Biochemical Engineering Journal (v.36, #3)

Biphenyl was successfully degraded by Burkholderia xenovorans LB400, initially described as Pseudomonas sp. LB400, in a solid–liquid two-phase partitioning bioreactor (TPPB). Solid–liquid TPPBs are comprised of an aqueous, cell containing phase, and a solid polymeric phase that partitions toxic and/or poorly soluble substrates (in this case biphenyl) based on maintaining a thermodynamic equilibrium. The employed polymer was Hytrel™, a thermoplastic polyester elastomer. The surface area available for mass transfer of biphenyl was limiting and resulted in mass transfer limited growth, as demonstrated experimentally by employing two different geometric shapes (cylinders with different aspect ratios) of the polymer phase with different specific surface area, while keeping all other parameters constant. The linear microbial growth rates were substantially higher when more polymer surface area was provided.The mass transfer coefficient of biphenyl from Hytrel™ to water was measured under experimental conditions, which allowed predicting the release rate based on the biphenyl concentration gradient. The partitioning behaviour of biphenyl between Hytrel™ and culture medium was measured as well, which allowed the development of a simple mechanistic model describing microbial growth based on known microbial properties in combination with substrate delivery from the solid polymer phase. The model was capable of describing the experimental data well and can be used to predict degradation rates for other geometric shapes of a solid delivery phase such as sheets or rods, which might be of operational advantage in various applications of TPPBs to the controlled uptake and release of other recalcitrant molecules.
Keywords: Availability; Mass transfer; Biokinetics; Modelling; Multiphase bioreactors; Burkholderia xenovorans LB400;

Palatinose production by free and Ca-alginate gel immobilized cells of Erwinia sp. by Haroldo Yukio Kawaguti; Hélia Harumi Sato (202-208).
Palatinose is a non-cariogenic disaccharide obtained from the enzymatic conversion of sucrose, used in food industries as a sugar substitute. Free and Ca-alginate immobilized cells of Erwinia sp. D12 were used to produce palatinose from sucrose. Palatinose production was studied in a repeated-batch process using different immobilized biocatalysts: whole cells, disrupted cells and glucosyltransferase. Successive batches were treated with the immobilized biocatalyst, but a decrease in palatinose production was observed. A continuous process using a packed-bed reactor was investigated, and found to produce 55–66% of palatinose during 17 days using immobilized cells treated with glutaraldehyde and a substrate flow speed of 0.56 ml min−1. However, immobilized cells in a packed-bed reactor failed to maintain the palatinose production for a prolonged period. The free cells showed a high conversion rate using batch fermentation, obtaining a palatinose yield of 77%. The cells remained viable for 16 cycles with high palatinose yields (65–77%). Free Erwinia sp. D12 cells supported high production levels in repeated-batch operations, and the results showed the potential for repeated reuse.
Keywords: Biocatalysis; Free-cells; Immobilized cells; Packed-bed bioreactor; Palatinose; Repeated-batch process;

Microbial fuel cell (MFC) attracts growing efforts as a kind of environmentally friendly biotechnology. This study examined effects of the bio-factors (anode inoculums species, inoculums concentration), as well as abio-factors (cathode electron acceptor and proton exchange material) on electricity production of a dual-chamber mediatorless MFC in fed-batch mode. MFCs inoculated with pure culture (Rhodoferax ferrireducens) and mixed culture (activated sludge) obtained the close peak voltages of around 0.18 V and had the similar coulombic yields of about 75 C using monosodium glutamate wastewater (MGW) as substrate. MFCs with different concentrations of inoculums (5%, 10% and 15%) also achieved the similar peak values of around 0.2 V after 30 days’ growth although they were different at the early stage. By replacing cathode oxygen-saturated solution with 10 mmol/L Fe(III)NTA or 10 mmol/L K3Fe(CN)6 solutions, voltage output nearly doubled (0.35 V). However the replacing of proton exchange membrane with salt bridge leaded to a marked decrease of voltage output. These results suggest that electricity production was more significantly influenced by cathode electron acceptor and proton exchange material, less affected by the inoculums species and inoculums concentration.
Keywords: Batch processing; Bioreactors; Biofilms; Glucose; Electricity production; Microbial fuel cell;

Study of Microcystis aeruginosa inhibition by electrochemical method by Yunfeng Xu; Ji Yang; Mingming Ou; Yalin Wang; Jinping Jia (215-220).
In recent years, algal blooms as a result of abnormal algae growth have been found accelerated eutrophication, being the source reason of producing odor and being obstacles of water purification systems. In order to control water quality and minimize the threat, the treatment of Microcystis aeruginosa (M. aeruginosa) by electrochemical method was investigated in this paper. Experiments were conducted to examine the effect of the operating conditions, such as different electrodes materials, current density and agitation on inhibition ratio of algae. The electrolysis was found to be quite effective for inhibiting M. aeruginosa and the results show that the higher current density could effectively improve the inhibition. The inhibition ratio of 91.51% could be obtained at current density of 12 mA cm−2. In addition, the changes of UV–vis spectra and fluorescence emission spectra further demonstrated the inhibitive behaviors of electrolysis on the growth of M. aeruginosa.
Keywords: Electrochemical method; Inhibition ratio; Microcystis aeruginosa; UV–vis; Fluorescence;

Selective recognition and large enrichment of dimethoate from tea leaves by molecularly imprinted polymers by Yongqin Lv; Zhixing Lin; Wei Feng; Xin Zhou; Tianwei Tan (221-229).
Molecularly imprinted polymers (MIPs), using dimethoate as the template molecule, were prepared for selective recognition and large enrichment of dimethoate from tea leaves. Six functional monomers were examined, which showed that MIP prepared by methyl methacrylate (MMA) had the largest imprinting factor of 7.9 for dimethoate. Molecular dynamics (MD) simulations were carried out for the six different molecular systems to investigate the recognition mechanism by predicting the interaction energies, the closest approach distances and the active site groups. The dynamic adsorption property of dimethoate on MMA-based-MIP is demonstrated to accord with the Langmuir isotherm. MIP has a marked selectivity for dimethoate compared to other structurally related organophosphorous pesticides. Molecularly imprinted solid phase extraction (MISPE) has a large selective enrichment factor of approximately 100 for dimethoate from tea leaves. It is a useful tool in the trace detection of environmental engineering.
Keywords: Molecular imprinting; Solid phase extraction; Dimethoate; Molecular dynamics simulation; Adsorption isotherm; Environmental engineering;

Utilization of wastes as adsorbents or as raw materials in the preparation of activated carbon for environmental applications is an interesting alternative to commercial activated carbon commonly used for pollutants removal. Physical and chemical characteristics of the raw material play an important role in the properties of the carbonized waste. In this work two components of mango pit (the outer husk: SEMAVE-H and the inner seed: SEMAVE-S) were studied. The materials without carbonization were characterized by ultimate and proximate analysis. Cellulose, hemicellulose and lignin were quantified by the Crampton–Maynard, neutral detergent fiber (NDF) and acid detergent fiber (ADF) methods. The thermal behavior was studied by thermogravimetry and adsorption isotherms of water vapor, methylene blue and nitrogen were measured. Optic microscopy, Boehm's titration and IR spectroscopy were also used. SEMAVE-H contained more cellulose (39.4%) than hemicellulose (15.6%) and the inverse was revealed in SEMAVE-S. The thermal behavior of SEMAVE-S was complex; four peaks appeared in the DTG curves being the more intensive at 234 and 348 °C. For the husk and the seed S sp  ≤ 100 m2  g−1 and the acid groups were the most abundant in both materials.
Keywords: Mango pit; Characterization; Cellulose; Lignin; Adsorption; Wastewater treatment;

Dynamic simulation of benzene vapor treatment by a two-phase partitioning bioscrubber by David R. Nielsen; Andrew J. Daugulis; P. James McLellan (239-249).
A dynamic, mechanistic model has been developed to predict the performance of a liquid–liquid, two-phase partitioning bioscrubber (TPPB) for removal, and subsequent biodegradation, of toxic volatile organic compounds (VOCs) from industrial waste gases under various conditions of practical relevance. TPPBs, which contain an immiscible and biocompatible organic liquid phase, allow enhanced biodegradation rates to be maintained by partitioning inhibitory substrates away from microorganisms. The system being considered involves the treatment of benzene vapors by Achromobacter xylosoxidans Y234 using n-hexadecane as the organic phase. The model incorporates the following dynamic elements: volatile substrate and oxygen absorption by both liquid phases, partitioning of dissolved substrate and oxygen between liquid phases, and microbial consumption of dissolved substrate and oxygen in the aqueous phase for both growth and maintenance. Part I focuses on the development of the model equations and estimation of relevant parameters. Using parametric sensitivity analysis, the relative influences of the parameters are identified under transient and steady state conditions. Both the organic phase volume fraction and its properties are predicted to have a significant influence on performance. Biocatalysts capable of maintaining high biodegradation rates under dilute substrate concentrations are predicted to be superior for use in TPPBs.
Keywords: Bioscrubber; Biofiltration; Bioremediation; Modeling; Multiphase bioreactors; Sensitivity analysis;

Dynamic simulation of benzene vapor treatment by a two-phase partitioning bioscrubber by David R. Nielsen; Andrew J. Daugulis; P. James McLellan (250-261).
The two-phase partitioning bioscrubber (TPPB) model presented in Part I has been validated using experimental data under constant and transient operating conditions for the treatment of benzene waste gases by Achromobacter xylosoxidans Y234 with n-hexadecane as an immiscible, organic phase. Model calibration was performed to account for observed enhancements of benzene biodegradation rates in biphasic media, postulating that direct benzene uptake from dispersed organic droplets increased substrate bioavailability. This led to the use of an ‘effective dissolved substrate concentration’ to model cell specific growth rates. Model predictions were greatly improved using this empirical modification. The characteristics of the organic phase, both in terms of the volume fraction selected and its high equilibrium solubility, are found to be of critical importance for minimizing effluent gas VOC concentrations and stabilizing performance during transient operation. The biokinetic parameters μ max and K S are also particularly important, greatly influencing the response of the TPPB both during and while recovering from transient periods. K S was found to be important for influencing steady-state benzene removal efficiencies under even invariant operation, leading to the conclusion that microorganisms which can maintain high rates of biological activity under very dilute substrate concentrations will make ideal biocatalysts in the TPPB.
Keywords: Bioscrubber; Biofiltration; Bioremediation; Modelling; Multiphase bioreactors;

Optimization of five process parameters (concentrations of cassava bagasse, enzyme, yeast extract, NH4Cl and inoculum) was attempted using a Box-Behnken design for the optimal production of l(+)-lactic acid by a mixed culture of Lactobacillus casei and Lactobacillus delbrueckii by simultaneous saccharification and fermentation. Maximum lactic acid yield of 81 g/L was obtained when 15% (w/v) cassava bagasse treated with 12.5 mL/L enzyme mixture was supplemented with 7.5 g/L yeast extract and 3 g/L NH4Cl and inoculated with 3 × 1010  CFU/L of lactobacilli and incubated for 60 h at 37 °C as static culture.
Keywords: Cassava bagasse; l(+)-Lactic acid; Lactobacillus; Response surface optimization; Saccharification; Fermentation;

Response surface modeling of vanillin production by Escherichia coli JM109pBB1 by Danilo De Faveri; Paolo Torre; Bahar Aliakbarian; José Manuel Domínguez; Patrizia Perego; Attilio Converti (268-275).
The increasing concern for healthy and nutritional products together with the high price of natural vanillin extracted from vanilla pods has stimulated the search for alternative means of natural vanillin production. The present work deals with a ferulic acid to vanillin bioconversion process using the recombinant strain Escherichia coli JM109pBB1. A set of batch bioconversion tests was carried out on aqueous ferulic acid solutions according to a 32 full factorial design selecting the starting biomass concentration (X 0) and the initial ferulic acid concentration (S 0) as independent variables. The experimental data were analyzed by the response surface methodology (RSM) using a quadratic model for predicting the optimal point. The best starting biomass concentration and initial ferulic acid concentration were found to be 380 mg L−1 and 2.95 mM, respectively. Under these conditions the model predicted a maximum vanillin concentration ( P v ) of 1.45 mM, a vanillin yield on consumed ferulic acid ( Y v ) of 0.57 mM mM−1, a vanillyl alcohol on consumed ferulic acid (Y al) of 0.39 mM mM−1, a volumetric vanillin productivity ( Q v ) of 0.124 mM h−1 and a specific vanillin productivity ( q v ) of 0.047 g g−1  h−1.
Keywords: Vanillin; Ferulic acid; Escherichia coli; Optimization; Experimental design; Response surface methodology;

Taken impurities in fermentation broth into account, effect of concentration of fish waste hydrolyzate (FWH) on lactic acid production was investigated in this study. The efficiency of the fermentation using 6.8% FWH as a nutrient source was competitive to that using 1.5% yeast extract (YE), however, with a result of much higher impurities in the fermentation broth due to the much larger amount of addition of FWH. Reducing the amount of FWH reduced the amount of impurities in fermentation broth, but the fermentation efficiency dropped significantly. In order not to make fermentation efficiency drop, spent cells obtained after each fermentation were combined with FWH and their joint effect on lactic acid production was investigated. Spent cells as sole nutrient source did not show good performance in lactic acid production. Contrarily, the combination of the two nutrient sources showed complementary effect and the fermentation with the combination had much higher efficiency than that of either of the two nutrient sources. When 1.7% FWH was combined with spent cells, the fermentation efficiency was similar to that using 1.5% YE and the impurities were also in appropriate values. Finally, the feasibility of the fermentation process with the combination was determined.
Keywords: Lactic acid; Fermentation; Nutrient; Impurities; Fish waste hydrolyzate; Spent cells;

PCB and AOX removal in mesophilic and thermophilic sewage sludge digestion by T. Benabdallah El-Hadj; J. Dosta; R. Torres; J. Mata-Álvarez (281-287).
In this study, a comparison of the biodegradation of adsorbed organic halogen compounds (AOX) and polychlorinated biphenyls (PCB) in thermophilic and mesophilic anaerobic digestion (seeded with waste activated sludge) at different hydraulic retention times (HRT 18, 22 and 26 days in the mesophilic digester and 8, 12, 18, 22 and 26 days in the thermophilic digester) was performed. Results obtained in this work showed an enhancement of both PCB and AOX biodegradation under thermophilic conditions. The total PCB removal efficiency was in the range of 59.4–83.5% under thermophilic conditions and 33.0–58.0% under mesophilic conditions. HRT played an important role in the digester performance since high working HRTs implied more reduction of the total PCB amount in the sludge. The total PCB content in the treated sludge under thermophilic conditions lied below the cut-off limit proposed in the 3rd draft of Directive presented to the European Commission [CEC, Working Document on Sludge (3rd Draft), Commission of the European Communities Directorate-General Environment, ENV.E.3/LM, Brussels, 27 April 2000]. Besides, a bioaccumulation of lightly chlorinated PCBs was detected in the mesophilic digester, which is in concordance with the theory that the PCBs are anaerobically biodegraded by means of a reductive dechlorination mechanism. On the other hand, the AOX removal efficiency was in the range of 40.4–50.3% for thermophilic conditions and 30.2–43.2% for mesophilic conditions. The AOX content in the treated sludge of both thermophilic and mesophilic digesters did not exceed the cut-off limit proposed in the 3rd draft [CEC, Working Document on Sludge (3rd Draft), Commission of the European Communities Directorate-General Environment, ENV.E.3/LM, Brussels, 27 April 2000]. Moreover, high HRTs promoted an improvement of the AOX removal capacity of the anaerobic digestion.
Keywords: Anaerobic digestion; AOX; Mesophilic; PCB; Sewage sludge; Thermophilic;

Enhanced biodegradation of caffeine by Pseudomonas sp. using response surface methodology by Swati Sucharita Dash; Sathyanarayana N. Gummadi (288-293).
Pseudomonas sp. NCIM 5235 capable of degrading high concentrations of caffeine (>5 g/l) has been previously isolated from the soil of coffee plantation area. The critical medium components viz., KH2PO4, Na2HPO4, caffeine and Fe2+ affecting the rate of caffeine degradation were determined by Plackett–Burman design. These critical parameters were further optimized using response surface methodology. The optimum concentrations of KH2PO4, Na2HPO4, caffeine and Fe2+ were found to be 3.4 g/l, 0.352 g/l, 6.4 g/l and 0.075% (w/v), respectively. Under optimal conditions the caffeine degradation rate was increased to 0.18 g/(l h) from 0.1 g/(l h). Although the approach used for optimization is conventional, the increase in the rate of degradation of caffeine is significant. This is the first report on a strain capable of growing at higher concentrations of caffeine (>5 g/l) at maximum rate of degradation (0.18 g/(l h)).
Keywords: Caffeine degradation; Pseudomonas; Medium optimization; Plackett–Burman design; Central composite design;

The effect of graphite surface roughness on the immobilization of Acidithiobacillus ferrooxidans was studied. It was found that the most appropriate surface roughness was obtained by polishing graphite discs with 1500 grit polishing paper. After 24 h of inoculation this roughness had coverage of 25 × 103  cells/mm2. The effects of different types of activated carbon fibers (felt versus fabric; both silicated and non-silicated) on the immobilization of A. ferrooxidans were also experimentally studied. In non-silicated samples the activated carbon fiber support with the greatest surface area per gram (felt) lead to the greatest number of immobilized microorganisms over a 12 h period (29 × 103  cells/mm2). The addition of SiO2 particles to the activated carbon fiber supports considerably enhanced cell adhesion on both activated carbon felt and fabric. Furthermore, the silicated felt exhibited the greatest number of immobilized A. ferrooxidans cells of all activated carbon fiber preparations studied (36 × 103  cells/mm2).
Keywords: Acidithiobacillus ferrooxidans; Immobilization; Graphite; Activated carbon;

The effects of temperature and light irradiation (photoperiod) on growth and production of caffeic acid derivatives (caftaric acid, chlorogenic acid and cichoric acid) were studied in adventitious root cultures of Echinacea purpurea. Biomass accumulation and production of caffeic acid derivatives was optimal under incubation temperature of 20 °C among the different incubation temperatures tested (10, 15, 20, 25 and 30 °C). Biomass of adventitious roots was highest in cultures grown under dark while accumulation of caffeic acid derivatives was optimum in the cultures grown under 3/21 h light and dark cultural regimes.
Keywords: Adventitious root cultures; Caffeic acid derivatives; Echinacea; Photoperiod effect; Temperature effect;

In this study, we discuss diffusion based oxygen and glucose supplies in cell culture chips of three different setups, where cells are regularly arranged and cultivated, using oxygen and glucose transport models on the basis of the previous work. In the first setup, oxygen is continuously supplied through a gas-permeable wall equipped with the chip, but glucose is supplied only at the start. In this case, the life span of the cultivated cells is governed by the balance between the initial amount and the consumption rate of glucose. Secondly, a setup, where oxygen and glucose are continuously supplied through a semi-permeable membrane, is discussed. In this case, oxygen supply is so critical that the membrane must be well designed. Finally, a setup with reserves for glucose supply, where oxygen is supplied through a gas-permeable wall, is discussed. In this case, we can find an optimal thickness of the medium filled in the cell culture chamber because an increase in the thickness is advantageous to glucose supply but is contrary disadvantageous to oxygen supply. In all cases, cautious design of a cell chip is needed, if the consumption rates of the cultivated cells for oxygen and glucose are higher than 10 g m−3  s−1, which are very likely for hepatocytes.
Keywords: Transport model; Cell chip; Numerical simulation; Semi-permeable membrane; Hepatocytes; Hemispheroids;