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Biochemical Engineering Journal (v.42, #2)

BEJ Keywords (pp. ii).

Editorial Board (pp. co2).

A predictive kinetic study of lipase-catalyzed ethanolysis reactions for the optimal reutilization of the biocatalyst by Carlos F. Torres; Tiziana Fornari; Daniel Tenllado; Francisco J. Señoráns; Guillermo Reglero (pp. 105-110).

A new application of the kinetics modeling for the optimal reutilization of an immobilized lipase from Pseudomonas cepacia in the ethanolysis of vegetable oils is presented. Two different rate expressions were explored to take into account the lipase inactivation. The methodology developed is based on the utilization of the pseudo reaction time that indicates how much longer the reaction mixture must remain in the reactor (actual reaction time) to achieve the conversion that would have been achieved if the enzyme had not been partially deactivated (pseudo reaction time).An initial batch of lipase was employed in 15 consecutive trials in order to quantitatively characterize the process over a range of lipase activity and to validate the ability of the methodology utilized to describe the kinetics of both ethanolysis and deactivation. Then, the model developed was employed to predict the time necessary to attain a desired conversion in subsequent reaction cycles in both, trials with the same batch of lipase with different degree of inactivation, and trials with a new batch of immobilized lipase with different specific activity.The reaction times predicted to attain a 38% disappearance of glyceryl ester bonds were experimentally verified by carrying out the corresponding ethanolysis reactions of 100g of sunflower oil. The agreement between the desired and experimentally attained conversions achieved validates the methodology developed to estimate reaction time in lipase-catalyzed ethanolysis reactions.

Keywords: Enzyme inactivation; Ethanolysis; Kinetics; Lipase; Pseudo reaction time

Supplementation of cobalt to UASB reactors by pulse dosing: CoCl₂ versus CoEDTA²⁻ pulses by Fernando G. Fermoso; Jan Bartacek; Li Ching Chung; Piet Lens (pp. 111-119).

The effect of chelation on the dosing strategy of cobalt to restore the performance of a cobalt limited methanol-fed bioreactor was investigated. Three upflow anaerobic sludge bed (UASB) reactors (30°C, pH 7.0) were operated with methanol as the substrate at an organic loading rate of 8.5gCODL⁻¹d⁻¹. One UASB reactor was supplied with several pulses of cobalt bound to EDTA, and its operation was compared to that of another UASB reactor to which several pulses of CoCl₂ were given. The addition of cobalt (5μmoles cobalt per litre of reactor volume) in the form of CoCl₂ creates a pool of cobalt in the granular sludge matrix due to the high cobalt retention (around 90%). The methanogens present in the granular sludge are able to use that cobalt pool for stable methane formation from methanol during the subsequent 15 days. When added as Co-EDTA²⁻, only around 8% of the cobalt added is retained. The small amount of retained cobalt in case of Co-EDTA²⁻ addition supports methylotrophic methanogenesis only a few operational days. Furthermore, the side-effects EDTA has on the granule matrix or microbial cells make EDTA an unsuitable ligand for cobalt dosage in full-scale applications.

Keywords: Cobalt; Dosing strategy; UASB; FISH; EDTA

Evaluation of a new packing material for H₂S removed by biofiltration by E. Dumont; Y. Andrès; P. Le Cloirec; F. Gaudin (pp. 120-127).

This study aims to evaluate the feasibility of using a new packing material (UP20) in treating H₂S. Three identical laboratory-scale biofilters, filled with, respectively, UP20 alone, pine bark, and a configuration made of two layers of pozzolan/UP20 (80/20, v/v), were used for critical comparison. Various concentrations of H₂S (up to 100ppmv) were used to determine the optimum biofilter performances. The superficial velocity of the polluted gas on each biofilter was 65mh⁻¹ (0.018ms⁻¹; gas flow rate 0.5Nm³h⁻¹) corresponding to an empty bed residence time of 57s. Changes in elimination capacity, removal efficiency, moisture content, temperature and pH were tracked during 95 days. The pressure drops along each biofilter were also measured by varying the gas flow rate from 0.5 to 4Nm³h⁻¹. After 63 days of operation, the loading rate was significantly increased to 10gm⁻³h⁻¹ and the UP20 biofilter retained a removal efficiency of more than 93%, indicating a strong ability to stimulate microbial activity (compared to 69% for the pine bark biofilter and 74% for the biofilter filled with a configuration of two layers of pozzolan/UP20). A Michaelis–Menten type equation was applied and the maximum removal rate ( V_m) and saturation constant ( K_s) were calculated. V_m was evaluated at35gH2Smbiofilter−3h−1 for UP20 (14 and15gH2Smbiofilter−3h−1 for pine bark and pozzolan/UP20, respectively). The saturation constant K_s was 70ppmv for UP20 (18ppmv for pine bark and 20ppmv for pozzolan/UP20) indicating that the new packing material will be effective in treating large pollutant concentrations. At low concentrations of pollutant, the results suggest that a biofilter with a configuration of two layers of pozzolan/UP20 is the most suitable choice for treating H₂S.

Keywords: Biofiltration; Packing material; Odorous compounds; Biodegradation; Pressure drops; Hydrogen sulphide

Production of lignin modifying enzymes on industrial waste material by solid-state cultivation of fungi by Erika Winquist; Ulla Moilanen; Aila Mettälä; Matti Leisola; Annele Hatakka (pp. 128-132).

In this study we investigated the use of three industrial waste materials as substrates for white-rot fungi in solid-state cultivation (SSC). The materials used were oat husks and waste from paper process industry: fibre sludge and combined fibre and de-inking sludge (FDS). The aim of the work was to find suitable fungi able to grow and produce lignin modifying enzymes, manganese peroxidase (MnP) and laccase, on the used waste materials. Fifteen strains were tested. The best enzyme producing strains were Trametes versicolor K 120a2 and Cerrena unicolor T 71. C. unicolor T 71 showed the greatest potential for industrial use because of its ability to produce significant amounts of both enzymes simultaneously in short time period. The highest enzyme activities (MnP 64nkat/g DW and laccase 178nkat/g DW) were obtained on the medium containing sole oat husks or oat husks supplemented with 20% (w/w) of FDS.

Keywords: Solid-state cultivation; Oat husks; Trametes versicolor; Cerrena unicolor; Manganese peroxidase; Laccase

Optimization of culture conditions to enhance cis-epoxysuccinate hydrolase production in Escherichia coli by response surface methodology by Haifeng Pan; Zhipeng Xie; Wenna Bao; Jianguo Zhang (pp. 133-138).

The effect of culture conditions on the cis-epoxysuccinate hydrolase (CESH) production in recombinant Escherichia coli BL21 was investigated using response surface methodology (RSM), which was based on rotatable central composite design. The optimization of seed conditions consisted of a total of 13 experiments involving 4 star points and 5 replicates at the central points, while the optimization of induction conditions consisted of a total of 31 experiments involving 8 star points and 7 replicates at the central points. The optimum predicted culture conditions for maximum expression of recombinant CESH were found to be comprised of 11.75h seed age, 4.13% (v/v) inoculation level, OD₆₀₀ 0.2 induction-starting time, 2.53% (w/v) lactose, 24.29°C post-induction temperature and 27.56h post-induction time, with a predicted CESH activity of 40,460U/g, which was very close to the experimental CESH activity of 40,129U/g resulting in 4.6-fold increment after optimization.

Keywords: Cis; -epoxysuccinate hydrolase; Optimization; Response surface methodology; Enzyme production; Fermentation; Lactose

A kinetic study of chlorinated solvent cometabolic biodegradation by propane-grown Rhodococcus sp. PB1 by Dario Frascari; Davide Pinelli; Massimo Nocentini; Emilia Baleani; Martina Cappelletti; Stefano Fedi (pp. 139-147).

An aerobic bacterial strain, named PB1, was microbiologically characterized and subjected to assays of cometabolic biodegradation of chlorinated aliphatic hydrocarbons (CAHs) aimed at (i) investigating its capacity to biodegrade vinyl chloride (VC), cis-1,2-dichloroethylene ( cis-DCE), 1,1,2-trichloroethane (1,1,2-TCA), chloroform (CF) and 1,1,2,2-tetrachloroethane (1,1,2,2-TeCA); and (ii) evaluating the suitability of a kinetic model with inhibition and transformation product toxicity to describe CAH biodegradation by PB1. The strain proved to be closely related to the genus Rhodococcus. It biodegraded VC, cis-DCE, 1,1,2-TCA and CF via aerobic cometabolism on propane, but not 1,1,2,2-TeCA. CAH biodegradation by PB1 was satisfactorily described by the tested kinetic model. The maximum-specific biodegradation rates were in the 1–11μmol/(mg_proteinday) range. The transformation capacities varied from 0.6 to 3μmol/mg_protein. Propane exerted a significant inhibition on both CF and cis-DCE biodegradation. CF, at 52μM, did not inhibit propane utilization. An analysis of different inhibition models showed that cis-DCE inhibition on propane utilization was satisfactorily simulated with the competitive model, with an inhibition constant equal to 70% of the cis-DCE half-saturation constant. A final assay specifically aimed at validating the kinetic model and verifying the estimates of the kinetic parameters yielded a satisfactory result. The results provide positive indications on the feasibility of utilizing strain PB1 for CAH biodegradation in aquifers and wastewaters.

Keywords: Biodegradation; Modeling; Aerobic processes; Kinetic parameters; Cometabolism; Chlorinated solvents

Antimicrobial activity of celery fruit isolates and SFE process modeling by Dušan Mišić; Irena Zizovic; Marko Stamenić; Ružica Ašanin; Mihailo Ristić; Slobodan D. Petrović; Dejan Skala (pp. 148-152).

The antimicrobial activity of celery fruit essential oil obtained by supercritical fluid extraction with carbon dioxide and by hydrodistillation was investigated against Staphylococcus aureus, Listeria monocytogenes and Listeria ivanovii strains, isolated from victuals of animal origin and from clinical specimens. The supercritical extract was moderately or much more effective than the essential oil obtained by hydrodistillation against almost all the investigated strains, except in the case of one Listeria strain originating in minced meat, where the MIC values were identical. For two S. aureus strains originating in minced meat, supercritical extract had very strong antimicrobial activity, with MIC values of ≤40μg/ml. The chemical composition of the supercritical extract as well as of the oil obtained by hydrodistillation of dried fruits was analyzed. Qualitative and quantitative analyses were performed using GC/FID and GC/MS. The supercritical extract was greatly superior to the oil obtained by hydrodistillation in terms of the concentration and quantity of the isolated bioactive compounds: sedanenolide, sedanolide and 3- n-butylphthalide. The yields of the supercritical extraction and hydrodistillation were 2.74 and 1.63wt.%, respectively. The process of supercritical fluid extraction was modeled using a model on the secretory structure scale. The modeling results showed good agreement with the experimental data.

Keywords: Supercrirical fluid extraction; Antimicrobial activity; Celery (; Apium graveolens; ); Modeling; Mass transfer; Food engineering

Kefir grains production—An approach for volume optimization of two-stage bioreactor system by Andreja Goršek; Marko Tramšek (pp. 153-158).

The main objective of the present study was to design a continuous two-stage bioreactor system for kefir grains production. Based on the experimental data of time-depended kefir grain mass increase and average pH profile during kefir grains batch propagation, parameters of predictive growth and exponential pH models were initially estimated. Afterwards, the non-linear programming (NLP) optimization problem for estimating the optimal volumes of two unequal in series connected continuously stirred tank bioreactors (CSTB) has been developed. The NLP problem is based on criterion of minimal total holding time (MTHT), kefir grain mass balances in CSTB and parameters of the growth and pH models. The results showed that novel kefir grains production plant with two-stage continuous operation, at capacity, q m_,KG,pr=3kgh⁻¹ and conversion, X_o,2=0.980, would primarily require investment in two CSTB with V₁=5.93m³ and V₂=5.62m³, respectively. Technological and economical authorization for a two-stage continuous bioreactor system was mainly confirmed with a more than five times lower total bioreactor volume compared to one-stage plant.

Keywords: Abbreviations; CSTB; continuously stirred tank bioreactor; GAMS; general algebraic modeling system; MTHT; minimal total holding time; NLP; non-linear programmingKefir grains production; Bioreactor design; Modeling; Optimization; Continuous operation mode

Kinetic modeling of glucose and fructose dissolution in 2-methyl 2-butanol by Jean-Marc Engasser; Françoise Chamouleau; Latifa Chebil; Mohamed Ghoul (pp. 159-165).

With the objective of developing simulation models for sugar bioconversion processes in non-aqueous media, the present study investigated the dissolution kinetics of glucose and fructose in 2-methyl 2-butanol at temperatures between 20°C and 80°C. For both sugars a two-phase dissolution process was observed, characterized by an initially fast dissolution lasting a few minutes, followed by a much slower dissolution phase extending up to 24h. The experimental results are described by a combined sugar dissolution and mutarotation kinetic model that considers the dissolution of the sugar anomeric form present in the solid particles, namely α-d-glucopyranose and ß-d-fructopyranose, and its subsequent mutarotation in solution. The initial dissolution step is assumed limited by the solute transport from the surface to the bulk solution, and the corresponding sugar transport coefficient evaluated from established mass transport correlations. The second slower dissolution phase is solely controlled by the sugar mutarotation rate, and modeled as a first-order reversible reaction. The determined values of the mutarotation rate and equilibrium constants can be related to the solution temperature by an Arrhenius and Van’t Hoff relationship, respectively.

Keywords: Glucose; Fructose; Solvent; Dissolution; Solubility; Mutarotation; Kinetics; Modeling

The leaching of pentlandite by Acidithiobacillus ferrooxidans with a biological–chemical process by Xiaojuan Yang; Xiaoxue Zhang; Yanli Fan; Hongyu Li (pp. 166-171).

The Fe³⁺ leaching solution which was produced by Acidithiobacillus ferrooxidas was used to leach pentlandite. The effects of several kinetic parameters including temperature, Fe³⁺ concentration and Cl⁻ on Ni recovery were investigated in a container independently. The results showed that the leaching rate of Ni was favored by the rise of temperature, the increase of Fe³⁺ concentration and the partial replacement of SO₄²⁻ by Cl⁻, all of which could prove the indirect mechanism for sulfides leaching. Then a bioleaching process comprising biooxidation and chemical leaching is proposed. In the biooxidation process, A. ferrooxidans was used to produce Fe³⁺ at 30°C, which was then fed to the chemical process to oxidize pentlandite at 50°C. This biological–chemical leaching process features the continuous production of Fe³⁺ in the bioreactor and the high temperature leaching of pentlandite in the chemical reactor. By using this process, Ni leaching rate of 83.8% was obtained at 5 days. According to the results, the present heap leaching process could be improved by the results obtained by this biological–chemical leaching process.

Keywords: Mesophiles; Biological–chemical process; Bioconversion; Kinetic parameters; Optimization; Acidity

Isolation and comparison of biosurfactants produced by Bacillus subtilis PT2 and Pseudomonas aeruginosa SP4 for microbial surfactant-enhanced oil recovery by Orathai Pornsunthorntawee; Nampon Arttaweeporn; Sarawut Paisanjit; Pastra Somboonthanate; Masahiko Abe; Ratana Rujiravanit; Sumaeth Chavadej (pp. 172-179).

In this present study, two types of biosurfactant-producing microorganisms, Bacillus subtilis PT2 and Pseudomonas aeruginosa SP4, were isolated from sludge oil and petroleum-contaminated soil in Thailand, respectively. The biosurfactant production was done by using a nutrient broth with palm oil as the carbon source. The microbial growths were investigated, and the best cultivation times for the biosurfactant production by B. subtilis PT2 and P. aeruginosa SP4 were found to be 51 and 48h, respectively. After the microbial cultivations at 37°C under the optimum conditions, the biosurfactants produced by B. subtilis PT2 and P. aeruginosa SP4 were found to reduce the surface tension of pure water to 26.4 and 28.3mN/m with the critical micelle concentrations (CMCs) of about 25 and 120mg/l, respectively. From the results of the oil recovery tests, the biosurfactant produced by B. subtilis PT2 exhibited oil recovery efficiency higher than that produced by P. aeruginosa SP4. Moreover, it was found that both biosurfactants could recover oil more effectively than three synthetic surfactants, including polyoxyethylene sorbitan monooleate (Tween 80), sodium dodecyl benzene sulfonate (SDBS), and sodium alkyl polypropylene oxide sulfate (Alfoterra 145-5PO).

Keywords: Biosurfactants; Bacillus subtilis; Pseudomonas aeruginosa; Oil recovery; Growth kinetics; Microbial growth

Ethanol production from cheese whey powder solution in a packed column bioreactor at different hydraulic residence times by Serpil Ozmihci; Fikret Kargi (pp. 180-185).

Cheese whey powder (CWP) solution containing 50gL⁻¹ total sugar was fermented to ethanol in a continuously operated packed column bioreactor (PCBR) using olive pits as support particles for cell attachment. Pure culture of Kluyveromyces marxianus (DSMZ 7239) was used in the PCBR for ethanol formation from lactose content of CWP solution. Sugar utilization and ethanol formation were investigated as function of the hydraulic residence time (HRT) between 17.6 and 64.4h. Sugar concentration decreased with increasing ethanol concentration along the height of the column. Percent sugar utilization increased while effluent sugar concentration was decreasing with HRT between 17.6 and 50h. Similarly, effluent ethanol concentration increased while ethanol productivity was decreasing with increasing HRT up to 50h. Further increases in HRT above 50h resulted in decreases in effluent ethanol concentration. The ethanol yield coefficient also increased with increasing HRT and reached the highest level of 0.54gEg⁻¹S at an HRT of 50h. Due to cell settling to the bottom of the column, high fermentation rates were obtained in the lower section of the system. Therefore, the system can be operated with a height of 36cm from the inlet to obtain high ethanol contents in the effluent with an HRT of 18h.

Keywords: Cheese whey powder; Ethanol; Fermentation; Hydraulic residence time; Packed column bioreactors; Yeast

Increasing biogas production by thermal (70°C) sludge pre-treatment prior to thermophilic anaerobic digestion by Ivet Ferrer; Sergio Ponsá; Felícitas Vázquez; Xavier Font (pp. 186-192).

The objective of this work was to investigate the effect of a low temperature pre-treatment (70°C) on the efficiency of thermophilic anaerobic digestion of primary and secondary waste sludge. Firstly, effect of sludge pre-treatment time (9, 24, 48 and 72h) was evaluated by the increase in volatile dissolved solids (VDSs), volatile fatty acids (VFAs) and biogas production in thermophilic batch tests. Secondly, semi-continuous process performance was studied in a lab-scale reactor (5L) working at 55°C and 10 days solid retention time. The 70°C pre-treatment showed an initial solubilization effect (increasing VDS by almost 10 times after 9h), followed by a progressive generation of VFA (from 0 to nearly 5gL⁻¹ after 72h). Biogas production increased up to 30% both in batch tests and in semi-continuous experiments. Our results suggest that a short period (9h) low temperature pre-treatment should be enough to enhance methane production through thermophilic anaerobic digestion of sludge.

Keywords: Anaerobic processes; Biosolid; Thermal pre-treatment; Thermophiles; Waste-water treatment; Waste treatment

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Biochemical Engineering Journal (v.42, #2)