design; Bioelectrochemical system; Anaerobic processes; Hydrogen; Fermentation; Waste treatment;

Microalgae (Nannochloropsis salina) biomass to lactic acid and lipid by Md. Mahabubur Rahman Talukder; Probir Das; Jin Chuan Wu (109-113).
► A method for lipid extraction and lactic acid production from microalgae biomass. ► The method gave 1.75-fold higher lipid yield. ► The microalgae biomass hydrolysate, a nutrients source for L. pentosus. ► Lactic acid yield reached 92.8%.A method for lactic acid production and lipid extraction from microalgae (Nannochloropsis salina) biomass was investigated. Microalgae biomass was acid (5% H2SO4) hydrolyzed at 120 °C for 1 h, and subsequently treated with hexane at 40 °C and 200 rpm to separate lipid from the hydrolysate. The sugar (glucose plus xylose) yield reached 64.3% and remained the same until the biomass loading of 15% (w/v) after which, it dropped. Lipid free hydrolysate was neutralized and used as fermentation medium for Lactobacillus pentosus for lactic acid production. Lactic acid yield reached 92.8% at sugar 3–25 g/l, 30 °C and shaking speed 150 rpm under anaerobic condition. The acid hydrolysis facilitated lipid extraction from microalgae biomass, and lipid yields with and without acid hydrolysis were 85.6% and 48.7%, respectively. Results suggest that the developed method can be successfully applied in lipid extraction and lactic acid production from microalgae biomass.
Keywords: Lactic acid; Microalgae; Lipid; Ion-exchange; Lactobacillus pentosus;

► Akirin was produced in Pichia pastoris using an aqueous two-phase semicontinuous process. ► Effective recycling and recovery of PEG4000 was obtained. ► Higher protein production when compared to the process with fresh organic phase. ► Reduction of the operation time and production costs by 25% and 27%, respectively.The yeast, Pichia pastoris, has been successfully used as an efficient system to produce heterologous proteins. The secretion of the recombinant Aedes albopictus mosquito akirin (AKR) from P. pastoris using an extractive bioconversion in an aqueous two-phase system (ATPS) fed-batch fermentation process allowed the conduction of vaccination trials to assay its protective efficacy against different arthropod vector species. Herein, we report the development of a semicontinuous process for the extractive bioconversion in an ATPS coupled with a simple and easy to scale up process for polymer recycling as an effective alternative to improve production of recombinant A. albopictus AKR in P. pastoris. Six repetitive batches of the extractive bioconversion were conducted in a semicontinuous process by replacing 2/3 of the culture and recycling the top polymeric phase every 72 h. Overall process in a 5-L fermentor yielded a protein concentration in the bottom phase of 0.46 ± 0.13 mg mL−1 and 6 g of total purified protein in 490 h with a reduction of 25% of the operation time and 27% of the operation cost when compared to the fed-batch ATPS process. These results improve the production process for mosquito AKR and suggest the possibilities to develop similar processes for the expression of other recombinant proteins in P. pastoris.
Keywords: Pichia pastoris; Akirin; Subolesin; Fermentation; Biotechnology; Vaccine;

Reactive diafiltration for assembly and formulation of virus-like particles by Mervyn W.O. Liew; Yap P. Chuan; Anton P.J. Middelberg (120-128).
► Rate of assembly buffer exchange dramatically affects VLP yield and quality. ► VLP assembly diafiltration reactor designed with optimized buffer exchange rate. ► Integration of assembly and formulation in a single diafiltration unit demonstrated. ► Diafiltration assembly and formulation gave high final VLP yield of 53%. ► Intensified VLP process via reduced process units and buffer consumption.An emerging paradigm to quickly and cost-effectively manufacture virus-like particle (VLP) biopharmaceuticals involves VLP subunit production in highly productive bacterial hosts, followed by assembly in a controlled cell-free environment. Study of cell-free VLP assembly has yet to progress beyond laboratory-based dialysis methods, which are slow and buffer intensive, and are thus difficult to scale. In this study, a scalable cell-free VLP assembly and formulation process using a single diafiltration unit was developed. A reactive constant volume diafiltration VLP assembly process was systematically and quantitatively designed to maximize VLP yield. Using a dilute concentration of capsomeres during VLP assembly minimized membrane-induced aggregation. Optimizing the assembly buffer exchange rate, and the rapid introduction of calcium ions to the assembly mix, facilitated the propitious assembly of VLPs. An absolute increase in VLP yield of 42–56%, compared to initial laboratory-scale batch diafiltration assembly processes, was achieved. The optimized process produced high-quality VLPs (PDI = 1.08 ± 0.01) at high yield (78%). The constant volume diafiltration method has the added advantage of being scalable and represents a high level of process intensification. The methodology reported in this study, based on quantitative analysis of the influences of process changes, is consistent with modern engineering approaches, and can ultimately lead to cheaper and quicker VLP manufacturing routes and hence the accelerated translation of products from laboratory settings.
Keywords: Virus-like particles; Self-assembly; Bioprocess design; Downstream processing; Process integration; Scale-up;

► Co-digestion of OFMSW wastes with slaughterhouse wastes was possible. ► High loadings produced some inhibition attributable to the co-substrates. ► The PTE content of digestates was unsuitable for agricultural food production. ► Co-substrates improved the nutrient balance of the final digestate product.The current work aimed to resolve some long-standing questions about the potential benefits and limitations of co-digestion of slaughterhouse wastes. To achieve this, a laboratory-scale trial was carried out using the mechanically recovered organic fraction of municipal solid waste mixed with either sheep blood or a mixture of pig intestines with flotation fat. Both of these co-substrates are difficult to digest in isolation because of their high nitrogen and lipid concentrations, and are regulated as Category 3 materials under the Animal By-Products Regulations (EC 1069/2009). The results showed that at an organic loading rate of 2 kg VS m−3  day−1 with the slaughterhouse material making up 20% of the load on a volatile solids basis the process could operate successfully. As the loading was increased to 4 kg VS m−3  day−1 signs of inhibition appeared with both co-substrates, however, and volumetric methane production was reduced to a point where co-digestion gave no process advantage. The main operational problem encountered was an increase in the concentration of volatile fatty acids in the digestate, particularly propionic acid: this was thought to be a result of ammonia toxicity. The concentration of potentially toxic elements in the digestate made it unsuitable for agricultural application for food production, although the increased nitrogen content made it more valuable as a fertiliser for non-food crop use.
Keywords: Anaerobic processes; Biodegradation; Waste treatment; Slaughterhouse waste; Product inhibition; Potentially toxic element;

► Nutrient ratio (NR) regulates microbial phosphofructokinase activity (PFK). ► Relevance of NR was found to be independent of the type of nutrients tested. ► NR–PFK–oxygen uptake link was studied for Aspergillus niger, Streptomyces species, Pichia pastoris, Escherichia coli. ► Optimization of NR significantly improved microbial specific productivity. ► PFK was modeled as a function of nutrient ratio, ATP and an inhibitor citrate.A comprehensive optimization of fermentation processes is non-trivial due to the interdependence of individually well-studied fermentation parameters. Two such important parameters are the oxygen uptake rate (OUR) and the ratio of two critical nutrients (CNR) in the broth. Since CNR is easily measurable, it was investigated for its efficacy in process optimization. Phosphofructokinase activity (PFK) was used as a supplementary marker of productivity. The strong correlations between the above parameters motivated optimization of fermentations using four industrially useful microbes: Escherichia coli, Pichia pastoris, Streptomyces species and Aspergillus niger. The relevance of CNR was independent of dissolved oxygen and nutrient sources, even though the nutrient release and its consequent availability depended upon the nutrient type and source. Planned changes in CNR lead to a 3 fold and a 3–5 fold increase in PFK and specific productivity respectively. The ATP and citrate levels beyond 2.5 mmol (g cell)−1 inhibited PFK. A mathematical model was developed to explain the relationship between PFK and its effectors: CNR, ATP, and citrate. By optimizing CNR, the respective maximum productivities were increased by a minimum of 34%, 100%, 57% and 30% compared to the respective controls for above processes.
Keywords: Critical nutrient ratio; Phosphofructokinase; Metabolite overproduction; Fermentation; Dissolved oxygen; Bioprocess design;

► Wet disk milling and hydrogen peroxide treatments of bagasse were investigated. ► HP followed by WDM (WDM–HP) treatment produced high bagasse delignification. ► WDM–HP treatment significantly increased glucose concentration in saccharification. ► WDM–HP-treated bagasse is a good substitute for cellulose in cellulase production.A pretreatment method using wet disk milling (WDM) and hydrogen peroxide (HP) was investigated to produce a saccharified solution with a high glucose concentration from sugarcane bagasse. The chemical composition of the bagasse was uninfluenced by WDM treatment alone, whereas a slight reduction in the contents of lignin and xylan was effected by HP treatment alone performed at neutral pH. The combination of WDM treatment and HP treatment (WDM–HP treatment) significantly increased the removal of lignin and xylan. A cellulose-rich material containing 87% glucan was obtained by performing the HP treatment at 70 °C using 2.5% biomass, after WDM treatment. By using 15% WDM, HP- and WDM–HP-treated bagasse, glucose concentrations of 28 g/L, 27 g/L, and 98 g/L, respectively, were obtained from the various saccharification processes, suggesting the feasibility of the WDM–HP treatment for the production of a saccharified solution with high glucose concentration. Moreover, WDM–HP-treated bagasse was shown to be a good substitute for cellulose in cellulase production by Acremonium cellulolyticus. Under the same conditions, the treated bagasse provided a cellulase activity of 8.2 FPU/mL compared with the value of 7.9 FPU/mL provided by the commercial cellulose, SF 40.
Keywords: Cellulose; Lignin; Enzyme production; Cellulase; Hydrogen peroxide; Wet disk milling;

Reversible immobilization of glucoamylase onto metal–ligand functionalized magnetic FeSBA-15 by Guanghui Zhao; Jianzhi Wang; Yanfeng Li; Huayu Huang; Xia Chen (159-166).
► Magnetic FeSBA-15 mesoporous silica was chosen as the base of support. ► Polymer chains (PGMA) were grafted on the outer surfaces of magnetic FeSBA-15. ► PGMA was functionalized with imidazole or iminodiacetic acid. ► Functionalizable and metal–chelate surfaces for reversible enzyme immobilization. ► The regenerated biocatalyst still reserved its original performance.Magnetic SBA-15 (FeSBA-15) was prepared via wet impregnation, calcination and reduction, and p(glycidylmethacrylate) (PGMA) was grafted on the surface of FeSBA-15 using surface-initiated atom transfer radical polymerization (SI-ATRP) for a prescribed time. The epoxy groups of the PGMA were reacted with Cu(II) metal–ligand complex (i.e., imidazole or iminodiacetic acid) to form metal–chelate brush. Subsequently, the functionalized FeSBA-15 as a regenerated support was used for enzyme immobilization. Glucoamylase was immobilized as a model enzyme on the regenerated supports through metal-ion affinity interactions. The quality of glucoamylase immobilized on the regenerated supports is defined by determining of the enzyme activity, thermal stability, and reusability. The results indicate that the metal–chelate brushes offer an efficient route to immobilize enzymes via metal-ion affinity interactions. The applicability of the regenerated supports in the current study is relevant for the conjugation of other enzymes beyond glucoamylase.
Keywords: Biocatalysis; Affinity; Immobilization; Glucoamylase; ATRP; Regeneration;

Empirical modeling of the effects of emerging pretreatment methods on anaerobic digestion of pulp mill biosolids by Seyedeh Neda Mehdizadeh; Cigdem Eskicioglu; Abbas S. Milani; Mithun Saha (167-177).
► Microwave and ultrasound improve anaerobic digestion of pulp mill waste sludge. ► Pretreatment intensity, digester temperature, sludge type statistically significant. ► Microwave at 125 °C and mesophilic digestion achieve optimum disposal scenario.Effects of sludge type, microwave temperature, sonication time, and anaerobic digester temperature were investigated on solubilization and methane production from pulp mill secondary and primary sludges by multi-level factorial designs. Analysis of variance showed that all main effects (microwave temperature, sludge type and digestion temperature) and two-way interactions have significant (P-value < 0.05) effects on cumulative methane production relative to control (not pretreated) digesters (CMPr) at elevated microwave temperatures (150–175 °C). On the other hand, at lower microwave temperatures (50, 75, and 100 °C), pretreatments did not significantly improve the CMPr. During the sonication, sonication time and digestion temperature significantly affected the CMPr with a P-value < 0.05. The variance analysis also determined that, for both pretreatment methods, the most important factor affecting the sludge solubilization, indicated by the relative to control soluble to total chemical oxygen demand ratio (SCOD/TCODr), was the sludge type. To select a pretreatment/digestion that maximizes both the rate as well as the extent of anaerobic biodegradation, the technique for order preference by similarity to ideal solution (TOPSIS) was applied and it was concluded that mesophilic anaerobic digestion of pulp mill secondary sludge irradiated at 125 °C can result in an optimal pretreatment scenario with SCOD/TCODr and CMPr of 6.1, and 1.7, respectively.
Keywords: Anaerobic processes; Batch processing; Biogas; Modeling; Sludge pretreatment;

Improved operating policy utilizing aerobic operation for fermentation process to produce bio-ethanol by Der-Ming Chang; Tzu-Hsing Wang; I-Lung Chien; Wen-Song Hwang (178-189).
► We study the optimal fermentation operation to produce bio-ethanol. ► A realistic model fitted from experimental data is used to obtain the optimal operation. ► Combined aerobic and anaerobic fed-batch operation demonstrated to improve alcohol production. ► Aeration policy should include a initial aerobic operation and then followed by the anaerobic operation ► The desired glucose feeding policy for this optimal fed-batch operation is also obtained.To enhance the productivity of the alcohol fermentation process, combined aerobic and anaerobic fed-batch operation could improve the growth of microbes and also achieve better alcohol production because of well-utilizing the characteristics of the microbial metabolism. Using a realistic model fitted from experimental data, result shows that the highest productivity can be achieved using a proposed fed-batch operation with high glucose concentration. The proposed operating policy combines an initial aerobic operation to increase the biomass formation in the early phase of the fed-batch operation and then followed by the anaerobic operation to accelerate the alcohol production in the later phase. The glucose feeding policy for this optimal fed-batch operation should try to maintain a lower optimal glucose concentration in the fermenter during initial aerobic phase and then to maintain another higher optimal glucose concentration during the next anaerobic phase. Toward the ending of the fed-batch, the glucose concentration in the fermenter should be increased further in order to speed up the productivity of ethanol after the feeding stopped.
Keywords: Ethanol; Fermentation; Aerobic processes; Optimization; Operating policy; Cybernetic model;

► Organic matter from winery wastewater was removed by aerobic granulation in a SBR. ► Frequent COD fluctuations of winery effluents were properly treated. ► A SBR control system based on dissolved oxygen and ORP slope was designed. ► The control of feast/famine ratio lead to spherical, dense and fast settling granules. ► Important energy savings were achieved by implementing the control system.Winery wastewater is characterized by a high variability of its organic content, and flexible systems such as granular sequencing batch reactors (GSBRs) are required for its treatment. Despite the high load capacity inherent to these systems, the operation with a predetermined cycle length is not suitable to withstand high COD fluctuations. In this study, a control system able to automatically control the HRT depending on the actual organic load has been designed. The relation between ORP slope and DO profiles with the COD consumption together with a proper periodic starvation time are the basis of the control strategy. GSBR operation using the designed system led to an organic matter removal up to 95% for a wide range of organic matter concentrations (1–8 g/L), optimizing cycle duration and avoiding unnecessary aeration costs. The reactor showed a high stability and a rapid response to sudden changes in the COD influent concentration. Biomass settleability remained almost constant around 95 m/h and aggregates showed a Feret diameter of 4–6 mm and sphericity up to 0.8.
Keywords: Aerobic processes; Bioreactors; Control; ORP slope; Starvation time; Wastewater treatment;

Viability study of biofilm-former strains from paper industry by flow cytometry with application to kinetic models by E. Torres; M. Ladero; P. López; A. Alcon; V. Santos; A. Blanco (199-206).
► Biofilm forming microorganisms were studied by means of flow cytometry. ► Cellular states behavior was applied to build a kinetic growth model. ► Different equations for characterization of growth models were studied. ► Kinetic relationships among cell subpopulations are key parameters to fit models.Biofouling causes important economic losses in the paper industry. To prevent or mitigate the effects of biofilms it is necessary to implement microbiological control programs that require the evaluation of biofilm growth kinetics taking into account the viability of microorganisms. The monitoring techniques needed for this purpose must be rapid to minimize both costs and the use of chemicals. This paper presents a study on the growth kinetics of two bacterial strains known for their capacity to produce biofilms in paper mills. After the validation of flow cytometry as a technique for microbial growth monitoring, two segregated kinetic models integrating cell physiological state data were implemented. The models developed can be used to devise microbial control programs.
Keywords: Flow cytometry; Biofilms; Kinetic models; Paper industry; Cell state; Cellular integrity or functionality;

Production of l-theanine using glutaminase encapsulated in carbon-coated mesoporous silica with high pH stability by Tetsuji Itoh; Yasuto Hoshikawa; Shun-ichi Matsuura; Junko Mizuguchi; Hiroyuki Arafune; Taka-aki Hanaoka; Fujio Mizukami; Akari Hayashi; Hirotomo Nishihara; Takashi Kyotani (207-214).
Display Omitted► The pore surface of SBA-15 was coated with an extremely thin layer of carbon. ► The stability of SBA-15 toward high pH has successfully increased by coating it with carbon. ► The Glu/carbon/SBA-15 catalyzed the reaction for production of l-theanine under high pH conditions.A new enzymatic process for production of l-theanine was presented using glutaminase combined with immobilization technique on carbon-coated mesoporous silica (carbon/SBA-15). For the attempt to enhance the durability against high pH conditions, the pore surface of mesoporous silica SBA-15 was coated with a thin layer of carbon. Carbon coating was done by addition of 2,3-dihydroxynaphthalene (DN) on the pore surface, followed by a dehydration reaction between surface silanol groups in SBA-15 and hydroxyl groups of DN molecules, and further carbonization of DN. A glutaminase was confined in nanospace (about 5 nm) of carbon/SBA-15. The resulting product, glutaminase/carbon/SBA-15, successfully catalyzed the reaction for production of l-theanine under high pH conditions.
Keywords: Carbon; Mesoporous silica; Enzyme immobilization;

Reaction kinetics of the degradation of chloroanilines and aniline by aerobic granule by Liang Zhu; Meile Lv; Xin Dai; Xiangyang Xu; Hanying Qi; Yanwen Yu (215-220).
► Reaction kinetics of aniline and chloroanilines by aerobic granules are studied in this paper. ► Formation of aerobic granule favours enhancing the degradability of toxic organic compounds. ► Controlling granular diameter is important for optimising the performance of bioreactor.The reaction kinetics of the degradation of ClAs and AN by aerobic granules were studied in a stable sequencing airlift bioreactor (SABR). Results showed that the degradation of ClAs and AN by aerobic granules followed the Haldane equation. The maximum substrate degradation rates (V max) for different pollutants were as follows: AN > 4-ClA > 2-ClA > 3-ClA > 3,4-DClA, and the order of inhibition constant (K i) was AN > 3-ClA > 4-ClA > 2-ClA > 3,4-DClA. The biodegradabilities of ClAs and AN was well relevance to their spatial parameters and thermodynamic coefficients. At the same time, the values of V max and K i increased along with the granular diameter (peaked at 1.25–2.0 mm), and then K i increased but V max decreased. The results demonstrated that the formation of aerobic granule favours enhancing the degradability of toxic organic compounds, and controlling the granular diameter is important for optimising the performance of bioreactors.
Keywords: Aerobic granule; Chloroaniline; Biodegradation; Kinetic parameter; Wastewater treatment; Bioreactors;

► The expression levels of core autophagy-related genes of rCHO cell lines were evaluated. ► The mRNA/protein level of Atg9A decreased in a later phase of culture. ► The controlled overexpression of Atg9A did not induce autophagy well. ► The combinatorial regulation could be a promising approach for autophagy induction.Autophagy, a self-eating process, has attracted attention as a new target for anti-cell death engineering of recombinant Chinese hamster ovary (rCHO) cells in order to improve culture performance. In an effort to obtain genetic targets for autophagy control, changes in the mRNA and protein expression of four core autophagy pathway genes (Ulk1, Beclin-1, Atg7, and Atg9A) have been investigated in serum-free suspension cultures of rCHO cell lines. Among the four target genes, Atg9A was the only gene showing decreased levels of mRNA and protein simultaneously when comparing the expression in the late period of the culture showing the maximum level of autophagy with that in the control time point (Day 4). In order to verify the potential of Atg9A as an autophagy induction target, Atg9A was overexpressed in rCHO cells using the Tet-Off System. However, Atg9A overexpression did not significantly influence the autophagy induction and culture longevity. Taken together, the results obtained here demonstrate that the downregulation of Atg9A is not the sole limiting factor for autophagy induction in serum-free suspension cultures of rCHO cells. This suggests that combinatorial regulation of the genes in the upstream autophagy pathway with Atg9A overexpression could be a promising approach for autophagy control.
Keywords: Autophagy; Atg9A; Inducible expression; rCHO cells;

Involvement of c-type cytochrome CymA in the electron transfer of anaerobic nitrobenzene reduction by Shewanella oneidensis MR-1 by Pei-Jie Cai; Xiang Xiao; Yan-Rong He; Wen-Wei Li; Lei Yu; Michael Hon-Wah Lam; Han-Qing Yu (227-230).
► Mtr respiration pathway is not involved in the NB bioreduction. ► CymA plays an important role in intracellular NB bioreduction by Shewanella. ► Some unknown protein are suggested to be involved in electron transfer. ► Novel electron transfer pathways of NB bioreducation are proposed.Effective bioreduction of nitrobenzene (NB) by electrochemically active bacteria, such as Shewanella and Geobacter, has been demonstrated. However, the mechanism behind such a bioreduction is unclear yet. In this work, the mechanism for anaerobic NB reduction by Shewanella oneidensis MR-1 was investigated at a gene level. The omcA-mtrCAB gene cluster, an important extracellular electron transfer chain in S. oneidensis MR-1 for the reduction of a variety of compounds, was found to be uninvolved in the NB bioreduction. Knockout of cymA, a tetraheme c-type cytochrome in the periplasmic space, led to a 67% loss in NB bioreduction efficiency in comparison with the wild strain, and caused accumulation of an intermediate, phenylhydroxylamine, at the initial stage of NB bioreduction. These results demonstrate that, unlike the previously reported pure extracellular reduction pathways for many compounds, intracellular reactions are involved in NB bioreduction by S. oneidensis MR-1, and CymA plays an important role in the electron transfer of the intracellular NB bioreduction. In addition, the participation of some unknown protein other than CymA in NB reduction is discovered, and the possible pathways of electron transfer in the NB reduction by S. oneidensis MR-1 are proposed.
Keywords: Anaerobic processes; Biodegradation; Nitrobenzene (NB); Waste-water treatment; Protein; Shewanella oneidensis MR-1;

► First characterization of different protocols for immobilization of Cryptosporidium antibodies. ► Study of both immobilization protocols, antibody concentrations and immobilization conditions. ► Useful to inform the design of future biosensor systems, using antibodies as the detection element, for Cryptosporidium. Cryptosporidium is a waterborne protozoan parasite, which is problematic for the water industry due to widespread environmental presence, low infectious dose and resistance to chlorine disinfection. To replace the existing, slow regulatory monitoring procedure, immunosensors have been proposed. The performance of such sensors is often limited by the antibody immobilization. The aim of this article was to determine the immobilization protocol offering the highest oocyst capture efficiency. Four methods were tested both under static and convective conditions: physisorption, cysteamine–glutaraldehyde linkage, 3-aminopropyl-triethoxysilane functionalization and protein G linkage. The protein G protocol was shown to present the highest recovery rates in both conditions. For the protein G protocol, different antibody concentrations were tested and it was concluded that there is little difference in the recovery rates when the concentration of IgG antibody is higher than 20 μg/mL. Additionally, operation under convective rather than static conditions increased the recovery for every protocol. These results can be useful to inform the design of future biosensor systems, using antibodies as the detection element, for Cryptosporidium.
Keywords: Immunosensors; Cryptosporidiosis; Protein G; Cryptosporidium parvum; Convective conditions; Static conditions;