Biochemical Engineering Journal (v.25, #3)

IFC (CO2).

BEJ Keywords (IV).

Specific growth rate determines the molecular mass of the alginate produced by Azotobacter vinelandii by Rubén Priego-Jimenéz; Carlos Peña; Octavio T. Ramírez; Enrique Galindo (187-193).
A mutant strain (SML2) of Azotobacter vinelandii, unable to produce alginases, was grown in exponentially fed-batch cultures under constant dissolved oxygen tension and using washed-cells inocula. Using this culture technique, a pseudo-stationary steady state was achieved at specific growth rates (μ) of 0.03 ± 0.009, 0.06 ± 0.013 and 0.09 ± 0.012 h−1. A batch culture, which developed a μ of 0.21 h−1, was also performed. The mean molecular mass (MMM) of the alginate increased as μ decreased, and this dependence was particularly pronounced at μ below 0.09 h−1. The MMM obtained at 0.03 h−1 was 15 times higher (1300 ± 150 kDa) than in batch culture (95 ± 8 kDa). At the lowest μ tested (0.03 h−1  ± 0.009 h−1), the alginate yield on biomass (Yp/x) was about twice that observed at μ above 0.06 ± 0.013 h−1. This study shows that the MMM of bacterial alginate can be maximized by a simple strategy of growth rate control. Furthermore, production of high MMM alginates might be a bacterial response to cope with substrate shortages.
Keywords: Alginate; Fed-batch culture; Molecular weight; Specific growth rate;

Production of Acinetobacter radioresistens lipase with repeated fed-batch culture by Chen-You Li; Shu-Jen Chen; Chu-Yuan Cheng; Teh-Liang Chen (195-199).
Acinetobacter radioresistens lipase was produced with repeated fed-batch culture using a balanced medium based on Tween 80 as the carbon source. A constant cell concentration was shown to be a prerequisite to extend the number of repeated cycles, and an adequate cell growth rate was critical for a high lipase yield. The pH-stat feeding was found to sustain cell growth at a rate too high to effect the lipase production. The DO-stat feeding, on the other hand, could be manipulated to allow adequate growth rate for efficient lipase production. The lipase production rate obtained could reach 42,000 U/h in a 2.5 l tank fermentor.
Keywords: Lipase fermentation; Acinetobacter radioresistens; Tween 80; Repeated fed-batch culture;

Aerobic granules with mean radius of 0.25 mm were cultivated in a sequencing batch reactor (SBR) fed with soybean-processing wastewater, and the gas holdup and oxygen transfer were investigated for this granule-based reactor. At the discontinuous point in the variation of gas holdup with superficial gas velocity at 1.73 cm/s, bubble flow regime was changed into churn-turbulent flow one. Gas holdup of the granule-based SBR was greater than that of a floc-based SBR, indicating that higher oxygen transfer efficiency could be achieved in the former. The volumetric gas–liquid oxygen transfer coefficient increased with increasing superficial gas velocity, but declined with increasing solid fraction. Observed Thiele modulus and effectiveness factor were effectively used to describe the efficiency of oxygen diffusion in the granules and the oxygen utilization rate of the reactor. With the porous pellet model, the oxygen concentration profiles and effectiveness factor for aerobic granules with different sizes were predicted. Effectiveness factor of 0.98 for the granules with radius of 0.25 mm suggests that the oxygen diffusion limitation could be neglected. For the granules with radius of 1.00 mm, a severe oxygen limitation occurred, as indicated by an effectiveness factor of 0.55.
Keywords: Aerobic granule; Effective diffusivity; Effectiveness factor; Gas holdup; Oxygen transfer coefficient;

Improved dynamic analysis on cell growth with substrate inhibition using two-phase models by Tsuey-Ping Chung; Cheng-Ying Wu; Ruey-Shin Juang (209-217).
A simple two-phase model, originated from the Haldane model, was presented to predict the behavior of batch culture operations, in which the substrates show an inhibition effect on biomass growth. The model was based on the two regions of metabolic activity: the lag phase and the log phase. The validity of this model was experimentally verified with respect to the growth of freely suspended Pseudomonas putida CCRC 14365 during the biodegradation of phenol. In contrast to the one-phase Haldane model, it was demonstrated that the proposed two-phase Haldane model much better predicted the dynamics of biomass growth including the transient region from the lag to the log phases.
Keywords: Two-phase model; Cell growth; Pseudomonas putida; Phenol biodegradation; Substrate inhibition;

The extraction of papain (MW 23 kDa, pI 9.6) from water was studied using reverse micelles containing sodium bis(2-ethylhexyl)sulfosuccinate (AOT). Forward extraction of papain was carried out in the pH range of 4.0–6.5 and a maximum extraction of 65–70% was observed at pH 6.3. Papain extracted into the micelles could be back extracted to only 30% into a conventional aqueous phase (high pH and high KCl concentrations). In order to improve the back extraction of papain, 8–10 vol% alcohols were added because it was known that alcohols have an effect on the micelle–micelle interactions. However, this way resulted in the loss of papain activity inspite of good back extraction yields (80–90%). The back extraction of papain encapsulated in AOT reverse micelles was then carried out by adding a counter-ionic surfactant tri-n-octylmethyl-ammonium chloride (TOMAC). This technique resulted in faster back extraction compared to conventional method. Around 80–90% of activity recovery was obtained after back extraction. The improved backward transfer was likely caused by electrostatic interaction between the oppositely charged surfactant molecules, which led to the collapse of reverse micelles.
Keywords: Reverse micelle; Back extraction; AOT; Papain; Activity recovery; TOMAC;

Pb(II) ions were found to be accumulated extracellularly on the surface of Botrytis cinerea. The rate and extent of accumulation were affected by pH, contact time and initial Pb(II) ion concentrations. The Pb(II) sorption capacities of heat inactivated, detergent, NaOH, DMSO and AcOH pretreated B. cinerea cells were determined as 107.10 ± 1.87, 57.50 ± 2.42, 51.73 ± 1.19, 46.63 ± 3.22 and 30.00 ± 2.23 mg/g, respectively, at initial Pb(II) ion concentration of 350 mg/dm3 and optimum conditions of pH 4.0 and contact time of 90 min. The biosorbent was regenerated using 10 mM HCl solution, with up to 97% recovery and reused five times in biosorption–desorption cycles successively. The influence of Cu(II), Cd(II) and Ni(II) co-cations on Pb(II) biosorption capacity of heat inactivated B. cinerea biomass in binary and multimetal systems was evaluated and biosorption capacity of the Pb(II) ions was found to be reduced by the presence of the other competing metal ions. Langmuir adsorption isotherm model was used to describe the biosorption of Pb(II) ions by B. cinerea. The nature of the possible cell–metal ions interactions was also evaluated by FTIR, SEM and EDAX analysis. These examinations indicated the involvement of –COOH, –OH and –NH groups in the biosorption process and that Pb(II) ions were accumulated as crystals looking like “billiard balls” over the surface of B. cinerea cells.
Keywords: Biosorption; Botrytis cinerea; Pb(II); Langmuir isotherm; Competitive biosorption;

Biosoftening of arecanut fiber for value added products by Akhila Rajan; Jayalakshmi Gopinadha Kurup; Tholath Emilia Abraham (237-242).
The arecanut husk fibers are predominantly composed of cellulose and varying proportions of hemicellulose, lignin, pectin and protopectin. Microbes that selectively remove lignin without loss of appreciable amounts of cellulose and fiber strength properties are extremely attractive for the biosoftening of arecanut fiber. The fungal growth occurs during the first 72 h of incubation and by one week, enzyme production reaches the maximum level, thereby improving color and softness of the fiber. Maximum MnP production of 233.2 IU ml−1 was observed at pH 7.0 for Phanerochaete chrysosporium and a maximum activity of 221.8 IU ml−1 was observed at pH 5.0 for Phanerochaete strain. Maximum improvement of color (brightness) and softness and maximum MnP production of 321.3 IU ml−1 was achieved in a medium lacking both carbon and nitrogen for P. chrysosporium whereas maximum improvement in color and softness and MnP production of 341.2 IU ml−1 was achieved in a medium lacking only carbon for Phanerochaete sp. The fiber treated with P. chrysosporium showed 35.1% and Phanerochaete sp. showed 25.7% increase in strength when compared to the untreated fiber. Elongation of the fiber treated with P. chrysosporium was 5.0% and that treated with Phanerochaete sp. was 4.2% compared to the control fiber which had an elongation of 3.7%. The biosoftened arecanut fibers can be exploited commercially for the production of furnishing fabrics, textiles etc by blending with cotton, viscose and polyester.
Keywords: Biosoftening; Lignin; Cellulose; Filamentous fungi; Arecanut fibers;

Kinetics of disinfection of Escherichia coli by catalytic ultrasonic irradiation with TiO2 by Mahmoud Farshbaf Dadjour; Chiaki Ogino; Susumu Matsumura; Nobuaki Shimizu (243-248).
The kinetics of disinfection of Escherichia coli, in the presence of a TiO2 photocatalyst, using an ultrasonic irradiation system was investigated. TiO2 was found to significantly improve the disinfection process. A 98% reduction in the concentrations of viable cells was obtained in the presence of TiO2 during a 30 min period of irradiation, while only a 13% reduction was observed when an ordinary ultrasonic irradiation system was used. The rate of cell killing was also higher in the presence of TiO2 compared with Al2O3. The rate of disinfection was proportional to the amount of TiO2 in the concentration range examined. Cell concentrations were decreased by an order of 5 within 10 min of irradiation in the presence of 2.0 g/ml TiO2. No significant effect of cell concentration on the cell-killing process was found in the range of 103 to 107  CFU/ml. The mechanism of cell killing was further investigated by examining the effects of OH radical scavengers, such as histidine and glutathione. The rate of disinfection was decreased in samples containing these radical scavengers, thus indicating that radicals are of major importance in this process.
Keywords: Cell disruption; Ultrasonic irradiation; Titanium dioxide; Hydroxyl radical; E. coli; Disinfection kinetics;