Biochemical Engineering Journal (v.45, #1)

BEJ Keywords (IV).

Optimization of biotechnological processes. The acetic acid fermentation. Part I: The proposed model by Jorge E. Jiménez-Hornero; Inés M. Santos-Dueñas; Isidoro García-García (1-6).
Vinegar is a food product of increasing significance by virtue of its widely variable origin and uses (particularly as a condiment or food preservative). The gastronomic value of vinegar has been appreciated for thousands of years. The growing social and economic significance of these products has fostered research into the most salient aspects of their production processes. The widespread use of submerged cultures in such processes has aroused an obvious interest in their modelling with a view to facilitating their design, control and optimization. Also, the availability of increasingly powerful utility and dedicated software tools has enabled a much rigorous approach to devising and application of more complex and accurate models for these purposes. This paper (Part I) reviews previous attempts at modelling acetic acid fermentation and proposes a new mathematical model for the process based on extensive experimental testing. The model introduces new equations and considers cell lysis during the process. Part II is devoted to study the key subject of parameter estimation and finally Part III deals with the optimization task. Though the wine vinegar process is being considered, many of the studied issues could be applied to other fermentations.
Keywords: Bioreactor systems; Kinetics; Modelling; Optimization; Acetification; Vinegar;

In part I of this series a mathematical model for acetic acid fermentation was reported. However, no kinetic model can be complete until its equation parameters are estimated. This inevitably entails a practical identifiability analysis intended to ascertain whether the parameters can be estimated in an unambiguous manner based not only on the sensitivity of the model to them, but also on the amount and quality of available experimental data for this purpose. Also, estimating the model parameters entails optimizing a specific objective function subject to the model equations as major constraints and to additional, minor constraints on variables and parameters. This approach usually leads to the formulation of a non-linear programming problem involving differential and algebraic constraints where the decision variables constitute the parameter set to be estimated. In the scope of modelling biotechnological processes, this problem is not usually dealt with in a proper way. This second paper reviews available models for practical identifiability assessment and parameter estimation with a view to their prospective application to the proposed model and its validation.
Keywords: Bioreactor systems; Kinetics; Modelling; Optimization; Acetification; Vinegar;

Optimization of biotechnological processes. The acetic acid fermentation. Part III: Dynamic optimization by Jorge E. Jiménez-Hornero; Inés M. Santos-Dueñas; Isidoro García-García (22-29).
Wine vinegar is obtained in a biotechnological process one of the crucial steps in which is the biological oxidation of the starting wine. Such a step is usually performed in a semi-continuous operation mode where a preset fraction of the culture medium is unloaded from the fermenter as product and the remainder left in it as inoculum to facilitate expeditious fermentation of the wine subsequently added to replenish the amount withdrawn. The overall performance of the fermenter can vary markedly depending on the particular operating conditions, and so can the quality of the product and the economy of the process as a result. Identifying the most suitable operating conditions therefore poses a typical optimization problem named as dynamic optimization or open-loop optimal control, which is solved by determining the time profiles for the control variables of the system in order to optimize a given cost function. Such a function represents the goal to be achieved as regards the specific needs of the problem. In Part III of this series the previously proposed model in Parts I and II has been used for addressing the dynamic optimization of the acetic fermentation process in terms of various objective functions, with special emphasis on productivity.
Keywords: Bioreactor systems; Kinetics; Modelling; Optimization; Acetification; Vinegar;

A bench-scale continuous flow system, consisting successively of an anaerobic hydrolysis column (column A), an anoxic column (column D) and two aerobic columns (columns B and F), was constructed to remove nitrogen and carbon simultaneously from terramycin crystallization mother solution (TCMS). Columns A and D were sludge-bed reactors, and columns B and F were biofilm reactors packed with polyethylene balls and soft fibers hung on plastic rings, respectively. Approximately 82% of the chemical oxygen demand (COD) and 81% total nitrogen were removed by the system when tap water diluted TCMS was continuously fed (dilution ratio, 1:4). Sulfide which was produced during anaerobic hydrolysis was used as part of electron donors for denitrification in column D. Comparison results show that porous polyethylene balls had a better COD and NH4 +-N removal performance than the fiber type biocarrier.
Keywords: Antibiotics wastewater; Simultaneous removal of COD and nitrogen; Nitrification; Denitrification;

Simplified denitrification system using ethanol released from non-porous polyethylene-film bag by Hiroaki Uemoto; Masahiro Suzuki; Masahiko Morita; Atsushi Watanabe; Hiraku Shinozaki (35-40).
A holding bag fabricated using non-porous low-density polyethylene (LDPE) film was examined to simplify a denitrification system for nitrate-containing wastewater without organic carbon. The LDPE-film bag filled with ethanol released ethanol slowly. The amount of the released ethanol could be controlled by changing the film thickness. A dual bag was constructed with an outer non-woven fabric bag on which Paracoccus denitrificans cells were immobilized and the inner LDPE-film bag filled with ethanol. When the dual bag was immersed in a nitrate solution under agitation of N2 gas or air, the nitrate content of the solution decreased irrespective of the agitating conditions. The maximum nitrogen removal rate was 5.94 g N d−1  m−2 of the outer bag's surface. This value corresponded to the volumetric nitrogen removal performance of 0.1 kg N m−3  d−1, though the packed ratio of the dual bag in the solution was below 10%. The dual bag could remove nitrogen from nitrate solution without an additional apparatus. The bioreactor using the dual bag would be extremely effective as an additional denitrification system, since they can be easily installed in the vacant spaces of various tanks.
Keywords: Denitrification; Wastewater treatment; Immobilized cell; Nitrate; Bioreactor; Polyethylene film;

Efficient oxygen transfer by surface aeration in shaken cylindrical containers for mammalian cell cultivation at volumetric scales up to 1000 L by Xiaowei Zhang; Cédric-Alain Bürki; Matthieu Stettler; Dario De Sanctis; Marco Perrone; Marco Discacciati; Nicola Parolini; Maria DeJesus; David L. Hacker; Alfio Quarteroni; Florian M. Wurm (41-47).
Cylindrical containers agitated by orbital shaking are being developed as simple and cost-effective bioreactor systems for the cultivation of mammalian cells. Here the oxygen transfer capacities of containers with nominal volumes from 50 mL to 2000 L were determined, and the operating parameters influencing oxygen transfer were investigated. In general, the shaking speed necessary for efficient oxygen transfer diminished as the container size increased. At shaking speeds suitable for the growth of shear-sensitive cells, k L a values between 10 and 30 h−1 were typically achieved in small-scale (<1 L nominal volume) containers at shaking speeds above 120 rpm. A k L a value of 8 h−1 was measured at 75 rpm in a 200-L container with a working volume that was 50% of the nominal volume. In a 2000-L container with a working volume of 1000 L, a moderate k L a of 3 h−1 was obtained with a shaking speed of only 47 rpm. The free-surface area in 50-mL and 30-L containers was determined by photographic image analysis and computational fluid dynamic (CFD) simulation, respectively. The results showed that the increase in k L a at higher shaking speeds was mainly due to an increased k L value, highlighting the dominant effect of free-surface turbulence on gas transfer in orbitally shaken containers. The results demonstrated the feasibility of orbital shaking technology for the cultivation of mammalian cells at scales up to 1000 L.
Keywords: Bioreactor; Agitation; Animal cell culture; Oxygen transfer; Scale-up; Aeration;

Polyacrylonitrile (PAN) nanofibrous membranes were prepared by electrospinning and activated by amidination reaction for immobilizing Candida rugosa lipase with covalent binding. The diameters of initial PAN nanofibers ranged from 150 to 300 nm. The lipase, which aggregated and formed small particles that attached onto the nanofiber surfaces, was observed using field emission scanning electron microscopy. The activity retention of the immobilized lipase was 87.5% of the free enzyme. The kinetic parameters, K m and V max, of the immobilized lipase were determined to be 0.548 mM and 31.2 U/mg, respectively. The effects of oil-to-water ratio, temperature, and pH on hydrolysis of soybean oil were investigated to determine the optimal reaction conditions. Under these optimal reaction conditions, the hydrolysis conversion of soybean oil was 72% after 10 min and 85% after 1.5 h. In examining reusability, the immobilized lipase retained 65% of its initial conversion after 20 additional batch reactions. These results suggest that the proposed scheme for immobilization of lipase has potential in industrial applications for oil hydrolysis.
Keywords: Nanofibrous membrane; Electrospinning; Lipase; Immobilized; Kinetic parameter; Hydrolysis;

Removal of 2,4-dichlorophenol by chitosan-immobilized laccase from Coriolus versicolor by Jianbo Zhang; Zhenqiang Xu; Hui Chen; Yueru Zong (54-59).
Laccase from Coriolus versicolor was immobilized on chitosan using glutaraldehyde as a cross-linking agent. After immobilization, laccase retained 52.2% of its original activity and was used to study 2,4-dichlorophenol (2,4-DCP) removal from aqueous solutions. The optimum pH for 2,4-DCP removal by the immobilized laccase was ∼5.5, which was lower than the optimal pH of 6.0 for free laccase catalysis. Immobilized laccase also had an atypically wide optimum temperature range for catalysis of 35–45 °C. Immobilized laccase could be used repeatedly, and its removal efficiency for 2,4-DCP remained above ∼50% for up to six usages.
Keywords: Immobilization; Enzyme activity; Catalysis; Optimum condition;

A surface with collagen type I coating was used as a tool mimicking three dimensional environment for studying the behaviors of rabbit chondrocyte aggregates found in an early culture phase. The morphology of cell aggregates was evaluated quantitatively in terms of a morphology-relating parameter of specific branching factor, Z s, determined under varied culture conditions. Branching in aggregates increased along with proceeding of culture from 5 to 6 days when seeded at a lower density of X 0  = 1.0 × 103  cells/cm2, giving Z s  = 0.24 tips/cell at 6 days, the value of which was 1.7 times than that obtained from the culture with a higher seeding density of X 0  = 1.0 × 104  cells/cm2 (Z s  = 0.14 tips/cell). This value significantly reduced to Z s  = 0.13 tips/cell in the culture with X 0  = 1.0 × 103  cells/cm2 by using the conditioned medium prepared from the foregone culture with X 0  = 1.0 × 104  cells/cm2, inducing the morphological architecture and collagen type II production of aggregated cells with the similarities to those in the standard culture seeded at 1.0 × 104  cells/cm2. The mRNA expressions of differentiation-marker genes (collagen types I and II) were confirmed to be regulated in accordance with the morphological analyses as well as the extracellular matrix formation of aggregates in the both cultures conducted with the standard and conditioned media. The present study suggested that soluble factor(s) secreted from the chondrocytes at the higher seeding density modulates the cell aggregation and the state of chondrogenic differentiation through encouraging cell–cell communications.
Keywords: Collagen-coated surface; Chondrocyte behaviors; Conditioned medium; Aggregate formation; Cell differentiation; Image analysis;

From our previous report, the speedy removal of some n-alkanes or branched-alkanes by Rhodococcus erythropolis NTU-1 was already demonstrated in aqueous MS medium. Therefore, this strain was further tested under saline conditions, as well as in pure sea water. Hexadecane of 2000 ppmv was used as a model carbon source in this report. Under saline conditions, the biodegradation was again confirmed with the cell aggregates formation during the cultivation. The formation of biofloccules, trapping significant amount of alkane, provides an easy physical removal of alkane, in addition to biodegradation. At an initial NaCl concentration 1.2% and 2.4%, 80–90% removal of alkane was achieved within 56 h and 65% removal at 3.6% NaCl concentration within 68 h. Addition of 0.24 g/L nutrient broth (NB) in the medium would further promote the biodegradation rate and cell aggregation phenomenon during the process and more than 80–95% removal of alkane was achieved within 56–68 h. In the case of sea water, alkane removal was about 50% after 140 h and 65% with NB addition. The results suggest that strain NTU-1 has considerable ability for the bioremediation processes of n-hexadecane in a marine environment.
Keywords: Aerobic digestion; Bioremediation; Biosorption; Shake-flask; Rhodococcus erythropolis; Sea water treatment;

Characterization of bacterial population of raw milk from bovine mastitis by culture-independent PCR–DGGE method by Ying Kuang; Kaori Tani; Aidan J. Synnott; Kazuhito Ohshima; Hidetoshi Higuchi; Hajime Nagahata; Yasunori Tanji (76-81).
Culture-independent PCR–DGGE fingerprinting was used to reveal the bacterial composition and diversity associated with raw milk of mastitis-infected cows from Hokkaido, Japan for the first time. All the mastitic milk samples were diagnosed as solely infected by coliforms using the classical microbiological method following on-farm culturing. The variation in community structure between each sample observed in our results indicated that the bovine mastitis-associated bacteria were host-specific. Klebsiella pseudomoniae, Lactococcus lactis, Staphylococcus aureus and members of the Escherichia genus were found to be widely distributed. Furthermore, more than one pathogen known to cause mastitis was found to be present in some milk samples. These pathogens are not only potential etiological agents but may also play a role in disrupting the natural microbial ecology in mastitic cows. This finding highlights the limitation of the traditional identification and characterization strategy, and the PCR–DGGE are shown to be a powerful tool for describing the bacterial flora and especially etiological agents in mastitic milk.
Keywords: Bovine mastitis; PCR–DGGE; Raw milk; Microbial population;

Biomolecule–metal interactions: Applications in extraction and separation techniques by Kamalika Roy; Kalpita Ghosh; Anupam Banerjee; Debashis Mukhopadhyay; Susanta Lahiri (82-85).
We have isolated and characterized an alkaloid, piperine and a protein arachin, from black pepper and groundnut respectively. Interactions of these compounds with various metal ions in trace scale from different domains of the periodic table were studied using radiometric method. It is revealed that piperine has high selectivity and specificity towards gold. The protein arachin shows high specificity towards mercury only. The high selectivity of these two bioreagents for the specific metal ions Au and Hg show that the bioreagents have considerable potential to replace the synthetic chemicals and sometime they are superior to synthetic one.
Keywords: Heavy metals; Arachin; Piperine; Adsorption; Extraction; Biosorption;