Biochemical Engineering Journal (v.6, #1)

Culture of red beet hairy roots by considering variation in sensitivity of tip meristems to hydraulic stress by Yoshio Hitaka; Yusuke Takahashi; Masahiro Kino-oka; Masahito Taya; Setsuji Tone (1-6).
In the culture of red beet hairy roots in shaking flasks, a period for acclimation without lateral root generation existed at the early stage, and the root tip meristems containing growing points (GPs) were found to be damaged under an elevated shear stress condition. The loading experiments of shear stress to the hairy roots revealed that the GPs subjected to the acclimation acquired tolerance to shear stress, retaining relatively high viability of GPs up to 0.6 N/m2 of loaded shear stress. Next, the hairy roots after culture for 50 h at 0.05 N/m2 of shear stress were exposed to conditions at various levels of shear stress in a single column reactor, and a relatively high growth rate was obtained in the vicinity of 1.0 N/m2 of shear stress. According to these results, two-stage cultures of hairy roots were then performed, which was comprised of a first stage for 50 h at 0.05 N/m2 of shear stress for the prevention of decay of the GPs caused by hydraulic stress and a second stage for 110 h at 1.0 N/m2 of shear stress for active elongation of the GPs with sufficient nutrient supply by regulation of the medium flow rate. The cell concentration ultimately reached 7.6 kg dry cells/m3, although no growth was observed in the case where the hairy roots did not undergo the first stage.
Keywords: Plant cell culture; Plant cell bioreactors; Packed bed bioreactors; Viability; Red beet hairy roots; Shear stress effect;

α-Amylase immobilized on bulk acoustic-wave sensor by UV-curing coating by Deliang He; Yan Cai; Wanzhi Wei; Lihua Nie; Shouzhuo Yao (7-11).
A new method for immobilization of α-amylase by UV-curing coating is proposed in this paper. The immobilization procedure of UV-curing coating on piezoelectric quartz crystal is simple and convenient, and causes less loss of enzymatic activity. The activity of the immobilized α-amylase is monitored by a technique based on bulk acoustic-wave (BAW) sensor. The frequency shift of BAW sensor can reflect the degree of hydrolysis of starch by the immobilized α-amylase. It is appropriate for the immobilized α-amylase to hydrolyze the soluble starch under pH 7.0 condition, which is similar to that of the free α-amylase. Kinetic parameters (the Michaelis constant, K m, and the maximum initial rate V max) of the enzymatic hydrolysis of starch by the immobilized α-amylase are estimated by using a linear method of Lineweaver–Burk plot. K m=12.7 mg ml−1 and V max=15.9 Hz min−1. And the experimental results show that the immobilized α-amylase entrapped by the UV-curing coating retains adequate enzymatic activity and can be reused more than 50 times under certain experimental conditions.
Keywords: α-Amylase; Immobilization; UV-curing coating; Bulk acoustic-wave sensor;

We have studied the autotrophic growth of the marine microalga, Isochrysis galbana Parke, in a batch photobioreactor, comparing five different culture media and analysing the influence of each on growth kinetics as well as on the fatty-acid composition and protein content of the biomass. All the experiments were performed at 15°C, with the culture medium at pH 8.0, a specific rate of air supply of 1 v v−1  min−1 and a continuous illumination of 40–43 W m−2. The results show no parallel between good nutritional characteristics and high values of the kinetic parameters. Nevertheless, a compromise between the nutritional factors and growth kinetics could be provided by Ukeles medium, which provided a biomass with a good composition in polyunsaturated fatty acids (quotient n3/n6=3.2), an adequate protein content (25.3%) and relatively high values, although not the highest registered, for maximum specific growth rate (μ m=0.018 h−1) and biomass productivity (1.9×10−3  kg m−3  h−1).
Keywords: Microalgae; Photobioreactor; Isochrysis galbana; Fatty acids; Protein; Culture medium;

Lysozyme separation by hollow-fibre ultrafiltration by Raja Ghosh; Saliya Sudarshana Silva; Zhanfeng Cui (19-24).
This paper discusses the purification of lysozyme from chicken egg white using hollow-fibre ultrafiltration (30 kDa MWCO, polysulphone membrane). Lysozyme is preferentially transmitted through the membrane while the membrane largely retains other egg white proteins. Improvement in system hydrodynamics resulted in an increase in permeate flux while lysozyme transmission remained unaffected, leading to higher productivity. The percentage purity of lysozyme obtained was generally insensitive to system hydrodynamics. The permeate flux and productivity increased with increase in transmembrane pressure (TMP) before levelling off around 0.7 bar. However, the TMP did not have any pronounced effect on the transmission and the purity of lysozyme. Experiments carried out in the diafiltration mode showed that moderately pure lysozyme (80–90%) could be obtained in an extended operation.
Keywords: Lysozyme; Protein fractionation; Separation; Ultrafiltration; Hollow fibres; Purification;

Single versus multiple bioreactor scale-up: economy for high-value products by S.A Rouf; M Moo-Young; J.M Scharer; P.L Douglas (25-31).
The economy of scaling-up a bioreactor by increasing the number of units was investigated with respect to an integrated flowsheet. For the production of t-PA from animal cells, a base case flowsheet using a single large bioreactor was compared to a multiple bioreactor case. Simulation of the complete flowsheets for the two cases showed that a multiple bioreactor approach to scale-up increases the return of investment (ROI) of the base process by 122%. This enormous increase in ROI results from the smaller size of the downstream units compared to the base case, since downstream processing accounts for about 80% of the total cost for high value products like t-PA. Proper scheduling of the downstream units allowed sharing of the equipment by the bioreactors. A breakdown of the equipment purchase cost showed that cost related to cell culture equipment increased from 14% for the base case to about 37% for the multiple bioreactor case. The contribution from chromatography columns to the total equipment purchase cost, on the other hand, decreased from 52 to 33%.
Keywords: Bioreactor scale-up; Integrated flowsheet; Economies of scale;

Studies were carried out for the production of aroma compounds in solid-state fermentation using factorial design and response surface methodology (RSM) experiments. Five agro-industrial residues were evaluated as substrate for cultivating a strain of Kluyveromyces marxianus. The results proved the feasibility of using cassava bagasse and giant palm bran (Opuntia ficus indica) as substrates to produce fruity aroma compounds by the yeast culture. In order to test the influence of the process parameters on the culture to produce volatile compounds, two statistical experimental designs were performed. The parameters studied were initial substrate pH, addition of glucose, cultivation temperature, initial substrate moisture and inoculum size. Using a 25 factorial design, addition of glucose and initial pH of the substrate was found statistically significant for aroma compounds production on palm bran. Although this experimental design showed that addition of glucose did not have a significant role with cassava bagasse, 22 factorial design revealed that glucose addition was significant at higher concentrations. Head-space analysis of the culture by gas chromatography showed the production of nine and eleven compounds from palm bran and cassava bagasse, respectively, which included alcohols, esters and aldehyde. In both the cases, two compounds remained unidentified and ethyl acetate, ethanol and acetaldehyde were the major compounds produced. Esters produced were responsible for the fruity aroma in both the cases. With palm bran, ethanol was the compound produced in highest concentration, and with cassava bagasse (both supplemented with 10% glucose), ethyl acetate was produced at highest concentration, accumulating 418 and 1395 μmol l−1 head-space g−1 substrate in 72 h, respectively.
Keywords: Kluyveromyces marxianus; Solid-state fermentation; Flavour compounds; Cassava bagasse; Palm bran;

The overall diffusion coefficients for several low molecular weight solutes, such as glucose, fructose, sucrose, lactose, and vitamin B12 have been determined in Ca-alginate membrane liquid-core capsules using the unsteady-state method following the release of solutes from the capsules to a well-stirred solution of limited volume. The diffusion coefficients obtained for saccharides were 5–20% lower than the corresponding diffusivity in water while for vitamin B12 about 50% that of water. The diffusion coefficients of the investigated capsules were not influenced by the change in alginate concentration in the capsule membrane from 0.5 to 1.0%. Lower diffusivities and higher deviations from the diffusivity in water were obtained for higher molecular weight solutes.
Keywords: Alginate; Diffusion; Glucose; Hydrogel capsule; Immobilisation; Mass transfer;

Phenol biodegradation by Pseudomonas putida DSM 548 in a batch reactor by Álvaro A.M.G Monteiro; Rui A.R Boaventura; Alı́rio E Rodrigues (45-49).
Phenol biodegradation in a batch reactor using a pure culture of Pseudomonas putida DSM 548 was studied. The purpose of the experiments was to determine the kinetics of biodegradation by measuring biomass growth rates and phenol concentration as a function of time in a batch reactor. The Haldane equation μ=μ m S/((K s+S+S 2)/K i) adequately describes cell growth with kinetic constants μ m=0.436 h−1, K s=6.19 mg l−1, K i=54.1 mg l−1. These values are in the range of those published in literature for pure or mixed cultures degrading phenol.
Keywords: Phenol biodegradation; Pseudomonas putida DSM 548; Batch reactor; Haldane equation;

This paper combines Sturm’s method with the tangent analysis method to solve a biochemical reaction involving multiplicity. This method can easily derive the necessary conditions for multiplicity. In addition, we find a starting bifurcation point for multiplicity which cannot be obtained by the tangent method alone. Moreover, a start-up strategy is suggested to obtain a high conversion and unique steady state in four selected kinetic models of biochemical reactions, with inhibition.
Keywords: Biochemical reaction; Multiplicity; Start-up; Inhibition;

Microbial production of 2-chloro-α-methylbenzyl alcohol and its adsorptive recovery by Yuri Hasegawa; Shuji Adachi; Ryuichi Matsuno (59-64).
An adsorptive process was combined with yeast-mediated production of chiral 2-chloro-α-methylbenzyl alcohol (o-Cl-1-PA) for effective product recovery and reuse of the reaction medium. Low temperature was suitable for long-term reactor operation, and continuous production using a shallow-bed reactor was achieved for at least 22 days while maintaining a high conversion. The appropriate size of the adsorption column for product recovery from the reactor effluent was estimated through measurement of breakthrough curves of o-Cl-1-PA in a packed bed of the resin at various adsorbate concentrations and feed flow rates. Using the adsorption column, 98% of the product and the residual substrate were recovered from the reactor effluent, and the effluent from the adsorption column was successfully reused as the reaction medium after microfiltration to save the medium consumption.
Keywords: Asymmetric reduction; Bioconversion; Chiral systems; Immobilized cells; Packed bed bioreactor; Yeast;

In order to characterize the contributions of respiratory and photosynthetic actions to energy conversions, the mixotrophic cells of Marchantia polymorpha were cultivated in the medium containing 10 kg/m3 glucose as an organic carbon source. The cultures were conducted with the supply of ordinary air (0.03% CO2) at constant incident light intensities of 50 and 180 W/m2. From the results of metabolic analysis, it was found that the cell yield based on ATP synthesis was estimated to be 6.3×10−3  kg-dry cells/mol-ATP in these cultures. Under the examined conditions, energy conversion efficiency through respiration was larger than that through photosynthesis, and efficiency of overall energy conversion to ATP was maximized when the sum of energies from glucose and light captured by the cells was ≈7.2×105  J/(h kg-dry cells). Taking into account the efficiency of overall energy conversion, a batch culture of M. polymorpha in a bioreactor was carried out by regulating incident light intensity ranging from 9 to 58 W/m2. In the culture with light regulation, the cell yield of 6.2×10−9  kg-dry cells/J was achieved on the basis of energy provided to the system throughout the culture, and this value was 2.3 and 9.3 times as large as those obtained in the cultures under constant incident light intensities of 50 and 180 W/m2, respectively.
Keywords: Plant cell culture; Bioreactors; Photosynthesis; Marchantia polymorpha; Metabolic flux analysis; Energy conversion efficiency;

Viability of plant hairy roots is sustained without propagation in low sugar medium kept at ambient temperature by Hirofumi Nagatome; Takeo Yamamoto; Masahito Taya; Nobukazu Tanaka (75-80).
The effect of sucrose concentration in the medium on the growth and resumption ability to form lateral roots was investigated using the hairy roots of pak-bung and tobacco. It was found that the growth evaluated by root tip elongation of pak-bung and tobacco hairy roots was suppressed in the medium having an initial sucrose concentration of <2.5 kg/m3, and that the resumption abilities of both the hairy roots could be preserved when the hairy roots were kept at an initial sucrose concentration of 2.5 kg/m3 under ambient temperature conditions. The values of maintenance energy for pak-bung and tobacco hairy roots were determined to be 0.11 and 0.12 per day, respectively, from the total sugar consumption rates. Under the oligotrophic condition of the sucrose concentration of 2.5 kg/m3, the hairy roots were considered to exist as resting cells with maintenance metabolism, and the minimum demand for the energy source to ensure survival of the cells was met because the cells hardly multiplied and sugar consumption was not significant. In addition, long-term storage of pak-bung hairy roots in the liquid medium with 2.5 kg/m3 sucrose was performed at 25°C. It was demonstrated that the hairy roots could maintain their resumption abilities without a serious loss of viability over 600 days and that the number of budding lateral roots per unit length of the main roots remained a value of 72 roots/m after the 600-day storage.
Keywords: Plant cell culture; Long-term storage; Hairy roots; Tobacco; Pak-bung; Low sugar concentration;