Biochemical Engineering Journal (v.33, #3)

BEJ Keywords (IV).

The influence of initial pH value, various nitrogen sources, plant oils, and modes of propagation (shake-flask and static culture) on the production of biomass, exopolysaccharide (EPS), adenosine and, in particular, cordycepin, by Cordyceps militaris CCRC 32219 were investigated. Optimal conditions for mycelial growth, EPS and cordycepin production were observed at relatively low pH. Amongst organic sources, yeast extract (YE) was favorable for EPS and cordycepin production, while corn steep powder (CSP) was favorable for adenosine production. A lower C/N ratio was favorable for adenosine and cordycepin production; however, too low a C/N ratio led to diminished production. All plant oils tested stimulate mycelial growth and EPS production of C. militaris, but they did not show much effect on the adenosine and cordycepin production. A two-stage fermentation process by combining shake-flask fermentation with static culture significantly enhanced cordycepin production. A Box–Behnken experimental design was employed to optimize the production of cordycepin, which showed that the optimum conditions to produce cordycepin by C. militaris CCRC 32219 were at pH 6, YE concentration of 45 g/l and 8.0 day of the shake culture followed by 16 days of the static culture. Under the optimized conditions, the maximum production (2214.5 mg/l) of cordycepin was obtained, which is much higher than those reported up to date.
Keywords: Cordyceps militaris; Cordycepin; Adenosine; Polysaccharide; Response surface design;

Statistically based experimental designs were applied for the fermentation optimization of polysaccharide and ergosterol production from Agaricus brasiliensis in a stirred-fermenter. Culture temperature, agitation speed and initial pH were identified to have significant effects on polysaccharide and ergosterol production by a Plackett–Burman design. These three significant factors were subsequently optimized using steepest ascent method and Box–Behnken design. The validity of the optimum conditions was verified by separate experiments in which the polysaccharide and ergosterol yields were increased by 57% and 43%, respectively, as compared to those under unoptimized fermentation conditions.
Keywords: Agaricus brasiliensis; Ergosterol; Experimental designs; Fermentation; Optimization; Polysaccharide;

Application of seaweeds for the removal of lead from aqueous solution by R. Senthilkumar; K. Vijayaraghavan; M. Thilakavathi; P.V.R. Iyer; M. Velan (211-216).
Ten different seaweed species were compared on the basis of lead uptake at different pH conditions. The brown seaweed, Turbinaria conoides, exhibited maximum lead uptake (at pH 4.5) and hence was selected for further studies. Sorption isotherms, obtained at different pH (4–5) and temperature (25–35 °C) conditions were fitted using Langmuir and Sips models. According to the Langmuir model, the maximum lead uptake of 439.4 mg/g was obtained at optimum pH (4.5) and temperature (30 °C). The Sips model better described the sorption isotherms with high correlation coefficients at all conditions examined. Various thermodynamic parameters such as ΔG°, ΔH° and ΔS° were calculated indicating that the present system was a spontaneous and endothermic process. Through potentiometric titrations, number of binding sites (carboxyl groups) and pK 1 were determined as 4.1 mmol/g and 4.4, respectively. The influence of co-ions (Na+, K+, Mg2+ and Ca2+) on lead uptake was well pronounced in the case of divalent ions compared to monovalent ions. The solution of 0.1 M HCl successfully eluted all lead ions from lead-loaded T. conoides biomass. The regeneration experiments revealed that the alga could be successfully reused for five cycles without any loss in lead biosorption capacity. A glass column (2 cm i.d. and 35 cm height) was used to study the continuous lead biosorption performance of T. conoides. At 25 cm (bed height), 5 ml/min (flow rate) and 100 mg/l (initial lead concentration), T. conoides exhibited lead uptake of 220.1 mg/g. The column was successfully eluted using 0.1 M HCl, with elution efficiency of 99.7%.
Keywords: Biosorption; Lead; Seaweed; Turbinaria conoides; Regeneration;

While an aeration tank in an activated sludge process is often operated with high dissolved oxygen (DO) concentration to ensure organic degradation and nitrification, it may be operated at low DO concentration to reduce energy consumption and achieve desired denitrification. The ASM1 (Activated Sludge Model No. 1) can be used to describe the activated sludge process if the nitrification and denitrification occur either during different phases or in different tanks, but it may encounter problems in simulating the denitrification phenomenon caused by low DO concentration in the aeration tank. In the present work, we developed a model integrating the ASM1 kinetics and a biofloc model to account for the actual anoxic and aerobic rates. Oxygen was assumed the only substrate of both bio-kinetically and flux limiting in the flocs and its dispersion coefficient was estimated as 1.2 × 10−4  m2  day−1 by using a set of measured effluent qualities of a full-scale wastewater treatment plant (WWTP) operating at low DO concentration (∼0.80 mg L−1) for 60 days. Simulation studies predicted the optimal DO level of 0.36 mg L−1 which would lead to minimum total nitrogen of 15.7 mg N L−1 and also showed the insignificance of the addition of carbon source for nitrogen removal for the operation under study. The developed model may be helpful for process engineers to predict the plant behaviors under various configurations or operating strategies.
Keywords: Aerobic processes; Dynamic simulation; Dissolved oxygen; Waste-water treatment; ASM1; Floc model;

Catalytic properties of cross-linked enzyme crystals in organic media by Hidetaka Noritomi; Akihiro Sasanuma; Satoru Kato; Kunio Nagahama (228-231).
The activity and the enantioselectivity of cross-linked enzyme crystals (CLECs) of subtilisin in the transesterification between N-acetyl-l-phenylalanine ethyl ester and n-propanol have been examined in various organic solvents. The activity of CLECs of subtilisin in decane was 780 times greater than that in triethylamine. CLECs of subtilisin preferred l-enantiomer in the transesterification between N-acetyl-phenylalanine ethyl ester and n-propanol, and the (k cat/K M)L/(k cat/K M)D ratio was 20 000 in cyclohexane.
Keywords: Cross-linked enzyme crystals; Organic solvent; Activity; Enantioselectivity;

Effect of cultivation conditions on cell-surface display of Flo1 fusion protein using sake yeast by Takanori Tanino; Erina Noguchi; Sakurako Kimura; Hiroshi Sahara; Youji Hata; Hideki Fukuda; Akihiko Kondo (232-237).
The cell-surface display of the Flo1p anchor system with a flocculation functional domain was examined under various cultivation conditions. As a model system, lipase from Rhizopus oryzae with the pro sequence was genetically fused to the Flo1 short (FS) anchor (FSProROL) and displayed on the sake yeast cell-surface under the control of the SED800 promoter (pSED800). The nutrients and carbon source in the culture media affected the display of the fusion protein FSProROL on the sake yeast cell-surface. The lipase activity in whole cells cultivated in poor media, without peptone and/or yeast extracts, were higher than those cultivated in rich media. In addition, glucose and maltose were effective carbon sources for increasing the lipase activity in whole cells, and the addition of di- or tri-saccharide as the carbon source reduced the release of the lipase activity into the culture supernatants. The initial glucose concentration was found to influence the total lipase activity and it mainly affected the lipase activity in whole cells. Under the optimum condition, sake yeast was found to show high cell density and high lipase activity in short time cultivation.
Keywords: Biocatalysis; Cell-surface display; Cultivation condition; Lipase; Optimisation; Yeast;

Kinetic modeling using S-systems and lin-log approaches by Feng-Sheng Wang; Chih-Lung Ko; Eberhard O. Voit (238-247).
We analyze the growth dynamics and production of an industrially interesting amino acid in a recombinant Escherichia coli strain, using in parallel two alternative modeling frameworks, namely S-systems and lin-log models. These models have identical steady-state solutions, but differ in their representation of transients. The models were parameterized with data from two bioprocesses that differed in their initial culture concentrations and then used to predict responses under yet a different initial culture regimen. Among the S-system models, several alternatives representing slightly different pathway structures, were tested. All were found to be capable of capturing the dynamics of all variables in the test systems and also of predicting the dynamic responses under new conditions. The lin-log models also captured the dynamics, but not as well as the S-system models. A probable reason for the inferior performance of lin-log models is their intrinsic property of not representing situations well where variable concentrations are moderately small.
Keywords: Parameter estimation; Biochemical Systems Theory; Dynamic modeling; Amino acid; Kinetic parameters; Fermentation; Bioprocess control;

Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate dehydrogenase production by Ângelo Samir Melim Miguel; Michele Vitolo; Adalberto Pessoa (248-252).
We examined glucose 6-phosphate dehydrogenase (G6PD) production by fed-batch cultivation, using a recombinant strain of Saccharomyces cerevisiae W303-181 overexpressing this enzyme. The cultivations were carried out in a 3 L fermenter at pH 5.7, 30 °C, 2.0 vvm aeration, 200 rpm agitation and an inoculum concentration of 1.0 g/L. The volume of the culture medium in the fed-batch process varied from 1.333 to 2.0 L, due to the addition of 15.0 g/L glucose solution during 5 h. Different feeding rates were studied (exponentially increasing and decreasing feeding rates), and the feeding profile was determined by values of the parameter K (time constant), namely: 0.2, 0.5 and 0.8 h−1. The best enzyme production (847 U/L) was obtained with an exponentially increasing feeding rate and K  = 0.2 h−1. The results attained also showed that this process is promising for G6PD production.
Keywords: Fed-batch; Glucose 6-phosphate dehydrogenase; Saccharomyces cerevisiae; Exponential feeding rates;

Modelling and dynamic simulation of a moving bed bioreactor using respirometry for the estimation of kinetic parameters by M. Plattes; D. Fiorelli; S. Gillé; C. Girard; E. Henry; F. Minette; O. O’Nagy; P.M. Schosseler (253-259).
Respirometry was used for the characterization of active autotrophic and heterotrophic biomass in a pilot scale moving bed bioreactor (MBBR). For this purpose biofilm samples attached to the carrier elements of the MBBR were transferred to a static gas/static liquid type respirometer with intermittent aeration. Known amounts of ammonia nitrogen and acetate were added to the respirometer. The dissolved oxygen (DO) decrease during the non-aeration phase was measured and used to calculate the oxygen uptake rate (OUR) of the active biomass. The resulting respirograms featured the typical endogenous and exogenous respiration phases and the shape of the respirogram was as expected from analogous respirometry with activated sludge. The OUR response was modelled with the activated sludge model ASM1 and the relevant kinetic parameter values for autotrophic and heterotrophic growth were adjusted. The adjusted parameter set and data originating from a 4-day long intensive measurement campaign were used for modelling and simulation of the pilot scale MBBR. The parameter estimation resulted in a good dynamic simulation of ammonia and nitrate variations in the effluent of the MBBR. Important MBBR properties including biofilm age, biofilm composition, and both attachment and detachment rate were extracted from the model.
Keywords: Biofilms; Biofilm age; Dynamic simulation; Kinetic parameters; Moving bed bioreactors; Respirometry;

The present study describes a new method of synthesis of an anionic biopolymer, iron doped calcium alginate beads (Fe-CA). The beads were characterized in terms of size, porosity and surface area. The adsorption of the anionic species depends mainly on the iron content of the beads. Thus, iron content in the Fe-CA beads has been determined employing radiometric technique using 59Fe radiotracer. It has been found that Fe-CA beads contain 37.8 mg Fe/g of wet alginate beads in the proposed method, which is much higher than the earlier reported methods, keeping the mechanical stability.
Keywords: Biopolymer; Iron doped calcium alginate; FeCl3; CaCl2; 59Fe; Radiotracer;

Scale-down and parallel operation of the riboflavin production process with Bacillus subtilis by Bettina Knorr; Heinrich Schlieker; Hans-Peter Hohmann; Dirk Weuster-Botz (263-274).
The establishment and the improvement of industrial bioprocesses calls for the selection of media compositions and process conditions in highly parallel experiments as well as for the intensified screening of new biocatalysts and improved production strains. This work presents for the first time the scale-down and the successful adaptation of an industrial riboflavin fed-batch production process with Bacillus subtilis to a fully automated setup with 48 parallel stirred bioreactors at a milliliter scale (10 mL). The feasibility of an intermittent feeding mode and a discontinuous at-line pH control for parallel cultivations over up to 53 h is demonstrated together with interlaced process analyses at a microliter scale for quasi-simultaneous at-line monitoring of biomass, substrate and product concentration. The discontinuous feeding mode necessitated an increased oxygen input, resulting in lower final biomass concentrations. However, the product yields and volumetric productivities in the milliliter setup were equivalent to the yields and productivities obtained during the reference cultivations at laboratory scale, which allows considering the automated system together with the developed schedule as a screening tool for high-throughput bioprocess design of the described production process.
Keywords: Bioprocess design; Scale-down; Bioreactors; Parallel operation; Fed-batch culture; Industrial riboflavin production;

Co-composting of sewage sludge:fats mixtures and characteristics of the lipases involved by Teresa Gea; Pau Ferrer; Gregorio Alvaro; Francisco Valero; Adriana Artola; Antoni Sánchez (275-283).
Co-composting of sewage sludge and animal fat mixtures was studied in order to determine the possibility of using this technology to recycle fat-enriched wastes. A maximum fat content of 30% in fat:sludge mixtures is recommended to achieve the international sanitation requirements on compost quality and to avoid an excessive thermophilic composting time. Under these conditions a fat content reduction of 85% was achieved. Biological activity was highly dependent on the moisture content as shown by the respiratory quotient values. Moisture content is a critical control factor because of the hydrophobic nature of fats and should be maintained above 40% in the composting of fats. Biological indices of the compost obtained after 69 days of process (maturity grade: IV; respiration index: 1.1 mg O2  g OM−1  h−1) indicated a high stability and maturity degree of the material. Lipases responsible for fat hydrolysis were monitored during the composting process and a sample from the thermophilic period was characterized in terms of stability in front of pH and temperature. Optimal conditions for lipase stability were found at 38.3 °C and pH 7.97, however, the maximum lipolytic activity was observed at thermophilic temperatures. Lipases from the thermophilic period were purified by anion exchange chromatography and visualised by SDS-PAGE. Two major bands were observed at molecular weights of 29 and 62 kDa. These bands could not be identified precisely by N-terminal sequence analysis.
Keywords: Aerobic processes; Biodegradation; Waste treatment; Fat; Lipase; Respiration index;

The polyelectrolyte complex (PEC) effect between hyaluronic acid (HA) and chitosan was explored to recover HA from fermentation broth. Chitosan was conjugated with the magnetic nanoparticles by co-precipitation method to facilitate its recovery. The magnetic chitosan particles (chitosan–magnetite) have an average size about 5 μm and point of zero charge (PZC) around 6.5. pH lower than PZC favored the HA capture. About 39 mg of HA was captured per gram of particles at pH 6. Nearly quantitative release of captured HA was achieved at pH 8. Although HA could not be directly isolated from Streptococcus zoopedemics fermentation broth by manipulating pH between 6 and 8, HA free of contaminant protein could be purified from the crude ethanol precipitate using chitosan–magnetite.
Keywords: Hyaluronic acid; Magnetic separation; Chitosan; Magnetite; Streptococcus zoopedemics;