Biochemical Engineering Journal (v.29, #3)

BEJ Keywords (II).

IFC (CO2).

Analysis of biodesulfurization of model oil system by the bacterium, strain RIPI-22 by M. Rashtchi; G.H. Mohebali; M.M. Akbarnejad; J. Towfighi; B. Rasekh; A. Keytash (169-173).
The execution of 4S process (sulfur-specific pathway) by growing cells of the newly isolated strain RIPI-22 and its resting cells in both aqueous reaction system and in two-phase system were investigated. The time for maximum desulfurization activity of the strain in resting state and in model oil system (hexadecane containing dibenzothiophene) was 7 h. Using taguchi design procedure the effects of cell density, pH and phase ratio on the desulfurization reaction were studied. The extent of desulfurization was hardly dependent on the pH where as the volume ratio of hydrocarbon-aqueous phase significantly affected the desulfurization activity.On the basis of the determined optimum desulfurizing conditions, the biodesulfurization pattern of DBT was studied and kinetic parameters were calculated.
Keywords: Biodesulfurization; Dibenzothiophene; Kinetic parameters; Optimization; Affinity; Hexadecane;

Evaluation of temperature and guanidine hydrochloride-induced protein–liposome interactions by using immobilized liposome chromatography by Noriko Yoshimoto; Makoto Yoshimoto; Kazuma Yasuhara; Toshinori Shimanouchi; Hiroshi Umakoshi; Ryoichi Kuboi (174-181).
Hydrophobic interactions between nine model proteins and net-neutral lipid bilayer membranes (liposomes) under stress conditions were quantitatively examined by using immobilized liposome chromatography (ILC). Small or large unilamellar liposomes were composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and immobilized in a gel matrix by utilizing covalent coupling between amino-containing lipids and activated gel beads or avidin–biotin biospecific binding. Retardation of bovine carbonic anhydrase (CAB) in ILC was pronounced at particular temperatures (50 and 60 °C) where the local hydrophobicity of theses protein molecules becomes sufficiently large. Protein-induced leakage of a hydrophilic dye (calcein) from immobilized liposomes interior was also drastically enhanced at particular temperatures where large retardation was observed. For other proteins examined, similar results were also observed. The specific capacity factor of the proteins characteristic for the ILC and the amount of calcein released from immobilized liposomes were successfully expressed as a function of the product of the local hydrophobicities of proteins and liposomes, regardless of protein species and the type of the stress conditions applied (denaturant and heating). These findings indicate that lipid membranes have an ability to non-specifically recognize local hydrophobicities of proteins to form stress-mediated supramolecular assemblies with proteins, which may have potential applications in bioprocesses such as protein refolding and separation. ILC was thus found to be a very useful method for the quantitative detection of dynamic protein–liposome interactions triggered by stress conditions.
Keywords: Bioseperations; Biosensors; Immobilized liposome chromatography; Membrane stress biotechnology; Protein; Protein denaturation;

Computational fluid dynamics simulation was used to estimate the flow and ferric iron concentration profiles around a single cell or pairs of cells of A. ferrooxidans, immobilized on the surface of a sulfide crystal. It has been shown that there are significant concentration gradients of ferric iron concentration between the surface of the crystal and the liquid bulk. The difference between the bulk and surface ferric ions concentrations can reach several hundred mg/L. These results can explain the difference between the chemical and biological oxidation rates of metal sulfides in liquid media containing iron ions.
Keywords: Computational fluid dynamics; Acidithiobacillus ferrooxidans; Bioleaching; Mass transfer;

Efficiency of enzymatic and non-enzymatic catalysts in the synthesis of insoluble polyphenol and conductive polyaniline in water by Néstor Raúl Curvetto; Débora Figlas; Adriana Brandolin; Silvana Beatriz Saidman; Elsa Haydeé Rueda; María Luján Ferreira (191-203).
The present work analyzes the potential use of white-rot fungi (WRF) and hematin for phenol and aniline polymerization, as a low-cost alternative to horseradish peroxidase (HRPC). The objective is to evaluate the capability of these catalysts to produce tailor-made aniline as well as to eliminate phenols by precipitation from aqueous solution. 4-Aminoantypirine (4AAP) was used to test phenoxide formation by crude protein preparations of white-rot fungi at selected conditions. The crude extracts of Pleurotus sajor-caju (PSC) were selected because of the promising values obtained for the phenoxide formation rate. HRPC/H2O2 and P. sajor-caju derived enzymes/H2O2 (PSC/H2O2) systems produced soluble polyaniline in the presence of polystyrene sulphonated (PES), with high aniline conversions at 45 °C. For the case of insoluble polyphenol production, the PSC-derived enzymes, in absence of hydrogen peroxide, produced insoluble polyphenol with similar efficiencies as those found with HRPC or hematin in a one step phenol treatment (near 40% phenol conversion). For the aniline process, at least 75% aniline conversion was obtained when using PSC enzymes at room temperature. After long reaction times, the lignin-modifying enzymes derived from PSC only produced a conductive form of polyaniline (PANI) at lower temperatures than those required when employing HRPC. Fungal enzymes look promising for eliminating aniline/phenol from wastewaters since the obtained results demonstrated that they are able to polymerizate and precipitate them from aqueous solutions.
Keywords: Polyaniline; Polyphenol; White-rot fungi; Peroxidases;

Membrane bioreactors have been increasingly applied for wastewater treatment during the last two decades. High energy requirements and membrane capital costs remains as their main drawback. A new strategy of operation is presented based on a continuous critical flux determination, preventing excessive cake-layer accumulation on the membrane surface. Reactor operation is divided in cycles of 500 s filtration followed by a short back-flush of 15 s. If cake-layer formation is detected during continuous operation, a decrease in flux or an increase in cross flow velocity is performed. The proposed approach keeps reactor operation oscillating around the critical flux, minimizing reactor maintenance and maximizing performance. An easy to operate statistical steady state determination tool for the trans-membrane pressure was used to detect cake-layer formation. The developed control approach was tested on two Anaerobic MBRs equipped with submerged membranes. Despite the existence of very different critical fluxes and cake-layer formation characteristics, proposed approach was able to keep pressure increase during filtration cycles below 20 mbar. The developed approach is an efficient tool for on-line control of cake-layer formation over the membranes, changing cross flow velocities by manipulating gas sparging in submerged MBRs.
Keywords: Membrane bioreactors; Anaerobic processes; Wastewater treatment; Control; Critical flux; Cake formation;

The performance of up-flow anaerobic sludge blanket (UASB) in combination with down-flow hanging sponge (DHS) system for sewage treatment at an average wastewater temperature of 15 °C has been investigated for 6 months. The results showed that a combined system operated at a total HRT of 10.7 h and total SRT of 88 days represents a cost effective sewage treatment process. The average CODtotal and BOD5 total concentrations measured in the final effluent of the total system (UASB + DHS) amounted to 43 and 3.0 mg/l, respectively, corresponding to the overall removal efficiency of 90% for CODtotal and 98% for BOD5 total. The total process provided a final effluent containing a low concentration of 12 mg/l for TSS.Eighty-six percent of ammonia was eliminated at space loading rate of 1.6 kg COD/m3  d and HRT of 2.7 h. The calculated nitrification rate of the DHS system according to the nitrate and nitrite production amounted to 0.18 kg/m3  d. The removal of F. coliform in the UASB reactor only amounted to 0.86 log10. On the other hand, the F. coliform concentration dropped substantially, i.e. by 2.6 log10 in the DHS system resulting only 2.7 × 103/100 ml in the final effluent.The calculated average sludge production for UASB operated at an HRT of 8.0 h amounted to 30 g TSS/d, corresponding to sludge yield coefficient of 0.2 g TSS/g total COD removed, while it was indeed very low only 6.0 g TSS/d corresponding to sludge yield coefficient of 0.09 g TSS/g total COD removed, for DHS system.The DHS profile results revealed that in the first and second segment of DHS system, the CODtotal, BOD5 total and TSS was eliminated, followed by the oxidation of ammonia in the next segments.
Keywords: Sewage; UASB; DHS; COD; Nitrification; F. coliform; DHS profile; OUR;

Enzymatic hydrolysis of triglycerides of soy deodorized distillate (DOD), using immobilized Candida rugosa lipase under supercritical carbon dioxide (SC-CO2) medium, was carried out. Optimization of the reaction parameters using response surface methodology based on Box-Behnken model at three levels of pressure (120–180 bar), temperature (40–60 °C) and moisture content (40–80% of triglyceride content) for maximum hydrolysis of triglycerides was arrived by multilinear regression of the experimental results. The optimum conditions for maximum degree of triglyceride hydrolysis (94%) were found to be: pressure of 180 bar, temperature of 43 °C and moisture content of 40% to the triglyceride content. Maximum degree of hydrolysis was achieved with short incubation time of 1.5 h under SC-CO2. Whereas conventional method of hydrolysis in hexane under similar reaction conditions of temperature, moisture and enzyme concentration, needs 5 h to achieve 88% of triglyceride hydrolysis.
Keywords: Soy deodorized distillate (DOD); Supercritical carbon dioxide (SC-CO2); Lipase; Immobilization; Zeolite; Optimization;

Phenol biodegradation by the yeast Candida tropicalis in the presence of m-cresol by Jiang Yan; Wen Jianping; Bai Jing; Wang Daoquan; Hu Zongding (227-234).
Biodegradation of phenol and m-cresol using a pure culture of Candida tropicalis was studied. The results showed that C. tropicalis could degrade 2000 mg l−1 phenol alone and 280 mg l−1 m-cresol alone within 66 and 52 h, respectively. The capacity of the strain to degrade phenol was obviously higher than that to degrade m-cresol. The presence of m-cresol intensely inhibited phenol biodegradation. Only 1000 mg l−1 phenol can be completely degraded in the presence of 280 mg l−1 m-cresol. On the contrary, the phenol of low concentration from 100 to 500 mg l−1 supplied a sole carbon and energy source for C. tropicalis in the initial phase of biodegradation and accelerated the assimilation of m-cresol, resulting in the fact that m-cresol biodegradation velocity was higher than that without phenol. Besides, the capacity of C. tropicalis for m-cresol biodegradation was increased up to 320 mg l−1 with the presence of 60–100 mg l−1 phenol. In addition, the intrinsic kinetics of cell growth and substrate degradation were investigated with phenol and m-cresol as single and mixed substrates in batch cultures. The results illustrated that the models proposed adequately described the dynamic behaviors of biodegradation by C. tropicalis.
Keywords: Biodegradation; Candida tropicalis; m-Cresol; Kinetics; Phenol;

Fed-batch production of recombinant fuculose-1-phosphate aldolase (FucA) by Escherichia coli XL1 Blue MRF′ (pTrcfuc) has been automated by using a simple feedback specific growth rate control strategy. Non-induced continuous cultures were conducted in order to characterize substrate consumption and carbon dioxide production yields and rates. In fed-batch cultures, substrate feeding rate was adjusted using on-line biomass estimation based on exhaust gas analysis and macroscopic mass balances. Overexpression of recombinant protein induced by isopropyl-β-d-thiogalactopyranoside (IPTG) under trc promoter did not affect significantly the control of specific growth rate during 7 h after induction. Growth and protein production curves were parallel until high level of protein expression started to inhibit cell growth. The proposed specific growth rate control strategy has been successfully applied to both non-induced and induced fed-batch cultures that do not exhibit severe growth rate depression.
Keywords: Escherichia coli; Recombinant aldolases; Fed-batch culture; Enzyme production; Modelling; Feedback control;

Effect of agitation and aeration on the reduction of pollutant load of olive mill wastewater by the white-rot fungus Panus tigrinus by Alessandro D’Annibale; Daniele Quaratino; Federico Federici; Massimiliano Fenice (243-249).
The white-rot fungus Panus tigrinus CBS 577.79 was cultivated both in mechanical (stirred tank, STR) and pneumatically (bubble column, BCB) agitated bioreactors and investigated for its ability to reduce the polluting load of olive-mill wastewater (OMW). Both aeration and agitation strongly influenced treatment efficiency. Best pollutants biodegradation performances were achieved in the bubble column bioreactor. Using this bioreactor, COD reduction, dephenolization and decoloration were 60.9, 97.2 and 75%, respectively. In contrast, lower depollution efficiency was generally observed in STR due to the possible occurrence of shear stress.
Keywords: Olive mill wastewater; Biodegradation; Lignin modifying enzymes; Bubble column bioreactor; Aeration; Agitation;

Anaerobic thermophilic digestion of cutting oil wastewater: Effect of co-substrate by M. Perez; R. Rodriguez-Cano; L.I. Romero; D. Sales (250-257).
This paper describes the thermophilic (55 °C) anaerobic biodegradation of a mixed feed composed of vinasses and cutting oil wastewater (COW) in a laboratory upflow anaerobic fixed-film reactor (UAFF) with a porous support medium. The experimental protocol was defined to examine the effect of increasing the percentage of cutting oil wastewater in the feed.The UAFF reactor was initially started-up with vinasses as the only carbon source at an organic loading rate of 22.3 kg COD/m3  day and HRT of 0.8 days using porous particles as the support (SIRAN). The percentage of organic matter composed of vinasses was subsequently reduced while increasing the amount of cutting oil until 100% of cutting oil wastewater was added in the feed. Four stages were considered in the study (0, 42.4, 66.6 and 100% COW). HRT was adjusted in order to maintain an approximately constant organic loading rate applied to the system. Under theses conditions, the UAFF reactor was subjected to a programme of steady-state operation with hydraulic retention times (HRT) in the range 0.8–0.15 days and organic loading rates (OLR) between 22.3 and 14.9 kg COD/m3  day in order to evaluate the treatment capacity of the system.The COD removal efficiency was found to be 87% COD and 94.6% TOC in the reactor when treating vinasses at 22.3 kg COD/m3  day. The volumetric methane level produced in the digester reached 0.45 m3/m3  day. After an operating period of 120 days, the reactor was fed with cutting oil wastewater (COW) as the only source of carbon. An OLR of 16.7 kg COD/m3  day was achieved with 85.8% COD removal efficiency (58.1%TOC) in the experimental UAFF reactor. Under these conditions the volumetric methane produced in the digester was negligible.Hence, COW can be removed, if not degraded, by anaerobic treatment in the presence of a biodegradable co-substrate. Wine vinasses degradation creates conditions for non-biological removal of COW constituents. More studies are necessary in order to test the mechanisms of organic removal when biodegradation apparently had ceased. Also, toxicity assays of COW are necessary to evaluate the toxicity to the methanogenic community.
Keywords: Anaerobic digestion; Anaerobic fixed-film reactor; Thermophilic; Wine distillery wastewater; Cutting oil wastewater;

Cellulosic exopolysaccharide produced by Zoogloea sp. as a film support for trypsin immobilisation by A.H.M. Cavalcante; L.B. Carvalho; M.G. Carneiro-da-Cunha (258-261).
Trypsin (E.C. 3.4.21.4) was covalently immobilised onto a membrane of a cellulosic exopolysaccharide produced by Zoogloea sp. in sugarcane molasses. Carbonyl groups were introduced into the matrix by sodium metaperiodate oxidation and the enzyme was immobilised either directly or through bovine serum albumin (BSA) as a spacer. The trypsin-membrane and trypsin–BSA-membrane retained, respectively, 37.2% and 9.16% of the specific activity of the native enzyme acting on N-benzoil-dl-arginine-p-nitroanilide (BAPNA). No activity decrease was observed in both preparations after seven reutilisations as well as they showed to be more thermal stable than the native enzyme. The trypsin–BSA-membrane presented the same initial activity (99%) after 54 days stored in 0.1 M Tris–HCl buffer, pH 8.0, at 4 °C but the trypsin-membrane lost 15% of activity. Furthermore, the trypsin–BSA-membrane lost 31% of activity after reuse at 9 days interval during 54 days of storage at 4 °C whereas the trypsin-membrane lost 69% of activity under the same conditions. These results showed an additional application for this biofilm, namely, to act as a reusable matrix for trypsin immobilisation and the presence of BSA improved the derivative performance.
Keywords: Trypsin; Immobilisation; Immobilised enzymes; Biofilm; Cellulose; Zoogloea sp;

Relation between growth, respirometric analysis and biopigments production from Monascus by solid-state fermentation by Júlio Cesar de Carvalho; Ashok Pandey; Bruno Oliva Oishi; Débora Brand; José Angel Rodriguez-Léon; Carlos Ricardo Soccol (262-269).
Solid-state fermentation was carried out to establish relation between growth, respirometric analysis and biopigments production from Monascus sp. in columns and in a drum-type bioreactor with forced air. In these reactors, the best aeration rate for biopigment production was 1 ml of air, per gram of wet substrate, per minute. The outlet air composition was determined using gas chromatography (GC), while the pigments produced were measured by spectrophotometry after extraction with ethanol. An ergosterol-dosage method was used to estimate biomass production; in this method, the sterol was extracted and measured by liquid cromatography (HPLC). The results showed that although pigments were a secondary metabolite, its production was proportional to the biomass produced that was estimated by ergosterol analysis, and therefore could be used to estimate biomass formed in the natural support (rice). Specific velocities for pigment and biomass production were estimated by a sigmoid model applied to the data and also with the aid of a computer program nominated FERSOL. Under ideal conditions in column fermentation, a maximum specific growth velocity of 0.039 h−1 and a specific pigment production velocity of 27.5 AU/g biomass h was obtained, at 140 h, with 500 AU/g dry fermentate after 12 days. The specific product formation velocity in the bioreactor was 4.7 AU/g h, at 240 h fermentation, and the total pigment production was 108.7 AU/g dry fermentate after 15 days.
Keywords: Monascus; Aeration; Solid-state fermentation; Bioreactor; Kinetic parameters; Natural pigments;

Isomaltulose is a structural isomer of sucrose commercially used in food industries. Glucosyltransferase produced by Erwinia sp. D12 catalyses an intramolecular transglucosylation of sucrose giving isomaltulose. An experimental Design and Response Surface Methodology were applied for the optimization of the nutrient concentration in the culture medium for enzyme production in shaken flasks at 200 rpm and 30 °C. A higher production of glucosyltransferase (7.47 Uml−1) was observed in the culture medium containing sugar cane molasses (160 gl−1), bacteriological peptone (20 gl−1) and yeast extract Prodex Lac SD® (15 gl−1) after 8 h, at 30 °C. The highest production of glucosyltransferase in the 6.6-l bioreactor (14.6 Uml−1) was obtained in the optimized culture medium after 10 h at 26 °C. When Erwinia sp. D12 cells were immobilized in sodium alginate, it was verified that sodium alginate solution A could be substituted by a cheaper one, sodium alginate solution B. Using a 40% cell suspension and 2% sodium alginate solution B for cell immobilization in a packed-bed reactor, 64.1% conversion of sucrose to isomaltulose was obtained. The packed-bed reactor with immobilized cells plus glutaraldehyde and polyethylenimine solutions remained in a pseudo-steady-state for 180 h.
Keywords: Experimental design; Immobilized cells; Isomaltulose; Optimization; Packed-bed reactor; Response surface methodology;

Application of microthermometry to measurement of microbial activity and inactivation process by inhibitor by Hideo Maruyama; Akira Suzuki; Hideshi Seki; Norio Inoue (278-283).
A rapid and simple technique (microthermometry) was developed for the measurement of microbe's metabolic activities and its inactivation process by an inhibitor. To analyze the results and to determine the parameters for the estimation of the activity and inactivation degree, a simple model was proposed. Yeast cells (Saccharomyces cerevisiae) were used as a microbe. A differential method using two bead-type thermistors as reference and measuring probes was employed for the detection of a temperature change caused by the heat of metabolism of added carbon source (glucose) by the yeast. Experiments were conducted in a 30 °C water bath under non-growth conditions (without nitrogen source). The simple thermal response model was applied to obtain two characteristic parameters for the estimation of the yeast activities, i.e., a metabolic heat production rate, ΔQ, and a metabolic heat inhibition rate, ΔK. The proposed model was well in agreement with the experimental results, and the curve fitting gave ΔQ or ΔK. In the case of the addition of glucose to yeast as a carbon source, ΔQ was proportional to the number of live cell (CFU). The slope of CFU versus ΔQ for yeast in exponential phase was larger by about two-fold than that in the stationary phase. In the case of the addition of gultaraldehyde as an inhibitor to the above system, the logarithmic value of ΔK increased with increasing the concentration of gultaraldehyde within the range of 0.02–6.2 wt.%. It was suggested that the two model parameters, ΔQ and ΔK, can be regarded as characteristics to estimate the activities of bacteria and the degree of their inactivation by the inhibitor.
Keywords: Yeast; Glucose; Microbial activity; Metabolic heat; Thermistor; Bioprocess monitoring;