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Biochemical Engineering Journal (v.36, #2)

Editorial Board (pp. co2).

Biochemical Engineering Journal (pp. iv).

Evolution of the specific power consumption and oxygen transfer rate in alginate-producing cultures of Azotobacter vinelandii conducted in shake flasks by Carlos Peña; Cyril P. Peter; Jochen Büchs; Enrique Galindo (pp. 73-80).

The evolution of the specific power consumption and the oxygen transfer rate occurring in shake flasks was studied in cultures of Azotobacter vinelandii, which synthesize alginate, a polymer producing non-Newtonian fermentation broths. Power consumption increased exponentially during the course of the fermentation (up to 1.4kWm⁻³) due to an increase in the viscosity of the culture broth. This increase in the viscosity was associated with alginate concentration and the mean molecular mass of the polymer, which reached a maximum of 550kDa after 50h of cultivation. At the end of the fermentation, when the viscosity and alginate concentration reached a maximum, a slight drop in the power consumption was observed. This was probably because the fluid was in the “out-of-phase” state due to the high viscosity. In order to explain the “out of phase” behavior, the actual shear rate in the shake flasks should be close to 90s⁻¹. Although the on-line measurements of oxygen transfer rate showed clear differences in the different filling volumes evaluated, both biomass growth and alginate production were very similar in the three conditions tested. Analysis of molecular mass distributions of the alginates suggests that, in the shake flasks cultures, DOT conditions can be more homogeneous than those present in a stirred fermentor without control of DOT and pH.

Keywords: Alginate; Molecular mass; Power input; Oxygen transfer rate

Production of a novel bioflocculant by culture of Klebsiella mobilis using dairy wastewater by Shu-Guang Wang; Wen-Xin Gong; Xian-Wei Liu; Lin Tian; Qin-Yan Yue; Bao-Yu Gao (pp. 81-86).

In this study, dairy wastewater, with 2% added ethanol (v/v), was used as a substrate for the production of a novel bioflocculant by Klebsiella mobilis. Using optimized conditions for the production of the novel bioflocculant, 2.58g of crude bioflocculant per liter broth was produced with a flocculating activity of 95.4%. Chemical analysis showed that the novel bioflocculant was a polysaccharide where no amino acids were detected. The bioflocculant possesses flocculating activity comparable or superior to that of bioflocculant produced using conventional substrate. The bioflocculant was effective in flocculating some disperse dyes in aqueous solutions, in particular, disperse Violet HFRL with a decolorization efficiency of 91%.

Keywords: Bioflocculant; Dairy wastewater; Klebsiella mobilis; Substrate

Nitrification–denitrification via nitrite in SBR using real-time control strategy when treating domestic wastewater by Changyong Wu; Zhiqiang Chen; Xiuhong Liu; Yongzhen Peng (pp. 87-92).

A lab-scale sequencing batch reactor (SBR) was employed to treat low C/N ratio municipal wastewater to investigate the stability of nitrification–denitrification via nitrite using a real-time control strategy to switch aeration on and off. Three parameters, pH, DO and ORP, were selected as control parameters in the SBR reactor operation. Nitrite accumulation was observed under real-time control conditions (NO₂⁻-N/NO_x⁻-N>0.8), implying that nitrification–denitrification mainly via nitrite was achieved by the process control strategy applied. The nitrite concentration in the SBR decreased sharply when the SBR system was operated using a fixed time aeration control strategy for 7 days. Ammonia nitrogen concentration (NH₄⁺-N) and temperature also affect nitrite accumulation. Ammonia shock loads limited nitrification due to alkalinity limitations, but the system recovered quickly from short-time high ammonia shock loads when the real-time control strategy was applied. Temperature was observed to affect nitrite accumulation rate when the real-time control strategy was used in the SBR reactor, with higher nitrite accumulation at 28±0.5°C (NO₂⁻-N/NO_x⁻-N=0.84), compared to the accumulation at the ambient average temperature of 20°C (NO₂⁻-N/NO_x⁻-N>0.61).

Keywords: SBR; Real-time control; Nitrification–denitrification via nitrite; Ammonia shocking; Temperature

Hyperbranched polymer conjugated lipase with enhanced activity and stability by Jun Ge; Ming Yan; Diannan Lu; Minlian Zhang; Zheng Liu (pp. 93-99).

Hyperbranched aromatic polyamides were synthesized from p-phenylenediamine and trimesic acid, and subjected to the conjugation of lipase using carbodiimide as a coupling reagent. The molecular weights of the conjugates were determined by size exclusion chromatography and gel electrophoresis, which showed that each polymer molecule could be covalently modified with a maximum of five to six lipase molecules. The K_m of the conjugate to substrate, p-nitrophenylpalmitate, was equal to native lipase while V_max was increased by 20%, suggesting an enhanced structural transition of lipase during catalysis conducted at the periphery of the polymer. Moreover, the conjugate exhibited a significantly enhanced stability at high temperature or in the presence of organic solvent, as compared to its native counterpart. This might due to the hydrophilic carboxylic groups of polymer that created a suitable microenvironment for lipase. These results indicate that hyperbranched polymer–enzyme conjugate, with the enhanced activity and stability, is promising for industrial biocatalysis.

Keywords: Bioconjugate; Enzyme modification; Hyperbranched polymer; Hyperbranched aromatic polyamide; Lipase; Enzyme stabilization

A two-phase kinetic model for fungal growth in solid-state cultivation by Zohreh Hamidi-Esfahani; Parisa Hejazi; Seyed Abbas Shojaosadati; Marisca Hoogschagen; Ebrahim Vasheghani-Farahani; Arjen Rinzema (pp. 100-107).

A new two-phase kinetic model including exponential and logistic models was applied to simulate the growth rate of fungi at various temperatures. The model parameters, expressed as a function of temperature, were determined from the oxygen consumption rate of Aspergillus niger during cultivation on wheat bran. The model can describe the whole growth curve including the lag phase and the cessation of growth in the latter stages of the cultivation with an adequate approximation. Furthermore, the model describes the growth rate of Aspergillus oryzae on wheat properly. Comparisons between the current, logistic and previous two-phase kinetic models indicate that the new model can predict growth rate of fungi more accurately.

Keywords: Solid-state cultivation (SSC); Two-phase growth kinetic model; Wheat bran; Aspergillus niger; Aspergillus oryzae

Fermentation of olive tree pruning acid-hydrolysates by Pachysolen tannophilus by Inmaculada Romero; Sebastián Sánchez; Manuel Moya; Eulogio Castro; Encarnación Ruiz; Vicente Bravo (pp. 108-115).

The influence of hydrolysis conditions on the fermentation of hydrolysates obtained from olive tree pruning (a renewable, low cost, largely available agricultural residue) is analyzed in this work. Hydrolysis was performed using sulfuric acid at atmospheric pressure (90°C) in a concentration range of 0.5–4N for 240min. The fermentation of hydrolysates was carried out by Pachysolen tannophilus in a discontinuous tank bioreactor at 30°C and pH 3.5. The main fermentation parameters determined in this study include maximum specific growth rate, biomass productivity, specific substrate consumption rate, specific ethanol production rate, and ethanol and xylitol yields. The results show that ethanol yields are much higher than xylitol yields under all the conditions tested. The maximum ethanol yield (0.38g/g) is reached with the hydrolysate obtained with 0.75N sulfuric acid. Under these conditions the conversion of the hemicellulose fraction is 92%.

Keywords: Olive tree pruning; Bioconversion; Fermentation; Pachysolen tannophilus; Ethanol; Bioreactor

Carbon source in aerobic denitrification by Katarzyna Bernat; Irena Wojnowska-Baryła (pp. 116-122).

The objective of this study was to demonstrate denitrification by activated sludge under aerobic conditions. Activated sludge was cultivated in a sequencing batch reactor (SBR) with municipal wastewater enriched by acetate. Poly-β-hydroxybutyrate was accumulated in activated sludge to 0.35g PHB/g VSS ( f_PHB=0.27Cmol/Cmol). Microorganisms had the opportunity to preserve reducing power from poly-β-hydroxybutyrate (PHB) stored in microbial cells. Activated sludge, cultivated in such conditions, was used in further experimental series. The duration of each series was 24h.Two types of synthetic wastewater were used in the presented studies: wastewater without organic compounds, and wastewater with acetate as the main carbon source. Ammonium was the only nitrogen source. Since, ammonium oxidation by activated sludge had to go before denitrification. It was shown that under aerobic, autotrophic conditions, the amount of nitrogen reduced by activated sludge was 4.54mgN_red/l, using intracellular PHB stored in microbial cells, as the only carbon source for denitrification. In wastewater with acetate, the amount of reduced nitrogen due to aerobic denitrification increased to 22.5mgN_red/l.The results indicted that PHB, stored in activated sludge, can be served as electron donor for aerobic denitrification. Denitrification under aerobic conditions using internal substrate is not as effective as in presence of the internal and external carbon sources but during denitrification, supported by endogenous substrates, the amount of organic compounds in wastewater required for the process can be limited.

Keywords: Nitrification; Aerobic denitrification; Poly-β-hydroxybutyrate; Activated sludge; Endogenous, and external carbon source

Cooperative effects of artificial chaperone and Mg²⁺ ions on alkaline phosphatase refolding by Fariba Khodagholi; Razieh Yazdanparast (pp. 123-130).

The refolding of GuHCl-denatured alkaline phosphatase (ALP) has been investigated by a new elegant strategy using the artificial-chaperone assisted two-step mechanism. However, using detergent and cyclodextrin, which are both poorly soluble in aqueous solutions, necessitates large sample dilutions to earn adequate refolding yield. In the present work, to overcome this obstacle, we studied the concomitant effects of the artificial chaperone and metal ions on ALP refolding at 0.2mg/ml concentration. The method presented is a high-yielding process with few operational steps which may result in a low process cost. Based on this approach, refolding of GuHCl-denatured ALP raised from 20%, in the absence of anti-aggregators, to about 50% in the presence of artificial chaperone and/or MgSO₄ and to 82% in the concomitant presence of the artificial chaperone and MgSO₄ while the extent of aggregation was totally quenched. Using different salts of Mg²⁺ resulted in different recovered yields probably due to their variable interactions with cyclodextrin. Under the same experimental conditions except for the metal ions, Zn²⁺ and Ca²⁺, worked neither alone nor in combination with the artificial chaperone showing that Mg²⁺ can work as an auxiliary agent because of its structural role.

Keywords: Alkaline phosphatase; Artificial chaperone; Magnesium ion; Metal ions

Adsorption properties of Cu(II) ions onto N-succinyl-chitosan and crosslinked N-succinyl-chitosan template resin by Shengling Sun; Qin Wang; Aiqin Wang (pp. 131-138).

In the current study, the adsorption behavior of N-succinyl-chitosan (NSC) for Cu(II) ion was investigated. The equilibrium adsorption data of NSC have been found to fit the Langmuir adsorption isotherm, and the saturation adsorption capacity is 2.74mmol/g. Based on the obtained optimum condition of the influences of the pH, the adsorption temperature and time, the degree of substitute (DS) of NSC and the dosage of crosslinking agent, a new perfect adsorbent material, crosslinked NSC resins with Cu(II) as template ions (crosslinked NSC template) was synthesized. The adsorption experiments indicated that the crosslinked NSC template and NSC can selectively adsorbed Cu(II) ions from the solution of Cu(II), Zn(II), Co(II), and Ni(II) ions coexistence, but the selectivity of the crosslinked NSC template is higher than that of NSC. Furthermore, the crosslinked NSC template has shown a good reusability. The FTIR analysis results revealed that the adsorption sites mainly occur on the carboxyl groups of the succinyl groups of the crosslinked NSC template in the Cu(II) adsorption process.

Keywords: Adsorption; N; -Succinyl-chitosan; Crosslinked; Resin; Cu(II)

Protein adsorption in supermacroporous cryogels with embedded nanoparticles by Kejian Yao; Shaochuan Shen; Junxian Yun; Lianghua Wang; Fang Chen; Xiaomei Yu (pp. 139-146).

Supermacroporous cryogel with embedded nano-size adsorbent particles is a new media suggested for separation and purification of biomolecules. In order to reveal the protein adsorption characteristics in this novel cryogel, several polyacrylamide-based cryogel beds with embedded Fe₃O₄ nanoparticles were prepared by radical cryo-copolymerization of acrylamide, N, N′-methylene-bis-acrylamide, allyl glycidyl ether and surfactant-stabilized Fe₃O₄ nanoparticles under freezing variation conditions in glass columns of inner diameters 16 and 26mm. Bovine serum albumin (BSA) was used as model protein to study the cryogel adsorption behaviors in the cryogels. Experimental measurements of the isothermal and kinetics adsorption of BSA were carried out and the protein adsorption capacities were determined. Breakthrough curves of BSA in NaAc–HAc buffer at different liquid flow rates were obtained to reveal the protein adsorption behaviors. Effect of liquid velocity, as well as the pore-structures and liquid dispersion on the protein breakthrough performance were investigated and analyzed.

Keywords: Supermacroporous cryogel; Nanoparticle; Bovine serum albumin; Protein; Isothermal adsorption; Dynamic adsorption; Breakthrough curve

Theoretical and experimental investigation of biodegradation of hydrocarbon polluted water in a three phase fluidized-bed bioreactor with PVC biofilm support by D. Mowla; M. Ahmadi (pp. 147-156).

The aerobic treatment of hydrocarbon polluted groundwater was performed experimentally in a three phase fluidized-bed bioreactor (FBBR) using mixed culture of living cells immobilized on PVC particles. The characteristic of the living cells immobilized in FBBR were identified and the kinetic parameters of the biochemical reaction were evaluated. A steady-state biofilm model was developed based on porous pellet model which considers the diffusion and reaction of hydrocarbons within biofilm, axial dispersion coefficient in bioreactor and external mass transfer resistance between biofilm and completely mixed liquid phase. The developed model assumes a first-order biochemical reaction within biofilm. Experimental results were used to test the validity of the proposed model. A reasonable agreement between the experimental and theoretical results was found.

Keywords: Three phase fluidized-bed; Biofilm thickness; Modeling; Reaction kinetics

Development of adaptive modeling techniques to describe the temperature-dependent kinetics of biotechnological processes by Elmer Ccopa Rivera; Aline C. Costa; Rafael R. Andrade; Daniel I.P. Atala; Francisco Maugeri; Rubens Maciel Filho (pp. 157-166).

Bioprocesses are quite difficult and expensive to model, since their operation involves microbial growth under constantly changing conditions, with impact on process kinetics and performance. Hence, there is a need and incentive for the improvement of methods for rapid development of simple, though realistic, mathematical models. In this work the modeling of biotechnological processes is studied with focus on developing methodologies that can be used whenever a re-estimation of parameters is necessary. The ethanol fermentation process is used as a case study. The performance of a hybrid neural model and a balance based model, both considering the effect of temperature on the kinetics, are evaluated not only by their accuracy in describing experimental data, but also by the difficulties involved in the adaptation of their parameters. Experiments are performed to develop the two models and further experiments (using sugar cane molasses from a different harvest and a different production medium) validate the methodologies for re-estimation of kinetic parameters.

Keywords: Bioreactors; Modeling; Optimization; Parameter estimation; Temperature effect; Artificial intelligence

Lipase-catalyzed irreversible transesterification of vegetable oils for fatty acid methyl esters production with dimethyl carbonate as the acyl acceptor by Er-Zheng Su; Min-Jie Zhang; Jian-Guo Zhang; Jian-Feng Gao; Dong-Zhi Wei (pp. 167-173).

Dimethyl carbonate (DMC) was used in the enzymatic transesterification of vegetable oils, which resulted in an irreversible reaction benefiting fatty acid methyl esters production. Among the tested lipases, Novozym435 (lipase B from Candida antarctica) led to a higher conversion for all tested vegetable oils in the initial screening. DMC used as the acyl acceptor, the conversions of cottonseed oil, soybean oil and rapeseed oil were two to three times higher than those of conventional acyl acceptors (methanol and methyl acetate). Using cottonseed oil as the feedstock, a very high conversion of 96.4% could attain under the optimized conditions. This optimal condition was further applied to other vegetable oils. All of them showed very high conversion except sesame oil. Although the Novozym435 activity was impaired severely by the bound glycerol, it could remain about 80% activity after five batch reactions if only it was washed with acetone after each batch.

Keywords: Dimethyl carbonate (DMC); Acyl acceptor; Fatty acid methyl esters (FAMES); Novozym435; Transesterification; Cottonseed oil

Removal of heavy metal from industrial wastewater using modified activated coconut shell carbon by O.S. Amuda; A.A. Giwa; I.A. Bello (pp. 174-181).

The present study was undertaken to develop a cost effective biosorbent and to study the biosorption process involved in the adsorption of heavy metal-contaminated industrial wastewater using the developed biosorbent. Coconut shell carbon was modified with chitosan and/or oxidizing agent (phosphoric acid) to produce composite adsorbent. The adsorption efficiency of the adsorbent was evaluated by measuring the extent of adsorption of zinc (II) in synthetic beverage industrial wastewater. Operational parameters such as pH, agitation time and adsorbent concentration, initial ion concentration and particle size were also studied. Adsorption data fitted well with the Langmuir and Freundlich models. However, Langmuir isotherm displayed a better fitting model than Freundlich isotherm because of the higher correlation coefficient that the former exhibited, thus, indicating to the applicability of monolayer coverage of the zinc (II) on the surface of adsorbent.Desorption studies were carried out with NaOH and quantitative recovery of the metal was evident. The dominant sorption mechanism is ion exchange. The use of agricultural waste (coconut shell) and aquatic waste (chitin) to produce activated carbon potentially leads to the production of a highly effective adsorbent generated from less expensive raw materials that are from renewable resources.

Keywords: Coconut shell; Chitosan-coated carbons; Heavy metals; Industrial wastewater; Adsorption isotherms

Optimization of cell growth and carotenoid production of Xanthophyllomyces dendrorhous through statistical experiment design by Yuan Shuai Liu; Jian Yong Wu (pp. 182-189).

This study was to identify and optimize the major culture factors for both cell growth and carotenoid biosynthesis in Xanthophyllomyces dendrorhous through statistical experiment design and data analysis. A screening test was first conducted on seven culture factors including pH and six medium components using a Plackett–Burman design, from which glucose, ammonium sulfate and pH were identified as the significant factors affecting both cell growth and carotenoid biosynthesis. These significant factors were optimized by central composite design of experiments and response surface methodology, as 34.3g/L glucose, 2.95g/L ammonium sulfate and pH 5.85 for cell growth in the culture, and 19.3g/L glucose, 0.81g/L ammonium sulfate and pH 5.19 for carotenoid accumulation in the yeast cells, respectively. Then a two-stage culture process was exercised with these optimal conditions tailored for cell growth (first stage) and carotenoid accumulation (second stage), achieving 95.8% higher volumetric yield (18.6mg/L versus 9.5mg/L in the single-stage culture) and 22.6% higher volumetric productivity (2.33mg/(Lday) versus 1.90mg/(Lday)) of total carotenoid. The medium optimization results suggest that carotenoid biosynthesis in X. dendrorhous is promoted by high C/N ratio, low carbon and nitrogen concentrations, and a slightly acidic condition when the cell growth is suppressed.

Keywords: Xanthophyllomyces dendrorhous; Carotenoid; Optimization; Statistical design; Response surface methodology; Two-stage culture

Pore control with dextran generated from immobilized dextransucrase by Hidetaka Kawakita; Hirokazu Seto; Keisuke Ohto; Katsutoshi Inoue; Hiroyuki Harada (pp. 190-193).

Dextransucrase forms a complex with dextran during an enzymatic reaction with sucrose. Using its enzymatic character, dextransucrase was immobilized on the pore surface of an inorganic membrane at 1.5U/g-membrane, and subsequent permeation of a sucrose solution (25g/L) was used to produce dextran on its surface. The pressure loss in the sucrose solution permeating the enzyme-immobilized membrane increased exponentially, indicating that the produced dextran filled the pore. A solution containing latex particles was permeated through the dextran-containing membrane for determining pore filling with dextran, demonstrating that the rejection percentage of latex bead increased with increasing the amount of dextran produced.

Keywords: Dextransucrase; Dextran; Inorganic membrane; Latex bead; Separation

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Biochemical Engineering Journal (v.36, #2)

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Biochemical Engineering Journal (v.36, #2)