Biochemical Engineering Journal (v.70, #C)

BEJ Keywords (IV).

► The trophic links between syntrophic acetogens and homoacetogens was studied. ► The butyrate degraders were stimulated by homoacetogens with BES addition. ► The possible organic carbon flux under suppressed methanogenesis was estimated.Acetate production during anaerobic sludge treatment has significant economic and environmental benefits. In this study, trophic links between syntrophic acetogens and homoacetogens in the anaerobic acidogenic fermentation of sewage sludge were investigated using methanogenic inhibitor 2-bromoethanesulfonate (BES) to block the methanogenesis pathway and butyrate to enhance syntrophic acetogenesis. The Gibbs free energies (ΔG) of the butyrate-degrading and homoacetogenic processes were close to the thermodynamic threshold of the reaction activity (−15 kJ/mol). In addition, microbial quantification analysis revealed that the growth of syntrophic acetogenic bacteria and homoacetogens in the treatment incubations was higher than that of the control. The results indicated that hydrogen-producing butyrate degraders are stimulated with homoacetogens when methanogenesis was specifically inhibited.
Keywords: Waste treatment; Sewage sludge; Anaerobic digestion; Acetic acid; Biodegradation; Homoacetogens; Syntrophic acetogens;

Hexane biodegradation in two-liquid phase bioreactors: High-performance operation based on the use of hydrophobic biomass by Raúl Muñoz; Eleonora I.H.H. Gan; María Hernández; Guillermo Quijano (9-16).
► An innovative operation mode in two-liquid phase bioreactors (TLPBs) for the treatment of volatile organic compounds (VOC) was investigated. ► The novel operation mode was based on confining the biocatalytic activity in the non-aqueous phase. ► A stable elimination capacity (EC) of 21.0 ± 2.5 g m−3  h−1 (corresponding to a removal efficiency of 80%) was recorded for 26 days. ► Aqueous culture broth exchange by fresh mineral salt medium allowed maintaining a high and sustained VOC removal performance.An innovative operation mode in two-liquid phase bioreactors (TLPB) for the treatment of volatile organic compounds (VOC) was investigated. This mode was based on confining the biocatalytic activity exclusively in the non-aqueous phase (NAP) by using hydrophobic microorganisms. The TLPB was implemented in a 2.5 L stirred tank reactor using 10% (v/v) of silicone oil as NAP and hexane as model VOC. A stable elimination capacity (EC) of 21.0 ± 2.5 g m−3  h−1 (corresponding to a removal efficiency of 80%) was recorded for 26 days. The accumulation of inhibitory metabolites resulted in drastic drops in the elimination capacity (EC) and an unstable performance of the system, hexanol being identified as potential inhibitory metabolite. Aqueous culture broth exchange by fresh mineral salt medium at a dilution rate of 0.2 day−1 allowed maintaining a high and sustained VOC removal performance. Dissolved oxygen concentration measurements revealed that the oxidative metabolism was strongly stimulated by the aqueous broth exchange. The temporary blockage of the gas/water/NAP transfer pathway for O2 highlighted the paramount role of this pathway on the performance of the TLPB based on hydrophobic microorganisms.
Keywords: Biological gas treatment; Hexane; Hydrophobic microorganisms; Two-liquid phase bioreactors; Volatile organic compounds;

Combined process for 2,4-Dichlorophenoxyacetic acid treatment—Coupling of an electrochemical system with a biological treatment by Jean-Marie Fontmorin; Florence Fourcade; Florence Geneste; Didier Floner; Samuel Huguet; Abdeltif Amrane (17-22).
► An electrochemical pre-treatment based on a home-made flow-cell was implemented. ► An aerobic biological treatment was then carried out using activated sludge. ► Pretreatment led to a quick decrease of DOC during the biological process. ► The electrochemical pretreatment shortened the length of the biological treatment.A coupled process was studied for the removal of a chlorinated pesticide: 2,4-Dichlorophenoxyacetic acid (2,4-D). A home-made electrochemical flow cell was used for the pre-treatment and a biological treatment was then carried out using activated sludge supplied by a local wastewater treatment plant. 2,4-D was used as a target compound for the study. Several parameters were monitored during the biological treatment, like dissolved organic carbon (DOC), the target compound and the major by-product. Pretreatment led to a quick decrease of DOC during the biological process, since a 66% mineralization yield was measured after the second day, and 79% after the seventh day of culture. After two days of treatment, HPLC results revealed a total degradation of Chlorohydroquinone, the major by-product. The electrochemical pretreatment shortened the length of the biological treatment, since DOC measurements showed that in the case of non-pretreated 2,4-D, no mineralization was observed before day 7. These promising results should be subsequently confirmed on commercial 2,4-D-containing solutions and then on real effluents.
Keywords: 2,4-D; Carbon felt electrode; Electrochemical pre-treatment; Biodegradability; Activated sludge; Biological treatment;

Evidence of structural changes of an enzymatic extract entrapped into alginate beads by Cristián Omar Illanes; Evelina Quiroga; Gerardo Enrique Camí; Nelio Ariel Ochoa (23-28).
► We analyzed the structural changes of araujiain entrapped into alginate beads. ► A small red shift in the spectrum of the enzyme entrapped was observed indicating that Trp residues have more polarizable surroundings. ► A secondary structure with a high α-helical character was responsible for the highest activity of entrapped araujiain. ► Interactions between the enzyme extract and Ca alginate caused different structural behavior in araujiain. ► Entrapment of araujiain augments the thermal stability of both the enzyme extract and Ca alginate.In this work, we analyzed the structural changes of araujiain entrapped into alginate beads. Araujiain is an enzymatic preparation containing three known enzymatic fractions with each fraction individually presenting a similar catalytic performance. Fluorescence and infrared spectroscopy, thermal analysis and residual catalytic activity studies were carried out. A small red shift in the spectrum of araujiain was observed after the entrapment process. Changes in the polarity around the tryptophan (Trp) residues were associated with an enzyme conformational change. From the Fourier transform infrared spectroscopy (FTIR) analysis, it was demonstrated that interactions between the enzyme extract and Ca alginate caused different structural behavior in araujiain. According to the diffuse reflectance infrared Fourier transform spectroscopy (DRIFT) study, it was possible to conclude that a secondary structure with a high α-helical character was responsible for the highest activity of entrapped araujiain. Finally, from thermal analysis measurements, it was proved that entrapment of araujiain augments the thermal stability of both the enzyme extract and Ca alginate, indicating a possible interaction between enzyme extract and its support.
Keywords: Araujiain; Entrapment; Alginate beads; Conformational changes; Enzyme stabilization; Immobilization of enzymes;

Ultrasound-assisted intensification of bio-catalyzed synthesis of mono-N-alkyl aromatic amines by Hyacintha R. Lobo; Balvant S. Singh; Dipak V. Pinjari; Aniruddha B. Pandit; Ganapati S. Shankarling (29-34).
Display Omitted► Significant improvement of bio-catalyzed process for selective N-alkylation of aromatic amines. ► Mild, efficient and environmentally benign process. ► Recyclability of lipase shows good stability of bio-catalyst in ultrasound. ► Optimization and comparative studies reveal faster reaction in comparison to thermal method. ► Ultrasound emerges as an efficient alternative to conventional procedure in lipase-catalyzed reaction.Bio-catalyzed process for mono-N-alkylation of primary aromatic amines was considerably improved by the use of sonochemical energy. The optimized method exhibited good tolerance toward various aromatic primary amines as well alkylating agents. A comparative study was done by carrying out the bio-catalyzed reaction in the presence of ultrasound and non-ultrasound conditions both at room temperature and elevated temperature. The results showed that ultrasonication gave enhanced yields in shorter reaction times. The optimization studies also included variation in organic solvents, amount of catalyst and reaction temperature. These studies conclusively suggest that ultrasound technique improved the activity of catalyst thereby saving time and energy, improving the selectivity and yield of the product. In addition, the bio-degradable and non-toxic bio-catalyst was easily recycled till five consecutive runs.
Keywords: Ultrasound; Mono-N-alkylation; Enzymes; Bio-catalysis; Enzyme deactivation; Lipase;

Biodegradation kinetics of dibenzoate plasticizers and their metabolites by Azadeh Kermanshahi pour; Ranjan Roy; David G. Cooper; Milan Maric; Jim A. Nicell (35-45).
► Biodegradation kinetics of dibenzoate plasticizers was investigated. ► The effect of alkyl branches and ether function on biodegradation rate was studied. ► Dibenzoates were degraded to the monobenzoates as result of microbial hydrolysis. ► Removing the ether bond enhanced the biodegradation of the monobenzoates.The kinetics of the biodegradation of two commercial plasticizers, diethylene glycol dibenzoate (D(EG)DB) and dipropylene glycol dibenzoate (D(PG)DB), as well as two alternative plasticizers, 1,3-propanediol dibenzoate and 2,2-methyl-propyl-1,3-propanediol dibenzoate, were investigated in an aerated bioreactor. The experiments were conducted with resting cells of Rhodococcus rhodochrous, which had been grown with hexadecane as the substrate. The first step in the biodegradation was always the hydrolysis of an ester bond, releasing the corresponding monobenzoate and benzoic acid. Biodegradation of plasticizers and their associated metabolites were modeled using a Monod-type kinetic model. Significant differences between the biodegradation of commercial and alternative plasticizers were observed both in the biodegradation pathway and the biodegradation rates of monobenzoate metabolites. At a selected concentration of 0.4 g/L, the monobenzoates released from the biodegradation of 1,3-propanediol dibenzoate and 2,2-methyl-propyl-1,3-propanediol dibenzoate were degraded approximately 13 and 4 times more quickly, respectively, than the monobenzoate released from the biodegradation of D(PG)DB. The rapid biodegradation of monobenzoates released from microbial hydrolysis of alternative dibenzoate plasticizers was attributed to the lack of an ether bond in these compounds.
Keywords: Biodegradation; Plasticizers; Dibenzoate; Metabolites; Kinetics;

► Carboxyl ionic liquid modified SBA-15 is a good carrier for PPL immobilization. ► Structure and surface properties of the carriers affect the catalysis performance. ► After being covalently immobilized, the PPL exhibit the best stability.The mesoporous silica SBA-15 was modified by carboxyl-functionalized ionic liquid (COOH-IL-SBA). The prepared support was used to immobilize porcine pancreatic lipase (PPL) by physical adsorption (PPL-COOH-IL-SBA) and covalent attachment (PPL-CON-IL-SBA). Enzymatic properties of the immobilized PPL were investigated in the triacetin hydrolysis reaction. It was found that carboxyl functionalized ionic liquid modification of the support surface was an effective method to improve the properties of immobilized PPL. Incorporating into the functionalized SBA-15 made PPL more resistant to temperature and pH changes, compared with PPL immobilized on parent SBA-15 (PPL-SBA). Especially, after the covalent attachment to a functionalized support, the stability of PPL was improved obviously, which retained 81.25% and 52.50% of the original activity after incubation for 20 days and four times recycling, respectively, whereas PPL-SBA exhibited only 58.80% and 27.78% of the original activity under the same conditions. In addition, physical and chemical properties of the supports and immobilized PPL were characterized by small-angle X-ray powder diffraction (SAXRD), Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM), nitrogen adsorption, nuclear magnetic resonance (NMR) and thermogravimetry (TG). The images and data confirmed chemical modification in SBA-15 and PPL immobilization on the tested support.
Keywords: Carboxyl functionalized ionic liquid; Modification; Enzyme technology; Lipase; Immobilized enzymes; Biocatalysis;

Candida antarctica lipase A (CAL A) shows a superior activity in catalyzing syntheses of phytosteryl fatty acid (medium/long chain) esters, in which 93–98% yields could be achieved in 24 h.Display Omitted► β-Sitosteryl fatty acid esters (C2–C18) synthesized and identified by 1H NMR and FTIR. ► CAL A shows superior activity in catalyzing acylation of phytosterol to other lipases. ► CAL A achieves 6–14 times faster reaction rate over other lipases. ► 93–98% yield of β-sitosteryl esters achieved at 40–50 °C for 24 h.This work reported a novel approach to synthesize phytosterol (β-sitosterol as a model) fatty acid esters by employing Candida antarctica lipase A (CAL A) which shows a superior catalytic activity to other lipases. A series of β-sitosteryl fatty acid esters (C2–C18) have been successfully prepared with structural identification of products by 1H NMR and Fourier transform-infrared spectroscopy (FTIR). Compared to other immobilized lipases, CAL A achieves 6–14 times faster esterification of β-sitosterol with myristic acid. CAL A shows low activity toward short chain fatty acids (C2–C6), and remarkably high activity for medium and long chain ones (≥C8). Reaction time, temperature, enzyme load, substrate ratio and concentration, and solvent property are found to profoundly influence reaction rates. A pronounced correlation between enzyme activities and log  P values of solvents, among the solvents with a broad spectrum of log  P values, was observed. 93–98% yield of β-sitosteryl esters could be achieved with hexane as solvent, fatty acid (C8–C18)/β-sitosterol (1:1, mol:mol), 5–10% CAL A load at 40–50 °C for 24 h. This work demonstrated the promising potential of CAL A in bioprocess of phytosterols for value-added application.
Keywords: Phytosterol; β-Sitosterol; Fatty acids; Esterification; Candida antarctica lipase A (CAL A);

► HWRGWV demonstrates great potential for affinity purification of hIgM. ► DBC for hIgM with HWRGWV is highly dependent on linear flow rate. ► Final recovery and purity for hIgM purification is feedstock dependent. ► As high as 95% for both recovery and purity can be achieved for hIgM purification.Compared to immunoglobulin G purification with extensively studied affinity ligands such as protein A and protein G, little work has been done on affinity chromatographic purification of immunoglobulin M. Hexamer peptide ligand HWRGWV, previously shown to bind specifically to the Fc fragment of IgG, also demonstrated potential for IgM purification. This study presents further characterization and investigation of this ligand for its potential for purification of IgM. Different running conditions were employed in order to improve the recovery and purity of IgM. The final recovery and purity of the antibody is feedstock dependent, but can reach levels of both recovery and purity as high as 95%. The dependence of the recovery and purity on total loading amount and initial IgM concentration were investigated and discussed. Although relatively low dynamic binding capacities (DBC) in the range of 4.6–13.1 mg IgM/mL resin at linear flow rates from 173 to 35 cm/h were obtained for IgM compared to IgG because of the large molecular weight of IgM, the DBC value of HWRGWV for IgM is much greater than protein-based IgM affinity ligands found in the literature and is competitive with current commercially available affinity ligands, such as KAPTIVE-M, CaptureSelect IgM and Ultralink Immobilized Mannan Binding Protein.
Keywords: Affinity chromatography; Hexamer peptide; Human immunoglobulin; IgM; Dynamic binding capacity; Elution additive;

Effect of trace element supplementation on the mesophilic anaerobic digestion of foodwaste in batch trials: The influence of inoculum origin by Veronica Facchin; Cristina Cavinato; Francesco Fatone; Paolo Pavan; Franco Cecchi; David Bolzonella (71-77).
► The lack of trace elements may upset the anaerobic digestion process. ► The supplementation of Co, Mo, Ni, Se, W showed a process improvement. ► Mo showed to be the most beneficial element added.Batch anaerobic trials using a source-separated food waste as a substrate with inoculums of different origins were carried out under mesophilic conditions. Reactions were operated both with and without trace element (Co, Mo, Ni, Se, and W) supplementation. Supplementation with trace metals had either neutral or slightly negative effects with inoculums originating from reactors with a high background level of metals, such as those for the co-digestion of biowaste and waste activated sludge. For inoculums from reactors treating food waste only, which inherently contain low levels of trace metals, supplementation with these metals increased methane production. In particular, Mo concentrations in the range of 3–12 mg/kg dry matter and Se concentrations of 10 mg/kg dry matter increased methane production to as high as 30–40%. Supplementation with a metal mixture (Co, Mo, Ni, Se and W) increased the methane production to the range 45–65% for inoculums with low background concentrations of trace metals. These findings may have an important impact in the commercial production of methane from food waste.
Keywords: Anaerobic digestion; Food waste; Inoculum; Mesophilic; Trace elements;

► We described a more robust and productive high cell density cultures of E. coli. ► Double limitation of glucose and IPTG during induction phase avoids metabolic burden and acetic acid accumulation. ► Induction phase of high cell density cultures can be extended. ► Higher cell densities of induced cultures, specific product concentration, and thus volumetric productivity can be achieved. ► Novel production strategies in E. coli can be performed.The most common strategy to produce recombinant proteins using Escherichia coli as expression vector is fed-batch culture, since high cell density cultures strategies have successfully been applied. Several methodologies to limit the specific growth rate in order to control E. coli metabolism have been defined, demonstrating that cultures can be grown under glucose limitation up to high cell densities without accumulation of acetic acid. However, under induction conditions it has been observed that E. coli metabolism is reorganized again and leads to acetic acid accumulation, causing inhibition of cell growth and decreasing protein expression efficiency.We propose a double limitation strategy (glucose and IPTG) for E. coli fed-batch cultures to avoid the deregulation of the metabolism in the induction phase. Reducing the concentration of IPTG while keeping glucose growth limitation, the accumulation of acetic acid decreased. At an IPTG concentration of 0.03 mmol/g DCW no accumulation of acetic acid was observed during the induction phase, in contraposition to what has normally been observed.Although a slight reduction of protein expression rate was observed when applying this double limitation strategy, the bioprocess volumetric productivity was enhanced due to the capability to prolong the induction phase, reaching higher levels of protein production. Another advantage of this strategy is the reduction of media cost due to the lower level of IPTG used.
Keywords: Fed-batch culture; Escherichia coli; Bioprocess design; Substrate limitation; IPTG limitation; Acetic acid accumulation;

Enzymatic synthesis of Z-aspartame in liquefied amino acid substrates by Shinya Furukawa; Kazuhiro Hasegawa; Ichiro Fuke; Koji Kittaka; Terumitsu Nakakoba; Masahiro Goto; Noriho Kamiya (84-87).
► A new media for biotransformation aided by ionic liquefied amino acid substrates was designed. ► The amino acid ionic liquids play dual roles as substrates and reaction media. ► In this reaction media, effective peptide substrate concentration can be maximized. ► Thermolysin exhibited the catalytic activity in the presence of 10 vol% buffered water. ► The submolar range productivity in protease-catalyzed peptide synthesis was achieved.In this paper, we designed and validated a new media for biotransformation aided by ionic liquefied amino acid substrates. Thermolysin-catalyzed synthesis of Z-aspartame (N-carbobenzoxy-l-aspartame) in which ionic liquids play dual roles as both substrate and reaction medium was conducted. If the protease can retain catalytic activity in this new liquefied amino acid substrate/media, the effective substrate concentration can be maximized, leading to theoretically high yields. In fact, we observed a 34-fold enhancement in the catalytic activity compared with that obtained in a previous report. The yield of Z-aspartame reached at approximately 660 mM at 24 h with 50 mg mL−1 of the enzyme, thus submolar range productivity was achieved. The results suggest the successful construction of a high productivity reaction system for peptide synthesis.
Keywords: Peptide synthesis; Enzyme catalysis; Amino acid ionic liquid; High concentration reaction; Solvation;

► The time-dependent release of peptides from tryptic digestion of β-lg is studied. ► Physical accessibility to scissile bonds: responsible of trypsin-resistance areas. ► Secondary specificity of the enzyme determines the dynamics of peptide release. ► Best fitting results for β-lg degradation and peptide release: first-order kinetics.β-lg tryptic digestion leads to the release of a broad range of biologically active peptides. Understanding how these peptides are formed and the dynamics of the hydrolysis is of major relevance in order to control the quality of the final products. In this paper, the time-dependent release of peptides is reported and the dynamics of this release is discussed in terms of the physical accessibility of the enzyme to the scissile bonds and the secondary specificity of trypsin. In view of experimental data, a kinetic model based on first-order equations is proposed to simulate both β-lg degradation and peptide formation. The statistical data obtained seem to support the hypothesis that the hydrolysis mainly takes place via the proposed model. The results evaluated in this paper show the existence of areas within the intact protein with different susceptibility to tryptic attack. Whereas C- and N-terminal areas are easily digested, the internal part of the protein shows more resistance to hydrolysis and the release of final peptides within this region passes through the formation and subsequent degradation of intermediate peptides.
Keywords: Enzyme technology; Proteolysis; Modeling; Kinetic parameters; β-Lactoglobulin; Trypsin;

Immobilization of Candida rugosa lipase on hexagonal mesoporous silicas and selective esterification in nonaqueous medium by Wei hua Yu; Mei Fang; Dong shen Tong; Ping Shao; Tian ning Xu; Chun hui Zhou (97-105).
Display Omitted► Different strategies of modification of MSU-H mesoporous silica have been employed. ► Immobilization of Candida rugosa lipase (CRL) on MSU-H silicas was achieved. ► Bioconversion of conjugated linoleic acid (CLA) to its ethyl ester has been developed. ► The biocatalysts exhibit a high level of esterification activity and steady reuse. ► The mechanism of adsorption of CRL on silica has been presented. Candida rugosa lipases (CRLs) immobilized by physical adsorption, cross-linking and covalent binding methods on a MSU-H type mesoporous silica previously modified organically by different strategies, respectively, were examined as biocatalysts for esterification of conjugated linoleic acid (CLA) and ethanol in nonaqueous medium. MSU-H silica was modified by nonionic surfactant of triblock copolymer Pluronic P123, amino-functionalization and glutaraldehyde-grafting and confirmed by FT-IR analysis. Interaction mechanisms of CRLs and supports involve covalent and non-covalent interactions including electrostatic repulsion and hydrophobic interaction at pH 7. The immobilized CRLs containing surfactant were prepared by cross-linking via entrapping CRL aggregates inside the pores of silicas. The surfactant located inside the silicas could interfacially activate the immobilized CRLs and favored catalytic esterification. The biocatalyst containing 38 wt.% of surfactant afforded 1111.1 U/mg of specific activity about eight times higher than soluble CRL, and maximal 56.7% of total CLA esterification with 96.5% of 9c, 11t-CLA isomer esterification degree. The immobilized-CRL with 64.5 mg/g of loading amount of protein exhibited maximal hydrolytic activity of 2945.3 U/g-support for grafting glutaraldehyde. This derivative showed a high level of esterification activity and operational stability and remained 43.2–46.9% of total esterification for 32 h consecutive four runs.
Keywords: Biocatalysis; Lipase; Immobilization; Enzyme technology; Mesoporous silica; Conjugated linoleic acid;

Characterization of optimized production, purification and application of laccase from Ganoderma lucidum by Tamilvendan Manavalan; Arulmani Manavalan; Kalaichelvan P. Thangavelu; Klaus Heese (106-114).
► Laccase was produced under the optimized medium containing 3% (v/v) ethanol. ► Laccase production was enhanced 416 folds by the biomass tamarind shell. ► Purified laccase exhibits stability within a range of pH (4–7.5). ► Purified laccase exhibits stability within a range of temperature (30–60 °C). ► Laccase-mediated decolorization of different dyes without a redox mediator.We show for the first-time Ganoderma lucidum laccase enzyme production using medium containing 3% (v/v) ethanol, which enhanced the enzyme production up to 14.1 folds. A more than 400-folds increase could be achieved if grown in the presence of the novel lignocellulosic biomass tamarind shell plus ethanol (3%, v/v), CuSO4 (0.4 mM) and gallic acid (1 mM). A 38.3 kDa laccase enzyme was purified from the initial protein preparation with an overall yield of 32% using Sephadex G-100 and DEAE-cellulose column chromatography. The enzyme was identified through MALDI-TOF/TOF tandem mass spectrometry (MS/MS) as G. lucidum laccase-3. This enzyme exerted its optimal activity at a pH of 5 and a temperature of 55 °C with ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) as an ideal substrate. The catalytic efficiencies (k cat/K m) determined for ABTS and guaiacol were 11.5 × 105 and 3.9 × 105  s−1  M−1, respectively. The G. lucidum laccase decolorized various textile dyes and industrial textile dye effluent up to 90% and 97%, respectively.
Keywords: Ganoderma lucidum; Laccase; Ethanol; Tamarind shell; Acid Fast Red A; Methyl Violet 2B; Remazol Yellow G;

► A novel reactor with average velocity (0.38–0.92 m/h) and periodic velocity (6 m/h). ► Omitting the three-phase separator. ► The sludge bed is in the periodic “expansion-sedimentation-expansion” state. After hybrid hydrolysis acidification the biodegradability improved greatly. ► The effluent quality of the pilot-scale SBR was much better than full-scale SBR plant.A pilot-scale hybrid hydrolysis acidification reactor (HHAR) with periodic water allocation mode operation followed by sequencing batch reactor (SBR) in anoxic and aerobic metabolic function was evaluated for the treatment of low-biodegradable combined industrial and domestic wastewater. The HHAR combines the advantages of both the UASB reactor and AF, omitting the three-phase separator. Furthermore, it has lower average up-flow velocity (0.38–0.92 m/h) and higher periodic up-flow velocity (6 m/h), which made the reactor keep higher MLSS concentration (more than 10,000 mg/L) and sludge-bed is in periodic “expansion-sedimentation-expansion” state. When HRT less than 10 h, the B/C variation was positive and reached the maximum value of 0.07 at 8 h. SBR with a total cycle period of 4.5 h was applied as the post-treatment process to remove residual COD, NH3-N and TN. At steady stage, the pilot-scale SBR effluent COD, NH3-N and TN concentration was 65, 0.75 and 17.71 mg/L, corresponding in this case to full-scale SBR plant effluent was 93, 16.4 and 34 mg/L. Comparison results indicated that the application of HHAR–SBR system to treat combined industrial and domestic wastewater can improve effluent quality significantly.
Keywords: Combined industrial and domestic wastewater; Low-biodegradable; Hydrolysis acidification; Periodic water allocation; Biodegradation; Wastewater treatment;

► A variety of techniques are available for estimating the extent of cell disruption. ► All methods tested could be fitted to simple exponential decay models. ► Cell counting is the most accurate method across the full range of disruption. ► Turbidity, particle sizing and UV absorbance were the more conservative indicators. ► Measuring sample turbidity or UV absorbance is suitable for online monitoring.A comparison of quantification techniques was performed on suspensions of Saccharomyces cerevisiae which had been disrupted with a high pressure homogenizer. The quantification techniques included cell counting, monitoring protein release, UV absorbance, turbidity, sample mass loss analysis, variations in viscosity and measuring the particle size distribution of the homogenate. It was found that all quantification techniques resulted in similar relationships between the measured extent of disruption and number of passes through the homogenizer. The data from all techniques (except particle sizing) could be fitted to simple exponential decay models at various homogenization pressures. Turbidity, particle sizing and UV absorbance generally gave more conservative estimates of the extent of cell disruption compared to protein release and cell counting. Measuring both the turbidity and monitoring the release of cellular metabolites using UV absorbance gave simple, reliable and reproducible measures of disruption and were identified as being the most applicable to on-line disruption monitoring.
Keywords: Cell disruption; Quantification; High pressure homogenization; Bioprocess monitoring; Downstream processing; Protein recovery;

► Soluble organic content from sludge increased gradually with mild temperature. ► Biochemical methane potential was greatly enhanced by mild thermal pretreatment. ► General microbial activity was improved by mild thermal pretreatment. ► Activities of key enzymes were heightened by mild thermal pretreatment. ► An increase of methanogen amount was observed due to mild thermal pretreatment.Excess sludge with low organic content always led to the failure of anaerobic digestion for methane production. Recently, the mild thermal pretreatment, which is usually operated at temperatures below 120 °C, has drawn much attention due to less energy consumption and no chemical addition. In this study the effect of mild thermal pretreatment (50–120 °C) on the solubilization and methane potential of excess sludge with a low concentration of organic matters was investigated. Experimental results showed that the concentration of soluble organic matters increased gradually with temperature during the mild thermal pretreatment of excess sludge. Biochemical methane potential experiments demonstrated that the potential of methane production from excess sludge was greatly enhanced by mild thermal pretreatment, and under the conditions of pretreatment temperature 100 °C and digestion time 20 d the methane yield was as high as 142.6 ± 2.5 mL/g of volatile solids. Mechanism investigation on the enhancement of methane production from excess sludge exhibited that the consumptions of sludge protein and carbohydrate, the adenosine 5′-triphosphate content of anaerobic microorganisms, the activities of key enzymes related to anaerobic digestion, and the amount of methanogens were all improved by mild thermal pretreatment, in correspondence with the production of methane.
Keywords: Excess sludge; Anaerobic processes; Waste treatment; Biogas; adenosine 5′-triphosphate; Enzyme activity;

Ability of a perfluoropolymer membrane to tolerate by-products of ethanol fermentation broth from dilute acid-pretreated rice straw by Kengo Sasaki; Fumio Matsuda; Tomohisa Hasunuma; Chiaki Ogino; Masakatsu Urairi; Kazuhito Yoshida; Akihiko Kondo (135-139).
► Ethanol was produced from dilute acid-pretreated rice straw. ► By-products during ethanol fermentation process were showed in detail. ► Perfluoropolymer membrane was applied to vapor permeation. ► Perfluoropolymer membrane tolerated individual by-products. ► Perfluoropolymer membrane tolerated fermentation broth from acid-pretreated rice straw.A perfluoropolymer (PFP) membrane has been prepared for use in vapor permeation to separate aqueous ethanol mixtures produced from rice straw with xylose-assimilating recombinant Saccharomyces cerevisiae. PFP membranes commonly have been used for dehydration process and possess good selectivity and high permeances. The effects of by-products during dilute acid pretreatment, addition of yeast extract, and ethanol fermentation on PFP membrane performance were investigated. While feeding mixtures of ethanol (90 wt%) in water, to which individual by-products (0.1–2 g/L) were added, the PFP membrane demonstrated no clear change in permeation rate (439–507 g m−2  h−1) or separation factor (14.9–23.5) from 2 to 4 h of the process. The PFP membrane also showed no clear change in permeation rate (751–859 g m−2  h−1) or separation factor (12.5–13.8) while feeding the mixture (final ethanol conc.: 61 wt%) of ethanol and distillation of the fermentation broth using a suspended fraction of dilute acid-pretreated rice straw for 20 h. These results suggest that the PFP membrane can tolerate actual distillation liquids from ethanol fermentation broth obtained from lignocellulosic biomass pretreated with dilute acid.
Keywords: Ethanol; Fermentation; Vapor permeation; Bioconversion; Rice straw; Perfluoropolymer membrane;

CFD analysis of the turbulent flow in baffled shake flasks by Chao Li; Jian-Ye Xia; Ju Chu; Yong-Hong Wang; Ying-Ping Zhuang; Si-Liang Zhang (140-150).
► CFD method was used to simulate the flow field in baffled flaks for the first time. ► Mass transfer and shear environment in baffled flasks were studied comprehensively. ► A new model was used to describe the flask movement. ► Cultivation of filamentous fungus was conducted to validate the simulating results.In this work, computational fluid dynamics (CFD) technique is used to simulate the complicated unsteady-state turbulent flow field formed in baffled flask. The baffled flask shows advantages both in mass transfer capacity and in shear formation in comparison with unbaffled flasks. Detailed investigations of power consumption, mass transfer and shear rate are carried out in baffled flasks under shaking frequencies ranging from 100 rpm to 250 rpm, and filling volumes from 50 mL to 150 mL. The results show that the specific power input and specific interface area are both greatly influenced by shaking frequency and filling volume. For the positive effect of shaking frequency on both mass transfer coefficient (k L ) and specific interface area (a), the volumetric mass transfer coefficient (k L a) increases greatly with shaking frequency. Results also show that filling volume has no significant effect on k L but negative effect on specific interface area. Shear force formed in baffled flask shows great dependent on shaking frequency, but it is insensitive to the filling volume. Based on these investigations, correlations linking these parameters are proposed. Finally, cultivations of filamentous fungus conducted in unbaffled and baffled flasks validated the simulating results.
Keywords: Shake flask; Hydrodynamics; Mass transfer; Mixing; Shear force; Power consumption;

Comparison between acetate and propionate as carbon sources for phosphorus removal in the aerobic/extended-idle regime by Tianjing Zeng; Dongbo Wang; Xiaoming Li; Yan Ding; DeXiang Liao; Qi Yang; Guangming Zeng (151-157).
► AEI regime is a new P removal regime without a strict anaerobic period. ► AEI regime drove an EBPR using acetate and propionate as the sole carbon source. ► Propionate-SBR had higher BPR than acetate-SBR after long-term acclimatization. ► The reason for higher BPR in propionate-SBR after long-term acclimatization. ► Stoichiometric relation of two carbon sources in AEI and A/O systems was compared.Lately, we have proved that biological phosphorus (P) removal can be achieved in the aerobic/extended-idle (AEI) regime using glucose as the sole carbon source, which might develop a potential simple strategy for simultaneous removal of P and organic substrates from wastewater. Since acetate and propionate are the two most common substrates present in real domestic wastewater, this paper further assesses the P removal performances in the AEI process using acetate and propionate as the sole carbon source. The results showed that 3.91 and 3.64 mg of P/g of total suspended solids were, respectively, removed in the acetate-reactor and propionate-reactor after 50 days, respectively. After 90 days P removal in the propionate-reactor increased to 4.91 mg P/g of total suspended solids whereas that in the acetate-reactor kept in the same level (3.98 mg/g). Though both acetate and propionate could be used as carbon sources for P removal in such a novel system, the latter was more effective after long-term operation. Further investigations showed that, after 90 days’ acclimatization, sludge poly-P content in the propionate-system was more than that in the acetate-system, which was the primarily reason for the propionate-system showing higher P removal.
Keywords: Acetic acid; Aerobic processes; Bioreactors; Enhanced biological phosphorus removal; Propionate; Waste-water treatment;

Application of polyethylene glycol immobilized Clostridium sp. LS2 for continuous hydrogen production from palm oil mill effluent in upflow anaerobic sludge blanket reactor by Lakhveer Singh; Muhammad Faisal Siddiqui; Anwar Ahmad; Mohd Hasbi Ab. Rahim; Mimi Sakinah; Zularisam A. Wahid (158-165).
► PEG was used to entrap Clostridium sp. LS2 for hydrogen production from POME. ► Various amount of PEG immobilized cells were tested in UASB reactor for hydrogen production. ► Evaluate the efficiency of immobilized UASB reactor for hydrogen production at different HRT. ► Effect of different OLR on hydrogen production in immobilized UASB reactor.A novel polyethylene glycol (PEG) gel was fabricated and used as a carrier to immobilize Clostridium sp. LS2 for continuous hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. Palm oil mill effluent (POME) was used as the substrate carbon source. The optimal amount of PEG-immobilized cells for anaerobic hydrogen production was 12% (w/v) in the UASB reactor. The UASB reactor containing immobilized cells was operated at varying hydraulic retention times (HRT) that ranged from 24 to 6 h at 3.3 g chemical oxygen demand (COD)/L/h organic loading rate (OLR), or at OLRs that ranged from 1.6 to 6.6 at 12 h HRT. The best volumetric hydrogen production rate of 336 mL H2/L/h (or 15.0 mmol/L/h) with a hydrogen yield of 0.35 L H2/g CODremoved was obtained at a HRT of 12 h and an OLR of 5.0 g COD/L/h. The average hydrogen content of biogas and COD reduction were 52% and 62%, respectively. The major soluble metabolites during hydrogen fermentation were butyric acid followed by acetic acid. It is concluded that the PEG-immobilized cell system developed in this work has great potential for continuous hydrogen production from real wastewater (POME) using the UASB reactor.
Keywords: Hydrogen; Polyethylene glycol (PEG); Immobilized cell; Biogas; Upflow anaerobic sludge blanket (UASB) reactor; Fermentation; Palm oil mill effluent wastewater;

Formation of soluble Cr(III) end-products and nanoparticles during Cr(VI) reduction by Bacillus cereus strain XMCr-6 by Guowen Dong; Yuanpeng Wang; Libo Gong; Minggong Wang; Haitao Wang; Ning He; Yanmei Zheng; Qingbiao Li (166-172).
Display Omitted► The strain XMCr-6 could reduce sufficient Cr(VI) completely within 48 h. ► The Cr(VI) reduction mechanism by XMCr-6 was enzyme-mediated in the cell debris. ► Small organic molecules compete with bacteria for reduced Cr (III) coordination. ► Role of small organic molecules in determining the types of Cr(III) end-products. ► Reduced Cr(III) could form discrete spherical Cr2O3 nanoparticles.The Bacillus cereus strain XMCr-6 was isolated from chromium-contaminated soils and was identified on the basis of biochemical characteristics and 16S rRNA gene sequence analysis. The bacterium exhibited complete reduction of 100 mg L−1 Cr(VI) within 48 h under aerobic conditions. The reduction of Cr(VI) by XMCr-6 was attributed to cell debris rather than cell free extract, and the reduction mechanism was found to be enzyme-mediated. It was demonstrated that the reduced Cr(III) could bind to cells by coordination with functional groups on the bacterial surfaces while it simultaneously formed soluble Cr(III) end-products through interactions with small organic molecules. Discrete spherical Cr2O3 nanoparticles were observed on the surface of the bacterial cells after Cr(VI) reduction by XMCr-6 in the absence of an appropriate chelating chemical. Our results offer insight into bacterial Cr(VI) reduction processes and will be valuable for understanding reduction mechanisms and Cr(VI) bioremediation.
Keywords: Chromate; Nanoparticles; Bioremediation; Environmental preservation; Waste treatment; Bioconversion;

Impact of microcarrier coverage on using permittivity for on-line monitoring high adherent Vero cell densities in perfusion bioreactors by Amal El Wajgali; Geoffrey Esteban; Frantz Fournier; Hervé Pinton; Annie Marc (173-179).
► High adherent cell density in perfused reactor can be followed by on-line permittivity. ► Low carrier level induces cell confluency and loss of correlation linearity. ► Measurement of cell diameter by image analysis allows total biovolume calculation. ► The decrease of cell size from the time of confluency induces lower permittivity. ► On-line permittivity detects the time of complete coverage of microcarriers.The paper presents the interest of on-line permittivity monitoring to estimate the density of Vero cells growing on microcarriers (MCs), even when high cell densities were reached in perfusion bioreactors (4.5 × 106  cells ml−1). Cultures were performed with various MCs concentrations in a reactor equipped with a settling tube. A linear correlation between on-line permittivity and off-line volumetric cell concentration was observed provided that MCs are not fully covered by cells. High permittivities such as 250 pF cm−1 could be measured without signal saturation of the Fogale Biomass system®. The correlation was no longer linear when cell density per carrier exceeded 100% cell confluency corresponding to 150 cells MC−1 (0.15 × 106  cells cm−2). This behaviour was attributed to the decrease of cell volume when cells saturated MCs surface. It mainly happened when low MCs concentration and continuous medium renewing were used. Therefore, permittivity sensor can be considered as a reliable tool to monitor on-line adherent cell densities not exceeding total cell confluency. Moreover, it could be useful to detect when cell confluency occurs.
Keywords: Perfusion bioreactors; Bioprocess monitoring; Animal cell culture; Microcarriers; Cell diameter; Multi-frequency permittivity;

Membrane chromatography for the purification of laccase from the supernatant of Pleurotus sapidus by Mayuratheepan Puthirasigamany; Monika Wirges; Tim Zeiner (180-187).
► Laccase from fermentation broth purified using membrane chromatography. ► Membrane chromatography modules with different functionalisation. ► Pre-treatment of the fermentation broth not required. ► Enzyme activity is retained and enrichment up to fifteen fold observed.Extracellular laccase from the fermentation broth of Pleurotus sapidus was purified using membrane chromatography. Five laccase iso-enzymes produced in submerged cultures were purified; the enzymes had isoelectric points (pI) ranging from 3.3 to 4.7 and a calculated molecular weight of 57.4 kDa. The fermentation broth had a low product titre of 80 μg/l. Hydrophobic interaction (HIC) and ion exchange (IEX) membranes chromatography modules were used in an ÄKTApurifier 100 liquid chromatography system for the dynamic experiments. The pre-treatment of the fermentation broth was not required. The enzyme activity was retained, and enrichment up to 15-fold was observed via activity assays and SDS-Page analysis.
Keywords: Membrane chromatography; Membrane adsorption; Downstream processing; Laccase; Pleurotus Sapidus;

Display Omitted► Trehalose inhibits the acid-induced denaturation of ferricytochrome c at pH 2.0. ► Trehalose induces the formation of molten globule state at pH 2.0. ► At trehalose concentrations <0.8 M, the unfolding transition occurs in three phases. ► At trehalose concentrations >0.8 M, the unfolding transition occurs in two phases. ► The unfolding rates decrease linearly with increasing trehalose concentration.It has been known that trehalose can counteract acid-induced protein denaturation and induce the formation of molten globule (MG) state, but the equilibrium and kinetic behaviors are still unclear. Herein, the counteraction of trehalose on the acid-induced unfolding of ferricytochrome c was studied at pH 2.0. Far-UV circular dichroism, Soret absorption and fluorescence spectra indicate that trehalose inhibits acid-induced protein denaturation and induces the formation of MG state. The kinetics of the counteracting effects of trehalose was investigated by stopped-flow fluorescence spectroscopy. It is found that the unfolding of ferricytochrome c displays three phases (i.e., fast, intermediate and slow phases) in the absence of trehalose. At trehalose <0.8 M, the unfolding transition still occurs in three phases, but at trehalose >0.8 M, it is transformed into a biphasic process without the fast phase. The rate constants for all the unfolding phases decrease linearly with increasing trehalose concentration. Meanwhile, the increase in the fluorescence intensity for the fast and intermediate phases diminishes with the increment of trehalose concentration. Nevertheless, there is little influence of trehalose on the fluorescence intensity change in the slow phase. The studies have provided new insight into the effects of trehalose on the stability of proteins.
Keywords: Protein; Biophysical chemistry; Protein denaturation; Kinetic parameters; Trehalose; Ferricytochrome c;

► The macroscopic model that takes into account phenomena of overflow metabolism is proposed. ► The model is validated with hybridoma cell fed batch cultures. ► The overflow metabolism model gives satisfactory predictions during all periods of cultures. ► The optimal feeding profiles aiming at maximizing biomass productivity are determined. ► The optimal feeding profile allows to maintain the cells close to the critical metabolism state.A macroscopic model that takes into account phenomena of overflow metabolism within glycolysis and glutaminolysis is proposed to simulate hybridoma HB-58 cell cultures. The model of central carbon metabolism is reduced to a set of macroscopic reactions. The macroscopic model describes three metabolism states: respiratory metabolism, overflow metabolism and critical metabolism. The model parameters and confidence intervals are obtained via a non linear least squares identification. It is validated with experimental data of fed-batch hybridoma cultures and successfully predicts the dynamics of cell growth and death, substrate consumption (glutamine and glucose) and metabolites production (lactate and ammonia). Based on a sensitivity analysis of the model outputs with respect to the parameters, a model reduction is proposed. Finally, the maximization of biomass productivity of hybridoma cell fed-batch cultures is analyzed. This model allows, on the one hand, quantitatively describing overflow metabolism in mammalian cell cultures and, on the other hand, will be valuable for monitoring and control of fed-batch cultures in order to optimize the process. This is illustrated in this contribution with the determination of optimal feeding profiles aiming at maximizing biomass productivity.
Keywords: Hybridoma cultures; Mammalian cell cultures; Overflow metabolism; Macroscopic modelling; Optimal feeding profiles; Bioprocess optimization;