Biochemical Engineering Journal (v.32, #3)

BEJ Keywords (II).

Filamentous foaming and bulking in activated sludge process are common problems in pulp and paper mill wastewater treatment processes. In this study, the predominant foaming and bulking causing filamentous microorganism in the pulp and paper mill effluent treatment system was identified. Its growth kinetics was studied and used as a theoretical basis to develop an optimal operational strategy for foaming and bulking preventive control. Filamentous actinomycetes, Nocardia amarae was found to be the predominant filamentous microorganisms. The kinetics study revealed that the Food to Microorganism (F/M) ratio was a critical factor affecting the excessive growth of N. amarae. Accordingly, a novel Feast-Fast Operation (FFO) strategy was developed, in which the sludge microbes underwent a repetitive switch between high and low F/M ratio during the course of wastewater treatment. Feast-to-Fast Loading Ratio (FFLR) was proposed and used as the parameter for optimization of FFO performance. At FFLR of 4 and 3.5, the sludge volume index (SVI) rapidly reduced to a healthy level of 70 ml g−1 and the systems were free from stable foam, while the biochemical oxygen demand (BOD) removal efficiency remained above 96.2%. The FFO strategy effectively suppressed filamentous overgrowth and improved the settleability of activated sludge without adversely affecting the BOD removal efficiency.
Keywords: Bulking control; Pulp and paper mill effluent; Waste-water treatment; Filamentous bacteria; Bioreactors; Optimization;

Low-cost fermentative medium for biosurfactant production by probiotic bacteria by L.R. Rodrigues; J.A. Teixeira; R. Oliveira (135-142).
Potential use of alternative fermentative medium for biosurfactant production by Lactococcus lactis 53 and Streptococcus thermophilus A was studied. Suitable models were established to describe the response of the experiments pertaining to glucose, lactose or sucrose consumption, cell growth and biosurfactant production. Synthetic media MRS and M17 broth were used as control experiments. When the synthetic media were replaced by cheaper alternative media, as cheese whey and molasses, fermentations were carried out effectively with high yields and productivities of biosurfactant. An increase about 1.2–1.5 times in the mass of produced biosurfactant per gram cell dry weight and 60–80% medium preparation costs reduction were achieved, for both strains.In conclusion, the results obtained showed that supplemented cheese whey and molasses media can be used as a relatively inexpensive and economical alternative to synthetic media for biosurfactant production by probiotic bacteria, thus an attractive alternative as many of the potential applications for biosurfactants depend on whether they can be produced economically.
Keywords: Biosurfactant; Probiotic bacteria; Low-cost fermentative medium;

The aim of this study was to examine the possible antioxidant capacities of tissue cultured mountain ginseng (TCMG) root extract in acclimatised and potted plants of Spathiphyllum cannifolium, stressed by the application of polyethylene glycol (PEG). Plants were treated with 0% PEG (control), 10% PEG and 10% PEG plus with TCMG (63.5 mg/kg day) for a period of 10 and 20 days. The TCMG root extract was supplemented (63.5 mg/kg everyday) and the levels of thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2) and antioxidant enzymes were estimated in PEG induced S. cannifolium. PEG treated plants showed an induced accumulation of TBARS resulting in an inhibition of CAT, GR, GPx and GST activity indicating a clear oxidative stress. A significant increase in the activities of SOD, APX, G-POD, GPx, was observed in the leaves supplemented with TCMG root extract in PEG induced S. cannifolium indicating the ability of TCMG root extracts to ameliorate stress conditions. PEG treated plants reduced photosynthetic efficiency (F v/F m), indicating a down-regulation of photosynthesis of plant. Net CO2 uptake (A), stomatal conductance (g), transpiration rate (E), relative water content (RWC) and leaf osmotic potential (LOP) were also reduced in PEG-treated plant. However, these parameters were well protected in plants treated with PEG plus TCMG root extract treated plant. These results confirm the effectiveness of TCMG root extract supplementation in detoxifying oxidative damage, which is produced excessively in PEG-induced S. cannifolium. Therefore, roots of Panax ginseng are considered as a good source of antioxidant and can be produced on a large scale and, probably useful for human health.
Keywords: Antioxidant enzymes; Photosynthetic efficiency; Polyethylene glycol; Tissue cultured mountain ginseng root;

Application of regularized alternating least squares and independent component analysis to HPLC-DAD data of Haematococcus pluvialis metabolites by Hiroyuki Yamamoto; Keishi Hada; Hideki Yamaji; Tomohisa Katsuda; Hiromu Ohno; Hideki Fukuda (149-156).
The analysis of data from analytical equipment will be an important factor in the execution of metabolomics. Self-modeling curve resolution (SMCR) is one of the theoretical techniques of chemometrics and has recently been applied to the data of hyphenated chromatography techniques. Alternating least squares (ALS) is a classical SMCR method. In ALS, however, different solutions are produced depending on randomly chosen initial values. Simulation in the present study showed that the use of a normalized constraint in calculating ALS was effective in avoiding this problem. We also improved the ALS algorithm by adding a regularized term (regularized ALS: RALS). Independent component analysis (ICA) is a comparatively new method and has been discussed very actively by information science researchers, but has still been applied only in very few cases to curve resolution problems in chemometrics studies. We applied RALS with a normalized constraint and ICA to the HPLC-DAD data of Haematococcus pluvialis metabolites and obtained a high accuracy of peak detection, suggesting that these curve resolution methods are useful for identification of metabolites in metabolomics.
Keywords: Metabolomics; Chemometrics; HPLC-DAD; Alternating least squares; Regularized term; Independent component analysis;

Transfer free energies (ΔG tr) of amino acids from water to aqueous electrolyte solutions have been determined from the solubility measurements, as a function of salt concentration at 298.15 K under atmospheric pressure. The investigated aqueous systems contain amino acids of zwitterionic glycine peptides: glycine (Gly), diglycine (Gly2), triglycine (Gly3), and tetraglycine (Gly4) and cyclic glycylglycine (c(GG)) with an electrolyte compound of potassium chloride (KCl), potassium bromide (KBr) or potassium acetate (KAc). The solubilities of glycine and diglycine in aqueous solution decrease with increasing the concentration of salts (salting-out effect), whereas those of triglycine and tetraglycine increase with increasing the concentration of salts (salting-in effect). Furthermore, salting-in effect was found in aqueous c(GG)/KBr system, while salting-out effect was observed in aqueous c(GG)/KCl or c(GG)/KAc system. The experimental results were used to estimate the transfer free energies (Δg tr) of the peptide backbone unit (–CH2C=ONH–) from water to the aqueous electrolyte solutions. We developed a new trail to determine the activity coefficients (γ) for aqueous and aqueous electrolyte solutions using an activity coefficient model, with which the total contribution of transfer free energy between solute and the solvent was calculated. We compared the difference between neglecting and using the activity coefficients term in predicting ΔG tr. Since the transfer free energy contribution is negative, interactions between the ionic salts and the peptide backbone unit of zwitterionic glycine peptides are favorable and thus the ionic salts destabilize these amino acids. It was also found that KBr stabilizes c(GG), whereas KCl and KAc destabilize c(GG). These results provide evidence for the existence of interactions between the amide unit and ionic salts, in aqueous solution, which may be of importance in maintaining protein structure as well as in protein–solute and protein–solvent interactions.
Keywords: Solubilities; Transfer free energies; Protein stability; Amino acids; Electrolyte solutions; Molecular interactions;

Relationship between morphology, rheology and polygalacturonase production by Aspergillus sojae ATCC 20235 in submerged cultures by Nihan Gögus; Canan Tari; Selale Oncü; Sevcan Unluturk; Figen Tokatli (171-178).
A full factorial statistical design, with the factors of, two taxonomically different strains, seven types of seed culture formulations (slants) and two types of fermentation media were used to investigate the effect of these parameters on the morphology and polygalacturonase production. The rheology of the final fermentation medium was analyzed and appropriate mathematical model was applied to calculate suspension viscosity. It was found that most fermentation broths showed non-Newtonian flow behavior. According to statistical analysis, factors of strain types and fermentation media and the interaction between them were found significant on the enzyme activity. The effect of seed culture formulations (slants) were found insignificant at the significance level of 1%. Interaction of slants with strain types and fermentation media were also found insignificant. Considering the morphology of the final culture, Aspergillus sojae with the desired pellet morphology in a complex media, inoculated with a seed culture prepared from molasses resulted in maximum polygalacturonase enzyme activity (0.2 U/ml) and lowest suspension viscosity with a broth rheology close to Newtonian flow behavior.
Keywords: Aspergillus sojae; Rhizopus oryzae; Polygalacturonase; Submerged fermentation; Fungal morphology; Rheology;

Purification and properties of a xylanase produced by Bacillus circulans BL53 on solid-state cultivation by Júlio Xandro Heck; Luís Henrique de Barros Soares; Plinho Francisco Hertz; Marco Antônio Záchia Ayub (179-184).
Xylanase from Amazon isolate Bacillus circulans BL53 grown on solid-state cultivation was purified to apparent homogeneity by ammonium sulphate fractioning, cation-exchange and gel filtration chromatography. A purification factor of 428-fold was achieved, with the purified enzyme presenting a specific activity of about 37 U mg−1 protein. The xylanase molecular weight was calculated as 38 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and its isoeletric point was determined as 8.8. Determination of pH and temperature for the maximum activity was obtained using a 22 factorial design over an extensive range of pH (5.0–8.0) and temperatures (40–80 °C). The enzyme follows Michaelis–Menten kinetics with K M and V max values of 9.9 mg xylan mL−1 and 25.25 μmol min−1, respectively. The purified enzyme hydrolyzes soybean hull, soybean fiber, rice straw, grape skin and sugar cane bagasse. Its activity was stimulated by Co2+, Mn2+ and protein disulphide reducing reagents, but strongly inhibited by Hg2+ ions and SDS.
Keywords: Xylanase; Hemicellulose; Solid-state cultivation; Bacillus circulans;

Effect of the enzymatic hydrolysis pretreatment of lipids-rich wastewater on the anaerobic biodigestion by Adriano A. Mendes; Ernandes B. Pereira; Heizir F. de Castro (185-190).
A low-cost commercially available lipase preparation from animal source was used to perform enzymatic hydrolysis pretreatment of lipid-rich wastewater from dairy industries. The hydrolysis was carried for different incubation time (4–24 h) and the pretreatment efficiency was verified by running comparative biodegradability tests (crude and treated effluents). All pretreated assays showed higher reaction rate in relation to crude wastewater assay, which was confirmed by the increased levels of biogas production and higher organic matter (COD) and color removal. The pretreatment was optimized for 12 h hydrolysis time, enabling high-biogas formation (445 ± 29 mL) and organic matter removal (78.2%). Promising results in relation to the biogas formation (354 ± 34 mL) were also found when hydrolysis and biodegradation steps were carried out simultaneously. The use of enzymatic treatment seemed to be a very promising alternative for treating wastewaters having high-fat contents, such as those from the dairy industry.
Keywords: Lipases; Lipids; Hydrolysis; Dairy wastewater; Biodegradability; Biogas;

An improved mutant was isolated from the cellulolytic fungus Stachybotrys sp. after nitrous acid mutagenesis. It was fed-batch cultivated on cellulose and its extracellular cellulases (mainly the endoglucanases and β-glucosidases) were analyzed. One β-glucosidase was purified to homogeneity after two steps, MonoQ and gel filtration and shown to be a dimeric protein. The molecular weight of each monomer is 85 kDa. Besides its aryl β-glucosidase activity towards salicin, methyl-umbellypheryl-β-d-glucoside (MUG) and p-nitrophenyl-β-d-glucoside (pNPG), it showed a true β-glucosidase activity since it splits cellobiose into two glucose monomers. The V max and the K m kinetics parameters with pNPG as substrate were 78 U/mg and 0.27 mM, respectively. The enzyme shows more affinity to pNPG than cellobiose and salicin whose apparent values of K m were, respectively, 2.22 and 37.14 mM. This enzyme exhibits its optimal activity at pH 5 and at 50 °C. Interestingly, this activity is not affected by denaturing gel conditions (SDS and β-mercaptoethanol) as long as it is not pre-heated. The N-terminal sequence of the purified enzyme showed a significant homology with the family 1 β-glucosidases of Trichoderma reesei and Humicola isolens even though these two enzymes are much smaller in size.
Keywords: Stachybotrys sp.; β-Glucosidase; Cellulase; Purification; Chromatography; Enzyme activity;

Oxygen uptake rate (OUR) during growth of Rhodococcus erythropolis IGTS8, a natural desulphurizing bacterium, is measured using two experimental methods for the same set of experiments performed in a 2 L work volume commercial bioreactor.The experimental OUR values obtained by the usual “dynamic method” were compared to those obtained by the “process method” after fitting the values of oxygen concentration measured during cell growth process.Experimental OUR values obtained from the ‘process method’ are always higher than those from the ‘dynamic method’. This discrepancy is explained by the cell economy principle: during the few minutes employed to measure OUR using the dynamic method, the cells do not use oxygen to synthesize 4S desulphurization route enzymes. The experimental values obtained by both methods are modelled to describe oxygen consumption using three parameters: consumption for growth (Y OX) and for maintenance ( m O 2 ), as usual, and the oxygen consumption to synthesize Dsz enzymes by means of a yield of oxygen into ‘biodesulphurization capability’ (Y OBDS). Oxygen concentration profile during growth is closely described using the final model proposed.
Keywords: Biodesulphurization; Oxygen uptake rate; Rhodococcus erythropolis; Kinetic parameters; Dissolved oxygen concentration modelling;

Ganoderic acids are valuable bioactive secondary metabolites produced by a traditional medicinal mushroom Ganoderma lucidum (“Ling-zhi” in Chinese and “Reishi” in Japanese). In this work, a fast and efficient method for the recovery and purification of ganoderic acid T (GA-T) and ganoderic acid Me (GA-Me) from triterpene-enriched extracts of G. lucidum mycelia was developed by using reversed phase HPLC (RP-HPLC) on a C18 column with an acidified methanol–water mobile phase in combination with ultraviolet (UV) detection and electrospray ionization mass spectrometry (ESI-MS). The presence of each targeted GA (GA-T and GA-Me) in its corresponding peak was easily identified and confirmed by UV and MS. The chemical structures of the purified GA-T and GA-Me were further confirmed by 1H NMR. The retention behaviors of the two GAs over a temperature range of 15–55 °C were also investigated. From the retention time data, van’t Hoff plots were obtained. The estimated enthalpy (ΔH) and entropy (ΔS) data suggest that the retention time difference between GA-T and GA-Me might be driven by an enthalpy difference. Furthermore, a semi-preparative HPLC purification was achieved on a semi-preparative C18 column using the conditions optimized for the analytical column. The method presented in this work can be a valuable tool for the rapid semi-preparative purification of targeted GAs, and it may also be applicable to some other natural products.
Keywords: Bioseparation; Purification; Natural products; RP-HPLC; Electrospray ionization mass spectrometry (ESI-MS);

A new immunoassay concept for the determination of aflatoxin B1 (AFB1) based bio-electrocatalytic reaction on micro-comb electrode was proposed. The micro-comb electrode was fabricated by means of self-assembling horseradish peroxidase (HRP) and AFB1 antibody molecules onto gold nanoparticles (nanogold) functionalized biorecognition surfaces. The presence of nanogold provided a congenial microenvironment for the immobilized biomolecules and decreased the electron transfer impedance, leading to a direct electrochemical behavior of the immobilized HRP. The formation of the antibody–antigen complex by a simple one-step immunoreaction between the immobilized anti-AFB1 and AFB1 in sample solution introduced a barrier of direct electrical communication between the immobilized HRP and the electrode surface, thus local conductivity variations could be detected by the HRP bio-electrocatalytic reaction in 0.02 M phosphate buffer solution (pH 7.0) containing 80 μM H2O2, 0.05 M KI and 0.15 M NaCl. Under optimal conditions, the proposed immune-biosensor exhibited a good conductometric response relative to AFB1 concentration in a linear range from 0.5 to 10 ng/ml with a relatively low detection limit of 0.1 ng/ml at 3δ. The developed immune-biosensor showed an acceptable accuracy compared with those obtained from ELISA. The inter-assay coefficients of variation are 7.1 and 4.6% at 1.0 and 8.0 ng/ml AFB1, respectively. The storage stability is acceptable in a pH 7.0 phosphate buffer solution at 4 °C for 12 days. Importantly, the proposed methodology could be extended to the detection of other antigens or biosecurities.
Keywords: Conductometric immune-biosensor; Bio-electrocatalytic signal; Gold nanoparticles; Aflatoxin B1;

Polyhydroxyalkanoate (PHA) copolymers consisting of short-chain-length (SCL) and medium-chain-length (MCL) 3-hydroxyalkanoates (3HA) were produced by recombinant Ralstonia eutropha PHB4 harboring a low-substrate-specificity PHA synthase PhaC2Ps from Pseudomonas stutzeri 1317. These polyesters, containing a wide range of chain length, were purified and characterized by acetone fractionation, nuclear magnetic resonance (NMR), gel-permeation chromatography (GPC), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and mechanical property studies. The physical properties of the copolymers are dependent largely with 3-hydroxyoctanoate (3HO) content in all PHA.
Keywords: Polyhydroxybutyrate (PHB); PHA synthase; Physical property; Fermentation; Biopolyester;

In the present work experiments were carried out to study the effect of free gossypol on the growth of Candida tropicalis ZAU-1, evaluate its ability in biodegrading free gossypol, analyze the time course of solid-state fermentation, and model the microbial growth by determining the kinetics of dry matter weight loss, total carbohydrate concentration and the free gossypol content during solid-state fermentation. Results showed that the biomass in inorganic salts glucose medium were unaffected by free gossypol at 500 and 1000 mg/l levels, compared with the control group (p  > 0.05); degradation of free gossypol reached 95.12% and 94.12%, respectively. A logistic equation (R 2  = 0.9922), describing the growth model of C. tropicalis ZAU-1 was obtained, with the maximum values of u m and X m at 0.0970 h−1 and 21.8631% of dry matter weight loss, respectively. A good-fit curvilinear regression model was achieved to describe the change pattern of total carbohydrate concentration (R 2  = 0.9910), and the biodegradation pattern of free gossypol (R 2  = 0.9825). These models could be used to predict the fermentation course by C. tropicalis ZAU-1 under solid-state fermentation.
Keywords: Solid-state fermentation; Biodegradation; Modeling; Free gossypol; Kinetics; Microbial growth;

Effect of two-steps substrate addition on steroids 11β-hydroxylation by Curvularia lunata CL-114 by Wen-Yu Lu; Lian-Xiang Du; Min Wang; Jian-Ping Wen; Bing Sun; Ya-Wen Guo (233-238).
In this paper, the effect of substrate 17α-hydroxypregn-4-ene-3,20-dione-21-acetate (RSA, Cortexolone-21-acetate) on the expression of cytochrome P450 and the production of hydrocortisone by Curvularia lunata CL-114 was studied. Meanwhile the effect of pH on the production of hydrocortisone was observed. Based on the effect of substrate RSA two-steps addition on cytochrome P450 expression and hydrocortisone production, a novel fermentation process was established as follows: 0.3 g/L RSA was added for the first time after 16 h of inoculation, followed by the second addition of 0.7 g/L RSA after 8 h later, then pH was regulated to constant 6.5 after another 8 h till the end of fermentation. The results showed that the novel process was much better than the original one on improving the induction of cytochrome P450 and production of hydrocortisone, and the hydrocortisone yield had achieved an improvement of 17.6% higher than that of the original process correspondingly.
Keywords: Curvularia lunata; Hydrocortisone; Cytochrome P450; Steroid; Induction;

The role of dissolved oxygen and function of agitation in hyaluronic acid fermentation by Wei-Chih Huang; Shu-Jen Chen; Teh-Liang Chen (239-243).
The role of dissolved oxygen (DO) and function of agitation in hyaluronic acid fermentation by Streptococcus zooepidemicus ATCC 39920 were explored. With controlled DO levels, it was found that the present strain grew as well under both aerobic and anaerobic conditions. Dissolved oxygen plays a role as a stimulant in the HA synthesis; the intrinsic factor affecting the efficiency of HA synthesis is DO level; and there existed a critical DO level of 5% air saturation for the HA synthesis. On the other hand, agitation functions to mix the broth, to enhance oxygen absorption, but not to release HA capsule. In addition, vigorous agitation would lengthen the operation time. It therefore suggests that the relevant criteria for scaling up the fermentor are to maintain DO level above the critical value, and to provide a mild agitation for homogeneity in the fermentor.
Keywords: Hyaluronic acid fermentation; Streptococcus zooepidemicus; Scaleup of fermentor; Critical dissolved oxygen concentration;

List of Reviewers (245-248).