Biochemical Engineering Journal (v.31, #1)

BEJ Keywords (II).

Optimization of culture conditions for CO2 fixation by a chemoautotrophic microorganism, strain YN-1 using factorial design by Kyung-Oh Kwak; Soo-Jung Jung; Seon-Yong Chung; Chang-Min Kang; Yang-il Huh; Sang-Ok Bae (1-7).
Culture conditions for CO2 fixation by a chemoautotrophic microorganism, strain YN-1, were optimized by statistical analysis using factorial design. Statistical analysis was performed by SAS software and then predicted the levels of four factors, H2 (X 1), O2 (X 2), CO2 (X 3) concentration and pH (X 4) which were required to obtain the optimum culture conditions. For the fractional factorial design 34−1, total 27 experimental runs by combination of each factor were carried out in a 5 L-jar fermentor, and the three-dimensional response surfaces were plotted to find out the optimum level of each factor for maximum CO2 fixation. Dry cell weight in optimized culture condition was predicted as 11.4 g/L in 48 h under the condition of 37.5 × 10−3  mol/min H2, 9.38 × 10−3  mol/min O2, 4.91 × 10−3  mol/min CO2, and pH 6.7. As compared with CO2 fixation rate before optimizing culture conditions, the improvement of CO2 fixation rate under optimized culture conditions was experimentally confirmed.
Keywords: Chemoautotrophic microorganism; CO2 fixation; Dry cell weight; Factorial design; SAS software;

Activity and stability of caffeine demethylases found in Pseudomonas putida IF-3 by Juan G. Beltrán; Richard L. Leask; Wayne A. Brown (8-13).
Resting cell suspensions and cell-free extracts of Pseudomonas putida IF-3 were tested to assess their ability to degrade caffeine, and to determine their capacity to retain activity at different temperatures. A method to quantify cell lysis using optical density was developed in order to allow the comparison of cell free extract and resting cell caffeine degradation rates on the same basis. Caffeine degradation rates for cell free extracts were found to be 2.4 μmol g cells−1  min−1; this rate is 55 times faster than previously reported P. putida data. Resting cells degraded caffeine 12 times faster than cell free extracts, at 22 °C. However, both systems were equivalently active at 50 °C. Resting cells were significantly more stable than cell free extracts, retaining their ability to degrade caffeine even at elevated temperatures. Cell free extracts lost all activity after 15 min at 55 °C.
Keywords: Caffeine; Decaffeination; Enzymes; Enzyme activity; Enzyme deactivation; Cell disruption;

Kinetic modelling of the thermal and pH inactivation of the free and an immobilized form of the β-galactosidase from Thermus sp. strain T2 has been carried out. A lacteous buffer containing 50 g L−1 lactose and a commonly used phosphate buffer 50 mM pH 7.2 were employed for the thermal and the pH inactivation studies, respectively. Temperatures changed between 60 and 90 °C; acid pH range from 3 to 5 and basic pH from 10 to 13. Thermal inactivation kinetics depended on the form of the enzyme: from 60 to 90 °C the free enzyme followed a two first-order reactions in series pathway while the immobilized enzyme shows that behaviour from 80 to 90 °C; at lower temperatures, a one first-order reaction pathway suffices. In both cases, the final enzyme retained a certain activity, higher in the case of the immobilized enzyme from 60 to 70 °C. At acid pH, biphasic or simple exponential (one first-order reaction) trends, both leading to an inactive species, were observed. In basic conditions, a kinetic model chosen was based on a two first-order reactions in series scheme. Kinetic parameters values reflect the stabilization got by immobilization, specially in acid conditions, which could be of interest for the industrial treatment of acid whey. The trend of the kinetic parameters in the pH inactivation was studied: hyperbolic or sigmoid trends with concentration of protonic or hydroxide species were observed. Kinetic model selection was performed by statistically robust multivariable non-linear regression techniques.
Keywords: β-Galactosidase; Immobilization; Thermus sp. T2; Stability; pH; Temperature; Kinetics;

The efficient release of intracellular β-galactosidase by ultrasonic disruption of cells treated with aqueous solutions of different pH has been investigated to improve the cavitational efficacy of the cell disruption process. Pretreatment of the cells at different pHs for various time intervals was studied. The maximum yield (95 ± 3 U/ml) was obtained by pretreatment of the cells at pH 4.4 for 6 h and subsequent ultrasonic cell disruption for 40 min. The maximum yield on ultrasonic disruption without pretreatment was 7.2 ± 0.6 U/ml. The energy efficiency of the disruption process for releasing β-galactosidase using different pre-treatments has been calculated and compared.
Keywords: Cell disruption; pH treatment; Cavitation; Enzyme release;

Enzymatic hydrolysis of castor oil: Process intensification studies by Meenal S. Puthli; Virendra K. Rathod; Aniruddha B. Pandit (31-41).
The usual methods of castor oil hydrolysis give impure product, i.e. ricinoleic acid. An alternative technique for production is the enzymatic hydrolysis of castor oil where the product is available as a light colored and odorless product. Usually lipase catalyzed enzymatic hydrolysis has been carried out in oil in water emulsions, which require rather high quantities of enzymes limiting the industrial scale operation due to very high enzyme costs. With an aim of finding the solution to this problem, the lipase catalyzed enzymatic hydrolysis reactions have been carried out with water in oil type emulsion where optimum utilization of enzyme can be obtained. The various parameters such as time study, reusability, non-aqueous to aqueous phase ratio, effects on interfacial area of the two phases and effect of enzyme dosing were studied in order to find out the optimum conditions for maximum castor oil hydrolysis.
Keywords: Castor oil; Ricinoleic acid; Lipase; Enzymatic hydrolysis; Immobilization; Product inhibition;

Different approaches to improving the textile dye degradation capacity of Trametes versicolor by Sílvia Romero; Paqui Blánquez; Glòria Caminal; Xavier Font; Montserrat Sarrà; Xavier Gabarrell; Teresa Vicent (42-47).
Trametes versicolor pellets were used in a pulsed fluidised bioreactor with laccase production media to decolourise the textile dye Grey Lanaset G. The effect of the enzyme on dye degradation was analysed. In a batch process, degrading the dye with the fungus results in a decolourisation percentage higher than 90% (initial dye concentration 150 mg/L) while the results were lower than 35% using enzymatic degradation. Although most of the decolourisation was initially due to an adsorption process, later on in both phases, the biomass and the culture broth became colourless. In order to check the possibility of improving the degradation capacity in the batch mode operation, after decolourising the initial dye solution, different pulse dye adding strategies were tested. Adding a large pulse resulted in fast enzymatic deactivation while adding a small pulse caused the system to operate below its optimum degradation capacity. There was a close correlation between the amount of laccase produced and the amount of dye degraded. The system worked in a bioreactor for one month without any operational problems. Finally, in the continuous mode the dye degradation has been demonstrated because it is possible to maintain the continuous production of the laccase enzyme.
Keywords: Trametes versicolor; Bioreactor; Laccase; Grey Lanaset G; Decolourisation;

A bench-scale anoxic–oxic (A/O) system was used to treat a synthetic starch wastewater for 1 year. The objectives were to improve nitrogen removal and reduce operating costs by using on-line sensors DO, pH and ORP. It is found that the DO in the first aerobic zone could be used to estimate influent ammonia load under constant aeration. The pH profiles and DO profiles along the length of aerobic zones give good indication of the nitrification, and the pH profiles in the aerobic zones and anoxic zones could be classified into two types: descending and rising type. Further, ORP value at the end of aerobic zone showed a good correlation with the effluent ammonia and nitrate nitrogen concentration, good effluent quality corresponded with the range of 40–60 mV of ORP in this study. ORP was also correlated well with nitrate concentration at the end of anoxic zone, and the maximum denitrification capacity could be achieved when ORP was maintained at about −90 mV. A fuzzy inference system for on-line aeration, nitrate recirculation flow and external carbon dosage control is developed on the basis of these observations and is implemented on the bench-scale system. Promising control performance is achieved.
Keywords: DO; pH; ORP; A/O process; Nitrogen removal;

The efficiency of oxygen transfer into the fermentation broths could be enhanced by adding in broths oxygen-vectors, such as hydrocarbons or fluorocarbons, without increasing the energy consumption for mixing or aeration. The experimental results obtained for simulated, Propionibacterium shermanii and Saccharomyces cerevisiae broths indicated the considerable increase of k L a and (k L a)v/(k L a)0 by adding n-dodecane, but the magnitude of this effect must be correlated with broths and biomass characteristics, especially with the cells affinity for oxygen-vector droplets. Thus, due to the higher affinity of yeasts cells for hydrocarbon droplets, the increase of oxygen mass transfer rate was lower than that recorded for simulated or bacterial broths.By means of the experimental data, some mathematical correlations describing the influences of the main parameters (apparent viscosity, concentration of biomass, specific power input, superficial air velocity) on k L a in presence of n-dodecane have been proposed for each considered fermentation systems. These equations have the general expression of k L a = α ( P a / V ) β v s γ η a δ or k L a = α ( P a / V ) β v s γ C x δ , the coefficients (β, γ and δ values being dependent on n-dodecane concentration, and offer a good agreement with the experiment, the average deviations being between ±9.6 and ±10.8%.
Keywords: Bioreactors; Oxygen transfer; Oxygen-vector; k L a; Simulated broths; Propionibacterium shermanii; Saccharomyces cerevisiae;

A mutant Fusarium maire K178 with high production of paclitaxel (taxol) was selected by protoplast mutation of UV radiation and diethyl sulfate (DES). The optimization of medium components was performed using both Plackett–Burman design and response surface methodology. Various nitrogen sources were initially compared and the Plackett–Burman design was employed for screening important components in trace elements. The results showed that NaOAc, NH4NO3 and MgSO4 were the most important components which were further investigated by response surface methodology. The optimal concentrations were: NaOAc 2.02 g/l, NH4NO3 7.84 g/l and MgSO4 0.68 g/l. After the strain improvement and optimization of the media the yield of taxol increased from 20 to 225.2 μg/l.
Keywords: Taxol; Mutant; Response surface methodology; Plackett–Burman design; Fermentation; Protoplast;

Physico-chemical and biological iron removal from potable water by A.G. Tekerlekopoulou; I.A. Vasiliadou; D.V. Vayenas (74-83).
A pilot-scale trickling filter was constructed and tested for iron removal from potable water. Iron removal was found to be caused by both biological and physico-chemical iron oxidation. The extent of each oxidation type was assessed. The system was inoculated with mixed culture and its performance was tested under continuous operation at 14 °C. Feed iron concentrations and volumetric flow rates (VFR), ranged between 1–4 mg/l and 1000–3000 ml/min (225–680 m3/m2day), respectively. First order kinetics was used to describe the physico-chemical iron oxidation while Monod-type kinetics was used to describe the net biological iron oxidation. An increase of VFR from 1000 to 3000 ml/min reduced the filter's physico-chemical removal efficiency from 93 to 80%, while it remained constant for all the iron feed concentrations (1–4 mg/l) that were tested at each VFR. Bio-oxidation improved filter efficiency by about 5–6%. In all cases there was very good agreement between model predictions and experimental data. A series of experiments were also performed in order to investigate the simultaneous ammonia and iron oxidation. It was found that ammonia influence on iron removal becomes substantial only for high iron and ammonia concentrations. The simplicity of the pilot-scale design, the lack of the need for an external mechanical aeration source and the ease for the design and prediction of the system operation offers a very attractive solution for iron removal from potable water.
Keywords: Aerobic processes; Biofilms; Modeling; Trickling filter; Iron removal; Potable water;

A hydrogen-based membrane biofilm reactor (MBfR), employing fibrous slag as a bacterial carrier, was developed for rapid and stable autohydrogenotrophic denitrification. This reactor allows hydrogen to be supplied through a gas-permeable membrane to the biofilm supported by fibrous slag. The estimation of hydrogen supply rate clearly demonstrated that hydrogen flux ( J H 2 ) is dependent on the gas pressure, leading to a possibility to control J H 2 by adjusting the pressure. A startup experiment to investigate denitrification rate with time clarified that denitrification rate of 4.35 g N/m2/day was achieved on day 10, exhibiting rapid startup for autohydrogenotrophic denitrification. Continuous denitrification experiment obviously indicated the effectiveness of the fibrous slag as a bacterial support; concretely, mean denitrification efficiency and rate after 70-days operation reached 99% and 6.58 g N/m2/day at a hydrogen pressure of 50 kPa, respectively, which results in the accomplishment of stable and high-speed denitrification. However, hydrogen utilization efficiency (HUE) was approximately 40%. This low efficiency allowed autotrophic sulfate-reducing bacteria (SRB) to grow in the fibrous-membrane matrix; eventually the HUE for sulfate reduction increased up to 28% on day 74. This result clearly indicates the significance of J H 2 control through the gas-permeable membrane for suppressing the occurrence of sulfate reduction.
Keywords: Autohydrogenotrophic denitrification; Biofilms; Environmental preservation; Hollow fibers; Hydrogen utilization efficiency (HUE); Membrane biofilm reactor (MBfR);

This article reports oxidative protein refolding assisted by artificial chaperones in reverse micelles formed by nonionic surfactant of sorbitan trioleate modified with Cibacron Blue F-3GA in n-hexane. The denatured/reduced lysozyme was used as a model protein and cetyltrimethylammonium bromide (CTAB) and β-cyclodextrin as the artificial chaperones. The use of the artificial chaperones has proved to increase the refolding efficiency of denatured–reduced lysozyme at the concentration range studied (3.5–5.9 mg/mL). Moreover, the artificial chaperones increased the refolding yield in a wide range of urea concentrations. However, the optimal urea concentration range was little affected by the presence of the artificial chaperones.
Keywords: Protein refolding; Lysozyme; Reverse micelles; Artificial chaperone; Nonionic surfactant; Cibacron Blue F-3GA;

Estimation of the self-purification capacity of biofilm formed in domestic sewer pipes by Yasunori Tanji; Rie Sakai; Kazuhiko Miyanaga; Hajime Unno (96-101).
To estimate the self-purification capacity of sewer pipe, six different types of concrete blocks were installed in a domestic sewer pipe for nine months. The concrete blocks used were plain, grain, porous, and wet concrete (no-hole, or perforated with holes of d  = 1 or 12 mm). After a 79-day exposure to sewage, a heterogeneous biofilm formed on the surface of each block. The self-purification capacities of the blocks were estimated by measuring the decrease in substrate concentration in artificial sewage. The analyzed substrates were dissolved oxygen (DO), total organic carbon (TOC), NH4 +, and NO3 . Wet concrete with holes (d  = 12 mm) showed the highest substrate consumption rates: DO = 460, TOC = 480, NH4–N = 87, and nitrogen as NO3–N = 170, in mg substrate/(m2  h). These results indicate that sewers have a considerable potential for removing organic material and nutrients, and modification of sewer surfaces may increase these activities.
Keywords: Self-purification; Wastewater treatment; Sewage pipes; Immobilization; Nitrification; Denitrification;

The Monod equation has been widely applied to describe microbial growth, but it has no any mechanistic basis. Based on the thermodynamics of microbial growth process, a general model for microbial growth was developed. The constants involved in the present model were defined with clear physical meanings. The model derived can be reduced to the Monod equation, Grau equation and Hill or Moser equation. Compared to the Michaelis–Menten constant with the equilibrium thermodynamic characteristics, it was shown that the Monod constant (K s) has non-equilibrium thermodynamic characteristics.
Keywords: Specific growth rate; Substrate concentration; Thermodynamics; Monod equation;