Biochemical Engineering Journal (v.35, #3)

Production of lactic acid from renewable materials by Rhizopus fungi by Zhan Ying Zhang; Bo Jin; Joan M. Kelly (251-263).
Lactic acid is a commonly occurring organic acid, which is valuable due to its wide use in food and food-related industries, and its potential for the production of biodegradable and biocompatible polylactate polymers. Lactic acid can be produced from renewable materials using various fungal species of the Rhizopus genus, which have advantages compared to the bacteria, including their amylolytic characteristics, low nutrient requirements and valuable fermentation by-product—fungal biomass. This paper reviews recent research in process engineering, metabolic and enzymetic mechanisms, and molecular biotechnology associated with lactic acid production by the Rhizopus fungi to get a better understanding of biochemical activities. The major process components: renewable materials, bioreactor systems and process modeling are reviewed. The role of key bioprocess parameters, such as nutrient composition, pH and growth morphology, involved in the production of lactic acid is discussed in detail. In addition, recent advances in simultaneous saccharification and fermentation, molecular genetic approaches, and enzymetic and metabolic pathways involved in the production of lactic acid by fungal strains are discussed.
Keywords: Lactic acid; Rhizopus species; Renewable materials; Simultaneous saccharification and fermentation; Starch; Enzymetic and metabolic pathway; Bioreactors; Immobilization; Lactate dehydrogenase;

Characterization of mass transport processes in IMAC chromatography by dynamics methods by R. Gutiérrez; E.M.M. Del Valle; M.A. Galán (264-272).
The characterization of mass transport processes in IMAC chromatography and the pH effect on the process have been done. To do that the adsorption equilibrium constant and adsorption rate constant for Cu, Zn-superoxide dismutase (Cu, Zn-SOD) on Cu-IDA-agarose matrix have been determined in batch and in a continuous operating systems (chromatographic column) by dynamic methods. The experiments were performed at pH of 7.00, 7.40 and 7.80 and, in case of chromatographic columns, flow rates of 1.56–2.66 ml/min. From the analysis of the experimental results it can be concluded that the controlling steps for the Cu, Zn-SOD adsorption on Cu(II)-IDA-agarose beads are both external and internal mass transfer processes, which was experimentally confirmed with the fact that the adsorption kinetic process is too fast. The medium pH affects equilibrium and mass transport parameters in the following way: a decrease in the medium pH towards the enzyme isoelectric point increases the adsorption equilibrium constant and both molecular and effective intraparticle diffusion coefficients; and decreases internal, external and global mass transfer resistances, and effective axial dispersion.
Keywords: Affinity chromatography; Purification; Mass transfer; Diffusion; Dynamic methods;

Optimization study of xanthan gum production using response surface methodology by S.K. Psomas; M. Liakopoulou-Kyriakides; D.A. Kyriakidis (273-280).
The cultural conditions for xanthan gum production by Xanthomonas campestris were investigated and optimized by response surface methodology, to maximize cell and xanthan production in batch experiments using a synthetic broth (Luria-Bertani plus glucose, LBG) without pH control. The individual and interactive effects of three independent variables (agitation rate (100–600 rpm), temperature (25–35 °C), time of cultivation (24–72 h)) on xanthan gum and biomass production were studied, using a face-centered composite design of experiments. A second ordered polynomial model was fitted and optimum conditions were estimated. Overall optimization allowed us to point out an optimal range of the three independent variables. Optimal xanthan gum production was found at 600 rpm 30 °C at 72 h and biomass at 600 rpm, 25 °C at 72 h. Furthermore, the consumption of substrate (glucose) and the variation of pH during the fermentation as well as the molecular weight and the pyruvate content of the formed polysaccharide are reported.
Keywords: Xanthan gum; Fermentation; Optimization; Agitation; Temperature; Time;

Modelling thermophilic cheese whey bioremediation in a one-stage process by M.R. Kosseva; A. Fatmawati; M. Palatova; C.A. Kent (281-288).
A one-stage bioremediation process for the treatment of cheese whey has been developed, involving use of a mixed population including Bacillus sp. isolated from fruit–vegetable wastes, at elevated temperatures (55, 60, 65 °C), in an aerated system. Based upon a reaction scheme which combined the activities of Lactococcus sp. and thermotolerant yeasts, found in unpasteurised whey, with that of a thermophilic Bacillus sp., we modelled this process using mass balances for a batch culture. We obtained adequate fits to experimental data by simulating the biodegradation of whey components and consequent biomass growth rates using models containing one “lumped” biomass and three “equivalent” substrates (lactose, lactic and acetic acids), with a further simplification of biomass specific growth rate into one constant, “pseudo-exponential” value over the batch. Values of “best fit” model parameters were generated to predict biomass specific growth rates. The average specific growth rate calculated from the models was 0.075 h−1 whilst the experimental one was 0.088 h−1. The results obtained suggest that temperature may be of greater importance to the biodegradation process than dissolved oxygen, as the composition of the microbial community changed with temperature within the range 55–65 °C. High COD removal efficiencies were achieved: up to 93% at 55 °C, and up to 70% at 65 °C. Our investigations suggest that modelling of complex bioreaction systems via “lumping” of key substrates and microbial species into a limited number of “equivalent clusters” is worthy of consideration as a possible means of facilitating rapid process development and practical process operation.
Keywords: Aerobic processes; Bioremediation; Dynamic modelling; Thermophiles; Whey;

During lactic acid fermentation, the positive effect of precultivating without pH control is now clearly established. At acidic pH, growth was inhibited. To account for this inhibition, the well-known Luedeking–Piret model was modified by introducing an additional term involving the undissociated form of the lactic acid, the main inhibitory species. The Henderson–Hasselbach equation was also considered in the model to describe the relationship between pH, also involved in growth inhibition, and both the dissociated and the undissociated forms of lactic acid. The model was found to describe satisfactory experimental growth and production data in a wide range of culture conditions. Indeed, whey permeate supplemented by a large range of nitrogen supplementations was considered and analysed, since the growth- and non-growth-associated part of the production can be easily deduced from the model. High supplementation of whey permeate, namely 20 g l−1 yeast extract and 10 g l−1 peptones, the usual seed culture supplementation, led to the main part of the lactic acid produced by a growth-associated mechanism (74%).
Keywords: Batch processing; Fermentation; Growth kinetics; Lactic acid; Modelling; Product inhibition;

Separation of human plasma proteins HSA and HIgG using high-capacity macroporous gel-filled membranes by Dharmesh M. Kanani; Elena Komkova; Tiffany Wong; Alicja Mika; Ronald F. Childs; Raja Ghosh (295-300).
This paper discusses the separation of human serum albumin (HSA), the most abundant protein present in plasma from human immunoglobulin G (HIgG) by membrane chromatography using a novel macroporous gel-filled membrane (designated Q Type 2). The membrane was prepared by anchoring a quaternary ammonium salt macroporous gel within the pores of a non-woven, polypropylene fabric. Factors affecting HSA binding were examined and operating conditions suitable for separating it from human plasma were identified. At an optimized condition, the HSA binding capacity of this novel membrane under saturating conditions was in the range of 290–300 mg/ml. This was not only significantly higher than binding capacities reported for other chromatographic membranes, but also higher than binding capacities of conventional gel based chromatographic media. The protein binding capacity was also largely insensitive to the superficial velocity, indicating the dominance of convective protein transport to and from the binding sites. The suitability of using this membrane for plasma fractionation was demonstrated by the separation of a simulated feed solution consisting of HSA and HIgG.
Keywords: Membrane; Membrane chromatography; Proteins; HSA; HIgG; Macroporous gel-filled; Anion-exchange; Bioseparation;

The influence of coating polystyrene tissue culture plates with different proteins on murine hybridoma cell growth and antibody production was investigated. Fibronectin, collagen I, bovine serum albumin and laminin were used to coat NUNC® and COSTAR® cell culture plates. Cell number and antibody concentration in culture fluids were quantified as indicators for cell viability, proliferation and productivity. Adhesive behaviour, morphology, expression of surface receptors of hybridoma cells and the presence of tyrosine-phosphorylated proteins in cell lysates were characterized by cell adhesion experiments, microscopy, flow cytometry and Western Blot analysis.It was shown that coatings with fibronectin (0.2 μg/ml) lead to a substantial improvement of cell growth by 50–70% and an increase of monoclonal antibody production by 100–120%.Collagen I coatings showed an improvement in cell growth by 30–70% and by 60% for the production of monoclonal antibodies. Coatings with BSA and laminin had minor effects on these parameters. It was found that the hybridoma cell lines used in this study did not express the α2-chain of the α2β1-integrin, which is responsible for binding to collagen and laminin.However, the presence of β1-integrin on the cell surface was shown, which should enable hybridoma cells to bind fibronectin. We propose, therefore, that fibronectin adsorption to cell culture materials may be a promising approach to enhance the production of monoclonal antibodies by cultivated hybridoma cells.
Keywords: Hybridoma cells; Tissue culture plates; Cell growth; Antibody production; Extracellular matrix; Fibronectin adsorption;

An attempt was made to examine the effect of applied pressure on membrane fouling that might influence the potential use of ultrafiltration (UF) membrane in treating as well as recovering the bioresources, namely protein and carbohydrate from complex feed like palm oil mill effluent (POME). POME was first subjected to physical pretreatment processes, consisting of depth and surface filtration in order to remove the total suspended solids (TSS). The pretreatment processes enabled the reduction of TSS, turbidity, total dissolved solid (TDS) and chemical oxygen demand (COD) up to 97.3%, 88.2%, 3.1% and 46.9%, respectively. Protein (45.3%) and carbohydrate (41.5%) that retained as insoluble matters together with suspended solids might be used as fertilizer or animal feed by-products. Then, polysulphone UF membrane of 20 kDa was used in the UF membrane study. This study indicated that the applied pressure imposed a direct effect on fouling, permeate flux, protein and carbohydrate recovery as well as wastewater treatment. In total, the permeate flux decreased with filtration time until it reached steady-state values. Beyond a certain applied pressure between 0.6 and 0.8 MPa, the increase in permeate flux with pressure was negligible and insignificance. The highest applied pressure (0.8 MPa) encouraged the formation of fouling up to 85.8% but at the same time enabled the recovery of protein and carbohydrate up to 61.4% and 76.4%, respectively. The highest reduction of TSS, turbidity, TDS and COD also occurred at 0.8 MPa up to 97.7%, 88.5%, 6.5% and 57.0%. The study revealed that it is possible to have appropriate control of applied pressure in order to favor fouling that would, in turn, lead to better rejection of other solutes present in the feed.
Keywords: Bioseparations; Protein and carbohydrate recovery; Ultrafiltration; Wastewater treatment; Fouling; Palm oil mill effluent (POME);

A partially purified Carica papaya (pCPL) is explored as an effective biocatalyst for the esterification resolution of (RS)-2-(chlorophenoxy)propionic acid (CPA) with trimethylsilylmethanol in anhydrous isooctane. In comparison with a crude Candida rugosa lipase (CRL), pCPL has higher enantioselectivity (E  > 100) for 2- and 4-chloro but not 3-chloro substituted CPA, in which more than an order-of-magnitude lower reactivity for the hindered (RS)-2-(2-chlorophenoxy)propionic acid is found for both lipases. The thermodynamic analysis indicates that the enantiomeric discrimination is mainly driven by the difference of activation enthalpy (ΔΔH) in the present reaction conditions. A linear enthalpy-entropy compensation effect between ΔΔH and the difference of activation entropy (ΔΔS) is demonstrated and elucidated from the regio-effect of 2-, 3- and 4-chloro substituent. The kinetic analysis indicates that pCPL is strongly inhibited by the acid substrate, but to a less extent by the alcohol substrate. With the disadvantage of decreasing the enzyme activity, the E value can be greatly enhanced when trimethylsilylmethanol concentration higher than 40 mM in isooctane is employed.
Keywords: Carica papaya lipase; Esterification; (R,S)-2-(Chlorophenoxy)propionic acid; Regio-substituent effect;

Laccase immobilization on copolymer of butyl acrylate and ethylene glycol dimethacrylate by Jolanta Bryjak; Paulina Kruczkiewicz; Adriana Rekuć; Wanda Peczyńska-Czoch (325-332).
Extracellular laccase produced by the wood-rotting fungus Cerrena unicolor was immobilized by covalent bonds formation on the copolymer of butyl acrylate and ethylene glycol dimethacrylate. The carrier had a fixed superstructure and three kinds of anchor groups: –NH2, –OH, and –COOH. Three procedures were used for the activation of the carrier: (i) glutaraldehyde, (ii) divinyl sulfone, and (iii) carbodiimide. It was found that laccase coupling to the carrier via glutaraldehyde yielded an enzyme-carrier preparation of very high activity and storage stability. Consideration was also given to the problem of how the pH, ionic strength, protein concentration and the presence of additives (syringaldazine, guaiacol, Cu2+) affect the coupling procedure via glutaraldehyde. Thermal- and pH-stability, as well as the activity profiles of the best enzyme-carrier preparation, was evaluated. The very high operational stability investigated in a packed bed reactor at 30 °C shows the potential of the preparation for practical use.
Keywords: Immobilization; Laccase; Acrylic carrier; Enzyme activity; Enzyme stability; Kinetic parameters;

Integrated process for production of surfactin by Tao Liu; Ludovic Montastruc; Frédérique Gancel; Ling Zhao; Iordan Nikov (333-340).
The work reported in this paper is aimed at studying the adsorption of surfactin from aqueous solution onto activated carbon. Among the factors, agitation rate, activated carbon particle-size, pH, temperature, initial adsorbate concentration, adsorbent amount and ionic strength of the solution were studied. Both adsorption equilibrium and kinetics showed that activated carbon acted as a suitable adsorbent for surfactin recovery. Two mechanisms represented by different kinetic models were examined, namely, the intraparticle diffusion one and the one involving chemisorption accompanied by surface coverage (conforming to the Elovich concept).
Keywords: Surfactin; Integrated processing; Adsorption; Modelling; Kinetic parameters;

This work presents modifications to the Hashimoto's hybrid simulated moving bed reactor (SMBR) system which was used to produce 55% high fructose syrup (HFS55). The purpose of this study is to develop a new SMBR system to overcome the disadvantages of Hashimoto system (3-zone SMB with seven reactors), i.e., low utility of reactors when feed being a 50/50 blend of glucose and fructose. Two different configurations of modified system were presented in this paper: the first configuration is 4-zone SMB with one reactor, while the other one consists of one additional reactor. Both of these configurations aim at improving the concentration and purity of glucose at the inlet of the reactor, which will lead to both high productivity and high purity of fructose in the product. A state-of-the-art optimization technique, viz., non-dominated sorting genetic algorithm (NSGA) is used in finding the optimal design and operating parameters for the modified reactive SMB and Varicol processes. Compared with the Hashimoto's system, high productivity and purity of fructose can be achieved in these new systems using less number of reactors.
Keywords: Fructose; Moving bed bioreactor; Bioprocess design; Optimization; Varicol; Pareto;

Separation of l-lysine from dilute aqueous solution using molecular imprinting technique by Reza Panahi; Ebrahim Vasheghani-Farahani; Seyed Abbas Shojaosadati (352-356).
In the present study, separation of l-lysine from dilute aqueous solution by solid-phase extraction based on molecular imprinting technique using a polar porogen was investigated. l-Lysine imprinted polymer (LLIP) was prepared by free radical solution polymerization of methacrylic acid and ethylene glycol dimethacrylate as functional and cross-linking monomers, in the presence of l-lysine as an imprint molecule, mixture of water and methanol as solvent and AIBN as an initiator. Non-imprinted polymer (NIP) as control was also prepared by the same procedure in the absence of template molecules. LLIP particles were applied to determine the optimum operational condition for l-lysine separation from dilute aqueous solution. In adsorption step, optimum pH and retention time were 7.8 and 90 min, while corresponding values in extraction step were 12 and 50 min, respectively. l and d-Lysine recovery by LLIP at optimum condition were found to be 96 and 58% with corresponding distribution coefficients of 8000 and 460, respectively. The retention capacity of LLIP was 27.26 mg  l-lys/g of polymer at optimum condition.
Keywords: Molecular imprinting; Lysine; Separation; Adsorption; Optimum condition; Retention capacity;

Water disinfection using the novel approach of ozone and a liquid whistle reactor by Rashmi Chand; David H. Bremner; Kyu C. Namkung; Phillip J. Collier; Parag R. Gogate (357-364).
A novel approach of ozone treatment assisted by a liquid whistle reactor (LWR), which generates hydrodynamic cavitation, has been explored for water disinfection using a simulated effluent containing Escherichia coli (E. coli), one of the dominant markers in faecal coliforms. A suspension having an E. coli concentration of approximately 108 to 109  CFU mL−1 was introduced into the LWR to examine the effect of hydrodynamic cavitation alone and in combination with ozone. Operating conditions of inlet pressure and ozone doses as well as time of ozonation for individual operation along with the combined operation have been varied with the aim of maximizing the extent of disinfection and arriving at an optimum strategy for treatment. It has been observed that nearly 75% disinfection can be achieved in about 3 h of treatment time using an optimized combination of hydrodynamic cavitation and ozonation. This combination has been found to be a cost-effective technique for achieving maximum disinfection compared to the individual operation of hydrodynamic cavitation (lower extent of disinfection) and ozonation (higher costs of treatment usually due to higher cost of ozone generation).
Keywords: Hydrodynamic cavitation; Ozonation; E. coli disinfection; Indigo method; Reactive oxidizing species;

Monitoring of sequencing batch reactor for nitrogen and phosphorus removal using neural networks by Sung Hun Hong; Min Woo Lee; Dae Sung Lee; Jong Moon Park (365-370).
The information of nutrient dynamics is essential for the precise control of effluent quality discharged from biological wastewater treatment processes. However, these variables can usually be determined with a significant time delay. Although the final effluent quality can be analyzed after this delay, it is often too late to make proper adjustments. In this paper, a neural network approach, a software sensor, was proposed for the real-time estimation of nutrient concentrations and overcoming the problem of delayed measurements. In order to improve the neural network performance, a split network structure applied separately for anaerobic and aerobic conditions was employed with dynamic modeling methods such as auto-regressive with exogenous inputs. The proposed methodology was applied to a bench-scale sequencing batch reactor (SBR) for biological nutrient removal. The extrapolation problem of neural networks was possible to be partially overcome with the aid of multiway principal component analysis because of its ability of detecting of abnormal situations which could generate extrapolation. Real-time estimation of PO4 3−, NO3 and NH4 + concentrations based on neural network was successfully carried out with the simple on-line information of the SBR system only.
Keywords: Sequencing batch reactor; Neural network; On-line monitoring; Multiway principal component analysis;

On-line monitoring of the enhanced biological phosphorus removal process using respirometry and titrimetry by A. Guisasola; M. Vargas; M. Marcelino; J. Lafuente; C. Casas; J.A. Baeza (371-379).
Enhanced biological phosphorus removal (EBPR) requires alternating anaerobic and aerobic (or anoxic) conditions to favour polyphosphate accumulating organisms (PAO) against other bacteria. Due to its economical and ecological relevance, EBPR is currently a focus of interest in many environmental labs. A sequencing batch reactor (SBR) is used in most lab-scale EBPR systems in order to enrich the sludge with PAO. The management of these reactors is mostly based on off-line measurements such as volatile fatty acids and phosphorus and, if available, internal storage polymer measurements (polyhydroxyalkanoates or glycogen). However, off-line monitoring of the SBR cycle implies low frequency data sampling and delay between sampling and availability of the results. This is an obstacle for a proper process monitoring and makes difficult the application of control strategies to the process. For this reason, the on-line monitoring of the SBR cycle would improve the daily process management, as well as facilitate the “on-time” detection of abnormal situations and the implementation of new control strategies. This work illustrates that titrimetry and respirometry provide enough information to monitor the EBPR process in lab-scale SBR. For this aim, the effect of EBPR on respirometry and titrimetry is theoretically discussed and several monitoring examples using a lab-scale SBR with a highly PAO-enriched sludge are shown.
Keywords: EBPR; Monitoring; PAO; Respirometry; Titrimetry;

In this work, an empirical correlation is proposed to describe k L a as a function of operating conditions (agitation and aeration rates) and of oil and surfactant volumetric fractions in a biotransformation medium, an oil-in-water dispersion. An interaction effect between the oil and the surfactant effects was found, since oil presence increased k L a in the absence of the surfactant but had an opposite effect when Tween 80 was available in the medium. The biotransformation of methyl ricinoleate (MR) into γ-decalactone (an aroma compound of industrial interest), by the yeast Yarrowia lipolytica, was carried out at different conditions of operation, to evaluate the influence of k L a on the production of the aroma. It was demonstrated that k L a had an influence on the aroma production; however, for the low hydrophobic substrate concentration used (1.08% v/v) and cellular density of 2.0 × 107  cells mL−1, a minimal k L a value of 70 h−1 was necessary to attain the maximal aroma production.
Keywords: Oxygen transfer; k L a; Biphasic medium; Yarrowia lipolytica; γ-Decalactone;