Biochemical Engineering Journal (v.112, #C)


Characterization of polyhydroxyalkanoates extracted from wastewater sludge under different environmental conditions by Sang-hyeop Cha; Jae-hyop Son; Yousuf Jamal; Mohd. Zafar; Hung-suck Park (1-12).
Polyhydroxyalkanoates (PHAs) are bioplastics that naturally accumulate in the microbial cells while performing organic substrate metabolism. PHAs bioconversion in microbial cells is affected by both growth environments (aerobic and anaerobic) and feeding systems (carbon and nutrient limitations). Sequential batch reactors (SBRs) were used in this research for producing PHAs; studies showed on an average 42% with a maximum of 63% PHAs yield under anaerobic–oxic conditions quantified by gas chromatography. Produced PHAs from phase 4 were thermally and physically characterized. Fourier transform infrared spectroscopy showed strong presence of carbonyl peaks at 1720 cm−1 in all PHAs. Gel permeation chromatography reported polydipersity index values in range of 2.4–3.6 showing non-uniformity of molecular weights in the slice of PHAs and differential scanning calorimetry reported melting point temperatures of 146–154 °C, confirming usefulness of produced PHAs in industrial applications. X-Ray Diffraction confirmed crystal structure in all PHAs with the most crystalline from SBR3. Thermogravimetric analysis further confirmed highest thermal degradation temperature of 283 °C for PHAs from SBR3. Different blends of wastewater fed to mixed sodium acetate acclimatized biomass further showed the importance of substrate carbon source for PHAs production in various growth environments.
Keywords: Polyhydroxyalkanoates; Wastewater sludge; Aerobic processes; Anaerobic processes; Bioconversion; Batch processing;

Development, identification and validation of a mathematical model of anaerobic digestion of sewage sludge focusing on H2S formation and transfer by F. Carrera-Chapela; A. Donoso-Bravo; D. Jeison; I. Díaz; J.A. Gonzalez; G. Ruiz-Filippi (13-19).
Nowadays, there are plenty of models of the anaerobic digestion process, however the impact of this odor emission on these models has received very little attention. Hydrogen sulfide (H2S) has been used as odor trace marker for odor dispersion studies which is formed by microbial action of sulfate reducing bacteria under anaerobic conditions. A mathematical model with focus on the H2S generation with a reduce number of parameters of five stages was developed. The model and parameters were calibrated and validated with experimental data from two pilot scale anaerobic reactors treating sewage sludge. The developed model is able to describe properly the dynamic behavior of this system, particularly, its gas phase composition with an accuracy of 90%, for hydrogen sulfide and carbon dioxide, and 60% for methane, within 99% of confidence.
Keywords: Anaerobic processes; Dynamic modelling; Dynamic simulation; Modelling; Waste-water treatment; Biogas;

Optimization and characterization of covalent immobilization of glucose oxidase for bioelectronic devices by Xue Wang; Sung Bae Kim; Dongwoo Khang; Hyug-Han Kim; Chang-Joon Kim (20-31).
Enzyme electrodes are widely applied to miniature implantable bioelectronic devices such as biofuel cells and biosensors. The main obstacle associated with miniaturization is the reduced surface area of electrodes for the accommodation of enzymes, leading to poor power output or detection signals. This study aimed to maximize the loading of glucose oxidase (GOx) on the surface of multi-walled carbon nanotubes (MWCNTs), thereby enhancing the generation of electric power or sensing signals. Because the concentrations of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), and glucose oxidase significantly affected the immobilization efficiency, these factors were optimized by the Box–Behnken design. The physically adsorbed enzyme was almost completely removed by washing the GOx-bound MWCNTs with buffer solution containing 5 g/L of Tween-20. Enzyme loading was found to be ∼3.3 ± 0.3 mg-GOx/mg-MWCNTs under the optimal conditions (430 mM NHS, 52 mM EDC and 8.7 mg/mL GOx). The formation of carboxyl group on the surface of MWCNTs and the covalent bonding between GOx and MWCNTs, and immobilized GOx were observed by FTIR and AFM, respectively. The biochemical analysis showed that the immobilized GOx possesses high activity for the conversion of glucose into gluconic acid. The cyclic voltammetry data showed that the anodic current density of electrodes loaded with the highest amount of GOx was much higher than those of electrodes loaded with smaller amounts of GOx.
Keywords: Immobilized enzyme; Multi-walled carbon nanotubes; Optimization; Covalent immobilization; Biosensor; Biofuel cell;

Subcutaneous (SC) delivery of biomacromolecular pharmaceuticals such as proteins often encounter barriers in the extracellular matrix, especially the hyaluronan (HA) network. In this study, chimeric hyaluronidases were designed, prepared and tested for assisting biopharmaceuticals in ID administration in mice as replacement of SC administration. The chimeras were hyaluronidase (rhPH20) conjugated with human serum albumin (rhPH20-HSA) and antibody Fc fragment (rhPH20-Fc). Expression of the new protein was undertaken in CHO cells cultured in a 5-L disposable bioreactor. Purification was carried out by a series of chromatographic methods to obtain high-purity products of 61 kDa (rhPH20), 79 kDa (rhPH20-HSA) and 190 kDa (rhPH20-Fc). The chimeric proteins rhPH20-HSA and rhPH20-Fc performed fairly well as spreading factors in short-term trypan blue intradermal (ID) infusion in comparison with recombinant hyaluronidase (rhPH20). They extended the channel opening from 24 h (rhPH20) to 85–120 h in vivo. The specific activity of rhPH20-Fc was 35,600 U/mg, higher than that of rhPH20-HSA (10,000 U/mg). Co-administration of rhPH20-Fc with two biomacromolecular pharmaceuticals, Stelara (150 KDa) and TNFRII-Fc-IL1ra (TFI, 250 kDa), through an ID route increased the bioavailability from 86% to 93% and from 64% and 97%, respectively, compared with rhPH20. The pharmacokinetic profile of ID administrated larger TFI was significantly improved through cooperation with the long-acting hyaluronidase.
Keywords: Recombinant DNA; Fed-batch culture; Purification; Biomedical; Long-acting hyaluronidase; Subcutaneous administration;

A case study in flux balance analysis: Lysine, a cephamycin C precursor, can also increase clavulanic acid production by André Pastrelo Cavallieri; Amanda Salvador Baptista; Carla Andréa Leite; Maria Lucia Gonsales da Costa Araujo (42-53).
Display Omitted Streptomyces clavuligerus is an excellent model for studying complex relationships involved in the biosynthesis of secondary metabolites. Despite the fact that several studies have investigated the mechanisms pertaining to the biosynthesis of two bioactive compounds produced by S. clavuligerus, cephamycin C (CephC) and clavulanic acid (CA), few have focused on metabolic flux analyses of this microorganism. It was proposed a novel representative metabolic model for S. clavuligerus addressing the simultaneous production of CephC and CA using flux balance analysis methodology (FBA). Although both are synthesized via different pathway, flux analysis showed that the production of these bioactives are connected. CephC production showed high dependency on N metabolism while CA production is more related with C metabolism. Adding lysine plus maltose increased CA, an interesting outcome since lysine is a CephC precursor. Notwithstanding, it was possible to propose a mechanism for this behavior by means of flux analysis. Furthermore, the results suggest that ATP yield maximization is the objective function that best suits the conditions under investigation.
Keywords: Antibiotic; Filamentous bacteria; Flux balance analysis; Growth kinetics; Lysine; Physiology;

Stabilization of lipase from Thermomyces lanuginosus by crosslinking in PEGylated polyurethane particles by polymerization: Application on fish oil ethanolysis by Eliane Pereira Cipolatti; Alexsandra Valério; Jorge L. Ninow; Débora de Oliveira; Benevides Costa Pessela (54-60).
Display OmittedThe adsorption of Thermomyces lanuginosus lipase (TLL) on PEGylated polyurethane particles as support permitted the development of several strategies to improve the properties of this commercial low-cost enzyme. The supports were synthesized by miniemulsion technique using isophoronediisocyanate (IPDI) and poly(ε-caprolactone) diol (PCL530) as monomers. The aqueous phase was composed of distilled water, surfactant sodium dodecyl sulfate (SDS), and poly(ethylene glycol) with different molar mass (PEG 400, 4000 or 6000). Polyethyleneimine (PEI) and trehalose were used to coat the PU-PEG polyurethane particles in order to increase the stability. In general, the coating with PEI (20%) allowed a greater stability of the derivatives. (100% of relative activity at 50 °C during 8 h). TLL immobilized on PEGylated polyurethane particles was efficiently used in the production of ethyl esters from fish oil compared to the free TLL (data not shown). The values of ethyl esters production of EPA and DHA were dependent on the support used for immobilization, which proved to be a determining factor in the activity. The highest selectivity obtained value was 45.8 for the PU-PEG4000-PEI20 derivative.
Keywords: Immobilized enzymes; Enzyme biocatalysis; Biocatalysis; Enzyme activity; Adsorption; Lipase;

FISHji: New ImageJ macros for the quantification of fluorescence in epifluorescence images by Sílvia Fontenete; Daniel Carvalho; Anália Lourenço; Nuno Guimarães; Pedro Madureira; Céu Figueiredo; Nuno Filipe Azevedo (61-69).
Fluorescence in situ hybridization (FISH) is based on the use of fluorescent staining dyes, however, the signal intensity of the images obtained by microscopy is seldom quantified with accuracy by the researcher. The development of innovative digital image processing programs and tools has been trying to overcome this problem, however, the determination of fluorescent intensity in microscopy images still has issues due to the lack of precision in the results and the complexity of existing software.
Keywords: Biomedical; RNA; LNA; Image analysis; Optimisation; Computational image processing;

Overexpression of pyrroloquinoline quinone biosynthetic genes affects l-sorbose production in Gluconobacter oxydans WSH-003 by Panpan Wang; Yu Xia; Jianghua Li; Zhen Kang; Jingwen Zhou; Jian Chen (70-77).
Display OmittedPyrroloquinoline quinone (PQQ) is a cofactor of various membrane-bound dehydrogenases. The amount of endogenous PQQ is generally regarded as a bottleneck to achieving higher catalytic efficiency of PQQ-dependent dehydrogenases. Proteins that biosynthesize PQQ in Gluconobacter oxydans WSH-003 are encoded by the pqqABCDE gene cluster and the tldD gene. In this study, PQQ overproduction in G. oxydans was attempted by overexpressing PQQ biosynthetic genes using the promoter of pqqA and the elongation factor TU (tufB). Overexpression of each single gene could enhance PQQ biosynthesis except for the tldD gene. Overexpression of pqqA, pqqB, pqqC, pqqD and pqqE with the pqqA promoter enhanced the extracellular PQQ concentration by 38.5%, 68.4%, 19.9%, 30.3% and 8.2%, respectively, whereas production was enhanced by 59.4%, 85.7%, 30.9%, 42.2% and 19.3%, respectively, using the tufB promoter. In addition, the results show that PQQ biosynthesis could be enhanced by overexpressing some of the individual genes in the gene cluster in G. oxydans and the PQQ levels were positively correlated with the efficiency of conversion of d-sorbitol to l-sorbose. The results demonstrated that cofactor engineering of PQQ in G. oxydans is beneficial for enhancing the production of quinoprotein-related products.
Keywords: Cofactor engineering; d-Sorbitol dehydrogenase; PQQ; PQQ transportation; Vitamin C;

Using an innovative pH-stat CO2 feeding strategy to enhance cell growth and C-phycocyanin production from Spirulina platensis by Chun-Yen Chen; Pei-Chun Kao; Chung Hong Tan; Pau Loke Show; Wai Yan Cheah; Wen-Lung Lee; Tau Chuan Ling; Jo-Shu Chang (78-85).
Spirulina platensis can mitigate CO2 emissions and simultaneously produce an antioxidant called C-phycocyanin (C-PC), which is a high value nutraceutical product. An innovative pH control system was used to cultivate S. platensis, in which CO2 feeding, instead of acid/alkaline titration, was used to control the pH of the culture. The optimum culture pH was 9.5. Compared to continuous CO2 feeding system, the pH control system improved the CO2 removal efficiency from 13.6% to 62.3%. The C-PC content and productivity were also enhanced to 16.8% and 0.17 g/L/d, respectively. Therefore, the proposed pH control system is economic and sustainable as it avoids the use of acid/alkaline and reduces the overall CO2 emissions. A two-stage C-PC purification process combining fractional precipitation and ion exchange chromatography could achieve the highest purity (A615/A280) of 4.33 with a 33% C-PC recovery.
Keywords: Microalgae; C-Phycocyanin; pH control system; Chromatography; Optimization; Purification;

Display OmittedBiotransformation of hydrophobic terpenes is limited by their toxicity and low bioavailability to biocatalyst cells. The metabolic activity of microorganisms, which is strongly dependent on environmental parameters, may also be affected by stressful conditions. The influence of pre-incubation of the fungus Chrysosporium pannorum in different stress conditions (organic solvents, medium pH and temperatures) on its activity in the oxidative bioconversion of α-pinene to verbenone and verbenol was examined. The total bioconversion activity increased over 2-fold after 15-min pre-treatment with 1.4-dioxane as an abiotic stress factor. Also, the change of the medium pH from the optimal 5.6 to 2.0 and 10 for 1 h before biotransformation enhanced product yield nearly 1.5-fold. Moreover, the use of dioxane, chloroform and ethanol and thermal stress at 50 °C caused changes in the oxidation product profile (predominance of trans-pinocarveol over trans-verbenol). A maximum increase in the yield of verbenol was observed when the biocatalyst was subjected to 15-min dioxane stress before 24-h biotransformation of 1% (v/v) pinene. The significance of the data presented is that abiotic stresses may improve the bioconversion activity or create changes in the proportion of the main biotransformation products.
Keywords: Abiotic stress; Aerobic process; Biocatalysis; Biotransformations; Filamentous fungi; Organic solvents;

A simple model to describe the performance of highly-loaded aerobic COD removal reactors by Jeremy Smitshuijzen; Julio Pérez; Olaf Duin; Mark C.M.van Loosdrecht (94-102).
Display OmittedHighly-loaded aerobic chemical oxygen demand (COD) removal reactors (also known as A-stage) include two main processes, COD removal by heterotrophic biomass and adsorption of COD on the activated sludge. A simple model to describe highly-loaded aerobic COD removal reactors has been developed. A one-year-set of measured data from a full scale wastewater treatment plant has been used to calibrate the efficiency of the adsorption and to evaluate the ability of the mathematical model to describe the measured data in both steady state and dynamic conditions. The approach lumped the efficiency of the settler and the adsorption with a simple but powerful approach which includes the use of the measured sludge retention time (SRT) and the settling efficiency. The effects of dynamics in terms of (i) seasonality (for water temperature, flow rate and concentration of pollutants), and (ii) daily variations in flow rate were investigated. Results showed how during winter the low water temperatures negatively impacted the efficiency of the A-stage, producing a higher COD concentration in the effluent, which eventually could impact the performance of the nitrogen removal in the B-stage. General guidelines for the application of the model to similar highly loaded A-stage reactor systems were provided.
Keywords: Adsorption; Biosorption; Dynamic simulation; AB process; A-stage; Modelling;

Table olive production is expanding worldwide. This production is associated with the generation of enormous amounts of polluting and difficult to handle wastewater streams. Thus, research on different technologies to decompose their organic load is currently needed. The economic benefit of the potential detoxification routes is expected to increase if processes for upgrading Table olive processing wastewaters are designed. This makes biological treatment of particular interest. The current review analyzes a relevant number of scientific studies dealing with promising technologies for biological treatment of table olive processing wastewaters from laboratory to pilot-scale systems. Concise details of the various technologies involved such as anaerobic digestion, lactic acid fermentation and fungal fermentation are determined. The most significant advances in the manufacturing of value-added products (e.g. biogas, platform chemicals, natural antioxidants) from table olive processing wastewaters through different microorganisms, bioreactor design modifications, and operational conditions are critically discussed. Future prospects of valorizing table olive wastewaters are presented.
Keywords: Table olive wastewaters; Bioprocess design; Lactic acid; Hydroxytyrosol; Biogas; Bioremediation;

Greenhouse gases from sequential batch membrane bioreactors: A pilot plant case study by Giorgio Mannina; Claudia Morici; Alida Cosenza; Daniele Di Trapani; Hallvard Ødegaard (114-122).
Display OmittedThe paper reports the results of nitrous oxide (N2O) emissions from aerobic and anoxic tank of a Sequential Batch Membrane Bioreactor (SB-MBR) pilot plant. The influence of salinity variation on N2O emission was analyzed by gradually increasing the inlet salt concentration from 0 to 10 g NaCl L−1.The observed results showed that the N2O concentration of the gaseous samples was strongly influenced by the salt concentration. This result was likely related to a worsening of the nitrification activity due to the effect of salinity on autotrophic bacteria. Dissolved oxygen concentration and salinity were found to be the key factors affecting N2O concentration in the gaseous samples withdrawn from the anoxic tank. Despite the fact that the N2O concentration in the anoxic tank was higher than in the aerobic one, it was found that the aerobic tank emitted around 25 times more N2O than the anoxic one.
Keywords: Global warming potential; Wastewater treatment; Environmental preservation; Membrane bioreactors; Sequencing batch reactor; Bioprocess monitoring;

Enzyme-containing silica inverse opals prepared by using water-soluble colloidal crystal templates: Characterization and application by Yanjun Jiang; Pengju Zheng; Dan Li; Liya Zhou; Lei Tian; Jinxia Wang; Biwei Yang; Xiaomei Wang; Xu Zhang; Jing Gao (123-129).
Display OmittedEnzyme-containing silica inverse opals are prepared for the first time by using water-soluble polyacrylamide (PAM) colloidal crystals as templates. Glucose oxidase-containing silica inverse opals (GOD@SIOs) are firstly fabricated and characterized by SEM, TEM, CLSM, and N2 adsorption-desorption. Compared with free GOD, the GOD@SIOs are more stable against extreme pH, heat, and chemical denaturants. GOD@SIOs can be used in fast and sensitive visual detection of glucose. In order to verify that this method is suitable for various enzymes, organophosphorus hydrolase (OPH)-containing silica inverse opals are also prepared and successfully applied as sensors for visual and spectrometric detection of organophosphorus based compounds. These enzyme-containing silica inverse opals offer a novel approach to in situ immobilize biomolecules.
Keywords: Immobilized enzymes; Enzyme biocatalysis; Enzymes; Biosensors; Inverse opals; Polyacrylamide;

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in a system with external cell recycle and limited nitrogen feeding during the production phase by Mélodi Schmidt; Jaciane Lutz Ienczak; Luci Kelin Quines; Kellen Zanfonato; Willibaldo Schmidell; Gláucia Maria Falcão de Aragão (130-135).
Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) is a biodegradable, biocompatible and thermoplastic biopolymer produced by microorganisms. Considering their high current production cost, the use of wastes and culture strategies designed to increase the process productivity has been studied. The aim of the present work was to evaluate the P(3HB-co-3HV) production from Cupriavidus necator in a repeated fed-batch system with external cell recycle, maintaining a controlled residual cell growth during the production phase by nitrogen feeding strategy. The glucose, at the feeding culture medium, simulated the carbon concentration usually found in agro-industrial wastes. Two cultures were performed: one conducted with nitrogen exhaustion and another with nitrogen feeding in limited concentrations, during the production phase. Propionic acid was added to allow 3HV production. In both cultures 73 % of P(3HB-co-3HV) was produced, but at the culture with nitrogen feeding, during the production phase, productivity reached 1.24 gP(3HB-co-3HV).L−1.h−1 and qP(3HB-co-3HV) remain at the maximum level (0.13 gP(3HB-co-3HV).gXr −1.h−1) during more time. The 3HV percentage was similar at the end of both cultures. It can be concluded that the system with external cell recycle and nitrogen feeding, during the production phase, is a good alternative to utilize agro-industrial wastes with moderate carbon concentration and to increase productivity of P(3HB-co-3HV).
Keywords: Biosynthesis; Fed-Batch culture; Glucose; Production kinetics; External cell recycle; High cell density;

Hydrolysis and oxidation of racemic esters into prochiral ketones catalyzed by a consortium of immobilized enzymes by Susana Velasco-Lozano; Javier Rocha-Martin; Ernesto Favela-Torres; Javier Calvo; José Berenguer; José Manuel Guisán; Fernando López-Gallego. (136-142).
Display OmittedOne-pot multi-step conversions are desirable to achieve more efficient and sustainable chemical processes. In this context, the immobilization of multi-enzyme systems allows the reusability of several stabilized biocatalysts working in cascade and orthogonal reactions to access more complex synthetic schemes. Herein, we have shown the one-pot tandem hydrolysis and oxidation of racemic esters (1-phenylethyl acetate) to yield quantitative conversion of prochiral ketones (acetophenone) catalyzed by a consortium of immobilized enzymes. Eukaryotic lipase and catalase, and microbial thermophilic alcohol dehydrogenase and NADH oxidase are covalently, irreversibly and individually immobilized onto four different carriers, achieving high immobilization yields (>95%) for all the enzymes, and residual activities >50% for both thermophilic alcohol dehydrogenase and NADH oxidase, 18% for the catalase and 10% for the lipase. This heterogeneous system efficiently recycles NAD+ with a maximum turnover frequency (TOF) of 294 h−1 and can be reused for up to 10 operational cycles, retaining more than 80% of its initial activity.
Keywords: System biocatalysis; Lipase; Alcohol dehydrogenase; Multi-enzyme systems; Protein stabilization;

Production and characterisation of exopolymer from Rhodococcus opacus by Magdalena Czemierska; Aleksandra Szcześ; Anna Pawlik; Adrian Wiater; Anna Jarosz-Wilkołazka (143-152).
Display OmittedScreening of Actinobacteria producing exopolymers with flocculating activity was carried out. An extracellular polymer with the highest flocculating activity extracted by Rhodococcus opacus was selected to characterisation. The water-soluble fraction of this exopolymer with molecular weight of about 760 kDa was found to be 64.6% polysaccharide and 9.44% protein. Chemical analysis showed the presence of reducing sugars, uronic acids, and amino sugars at concentrations of 184.79 μg/mg, 117.6 μg/mg, and 9.23 μg/mg, respectively. Additionally, the constituent sugars of the exopolymer were glucose, mannose, and galactose. The isoelectric point was measured at 2.5, and thermogravimetric analysis indicated the degradation temperature for this fraction at 275 °C. SEM microphotography showed a fibrillar structure with a sheet-like texture of the studied exopolymer. Infrared spectrophotometry analysis revealed that the exopolymer contained carboxyl, hydroxyl, acetyl, and carboxylate groups, preferred for the flocculation process. Additionally, the presence of these groups may facilitate the heavy metals adsorption and may influence carbonate minerals formation.
Keywords: Biophysical chemistry; Biosynthesis; Purification; Microbial growth;

Biofiltration of H2S in air—Experimental comparisons of original packing materials and modeling by Mouna Ben Jaber; Annabelle Couvert; Abdeltif Amrane; Franck Rouxel; Pierre Le Cloirec; Eric Dumont (153-160).
The treatment of hydrogen sulfide using a biofilter packed with expanded schist and topped with a layer of a synthetic nutritional material (UP20) was examined at a constant H2S concentration (100 ppmv). The impact of the empty bed residence time (EBRT) on process performances was clearly underlined by varying the polluted air flow from 4 to 20 m3  h−1 corresponding to a variation in the EBRT from 63 to 13 s. Complete H2S degradation was observed when the EBRT was higher than 51 s. Experimental data collected at various EBRTs (13–63 s) were fitted using the Ottengraf model equations. The αlump parameter value was found to be 26.4 g1/2  m−3/2  h−1. This single parameter, which enables the performance of the biofilter as a whole to be characterized whatever its composition (mixture or layers of different packing materials) and whatever the EBRT, is a powerful tool to compare packing materials and to design such bioreactors. The αlump value characterizing the performances of expanded schist coupled with a thin layer of UP20 was higher than the αlump values obtained for other packing materials (natural or synthetic) reported in previous studies.
Keywords: Biofilters; Modeling; Diffusion-Reaction; Waste treatment; Hydrogen sulfide; Odors;

High level expression of a recombinant xylanase by Pichia pastoris cultured in a bioreactor with methanol as the sole carbon source: Purification and biochemical characterization of the enzyme by Maribel Cayetano-Cruz; Ara Itzel Pérez de los Santos; Yolanda García-Huante; Alejandro Santiago-Hernández; Patricia Pavón-Orozco; Victor Eric López y López; María Eugenia Hidalgo-Lara (161-169).
Display OmittedThe xylanase gene xyn11A from Cellulomonas uda was expressed in Pichia pastoris under the control of an inducible promoter AOX1. The recombinant xylanase was named Xyn11AAOX1. The P. pastoris clone (C9) showing the highest xylanase activity was selected to evaluate the production of Xyn11AAOX1 in liquid cultures in a bioreactor. The culture was carried out by fed-batch fermentation using two strategies, one-stage method using methanol, and two-stage method using glucose and methanol as carbon sources. Interestingly, after 48 h of fermentation using one-stage method, a dry cell weight of 34 g/L and total protein concentration of 1.16 g/L were obtained, where Xyn11AAOX1 was the major enzyme secreted into the culture medium. Xyn11AAOX1 was purified from the culture supernatant of P. pastoris/pPICZαB −  xyn11A and showed an estimated molecular mass of 45 kDa. The optimal temperature and pH were 50 °C and 6.5, respectively. The K M and V max values were 4.5 mg/mL and 5000 U/mg protein, respectively. This is the first report on cultivating P. pastoris with methanol as the sole carbon source in a minimal salt medium in which the recombinant enzyme was obtained as the major enzyme secreted into the culture supernatant within a short fermentation time.
Keywords: Pichia pastoris; Methanol; Fed-batch culture; Enzyme production; Purification; Recombinant DNA;

Display OmittedBench-scale pot culture systems were used to investigate the effectiveness of phytoremediation (Testuca arundinacea), bioaugmentation (5 oil-degrading strains), and natural attenuation for remediating saline soil contaminated by Venezuela heavy crude oil. GC–MS was used to have a comparative characterization for chemical composition changes of aromatic groups (polycyclic aromatic hydrocarbons (PAHs), sulfur-containing heterocycles (SCHs) or polycyclic aromatic sulfur heterocycles (PASHs), and aromatic biomarkers) during different remediation processes. Bioaugmentation had faster startup (higher total petroleum hydrocarbon (TPH) removal efficiency on Day 30) while phytoremediation had higher TPH removal efficiency at the end of experiments (90 days). The 90-day TPH removal efficiency of phytoremediation (64.0 ± 1.6%) was significantly higher (p  < 0.05) than that of bioaugmentation (54.6 ± 1.3%) which was significantly higher (p  < 0.05) than that of natural attenuation (20.7 ± 2.8%). GC–MS analysis shows that the removal efficiencies of most of individual PAH and SCH compounds by phytoremediation were also significantly higher (p  < 0.05) than bioaugmentation which were significantly higher (p  < 0.05) than natural attenuation. GC–MS analysis also shows that the removal efficiencies of individual PAH and SCH compounds for all three treatments (phytoremediation, bioaugmentation, and natural attenuation) decreased with increases in ring number and degree of alkyl substitution. Overall, this study shows that phytoremediation with T. arundinacea and bioaugmentation with a halotolerant microbial consortium are two effective approaches for remediating saline soil contaminated by heavy crude oil.
Keywords: Bioremediation; Heavy crude oil spill; Saline soil; Biodegradation; Microbial growth; Waste treatment;

Process state classification of fed-batch fermentation based on process variables analysis by Jan Mareš; Jaromír Kukal; Pavel Hrnčiřík; Jan Náhlík (178-185).
The success of fermentation processes operated in the fed-batch mode depends, among other factors, on appropriate substrate feeding. Overfeeding should be avoided because even slightly higher concentrations may result in an inhibition or even poisoning of the microbial culture when toxic substrates are used. Therefore, bioprocess monitoring and control play a key role. This paper introduces a new bioprocess state classification methodology combining expert knowledge and automatic signal analysis, suitable for on-line application. A fed-batch cultivation of the strain Pseudomonas putida KT2442 grown on octanoic acid was used as a model process. The classification was performed in two steps – a manual classification done by an expert, and a subsequent automatic classification using the results obtained by the manual classification. The manual classification strategy was based on the analysis of time profiles of selected process variables related to substrate feeding, such as dissolved oxygen tension and substrate feeding rate. Three process states were recognized – normal feeding, overfeeding and underfeeding. Ridge regression was then applied to the data results of the manual classification in order to design an automatic classification strategy for easier on-line use. This strategy can distinguish between the normal and other feeding states, using a limited number of on-line measurable output variables and their time fluctuations.
Keywords: Bioprocess monitoring; Process state classification; Dissolved oxygen; Expert system; Fed-batch fermentation;

Display OmittedThe juice extracted from sweet sorghum stalks has been previously explored to produce ethanol and also to grow oleaginous yeasts and algae with the objective of producing microbial oil. In this paper we propose a different process route in order to produce ethanol and microbial oil in two consecutive fermentation steps. Ethanol is produced first followed by the growth of oleaginous yeast employing the residual carbon and nitrogen left from the first step. Two yeasts were compared for ethanol production. Trichosporon oleaginosus was cultivated for lipid production. The yeast selection for the first step was the most important factor for achieving a high ethanol yield and the effect of inorganic nitrogen addition was not significant. The remaining sugars consisted of a mixture of sucrose and fructose and no residual glucose was detected in any of the runs. In the second step T. oleaginosus DSM 11815 grew in the pooled juices remaining from the first step for 168 h and produced biomass with 28% lipid content. Glucose showed the highest uptake rate, sucrose was utilized until low glucose values prevailed, and fructose was slowly metabolized and a substantial amount remained. Although the two step process has flexibility in choosing the proper microorganism for each step, it is necessary to look for a rapid fructose uptake strain for both fermentations.
Keywords: Bioprocess design; Ethanol; Fermentation; Consolidated processing; Microbial oil; Sweet sorghum;

Growing interest surrounds isothermal PCR techniques which have great potential for miniaturization for mobile diagnostics. Particularly promising, Recombinase Polymerase Amplification (RPA), combines this advantage of isothermal PCR with simplicity and rapid amplification. A mathematical model is presented of Recombinase Polymerase Amplification (RPA) and compared to experimental data. This model identifies the rate limiting steps in the chemical process, the effects of stirring, and insights in to using RPA for quantitative measurement of initial DNA concentration. Experiments are shown in which DNA amplification occurs under conditions of Couette flow and conditions of rotational turbulent flow. Hand mixing has been shown to dramatically shorten amplification times but introduces unpredictable variability. In some cases, this variability manifests itself as human error induced false negatives, a serious problem for all potential applications. Mechanical stirring demonstrates similarly short delay times while retaining high repeatability and reduces the potential for human error.
Keywords: Recombinase Polymerase Amplification (RPA); Isothermal PCR; Couette flow; Mathematical model; Disease diagnostics;

The growth characteristic and interrelated microbial desulfurization activity of stain MP12 on both DBT and crude oil systems were investigated in order to desulfurize crude oil. The results showed that DMSO was the most suitable sulfur source for stain MP12 growth and expression of desulfurization activity, while initial 2-HBP had very significant influences on the growth and desulfurization activity. Moreover, a novel method using the production rate of sulfate was developed for rapid determination of desulfurization activity in crude oil system. Analysis of total 2-HBP and total sulfate molar balance suggested that the total 2-HBP buildup had a very good match with the total sulfate buildup during microbial desulfurization of crude oil. The desulfurization activity of high density cells could be increased sharply with an increase of agitation speed. In conclusion, the strain MP12 was able to effectively desulfurize recalcitrant organic sulfur compounds in both DBT and crude oil systems.
Keywords: Biodesulfurization; Microbial biocatalysis; Dibenzothiophene; Biocatalysis kinetics; Bioprocess control; Bioenergy;

Lipid production from biodiesel-derived crude glycerol by Rhodosporidium fluviale DMKU-RK253 using temperature shift with high cell density by Pirapan Polburee; Wichien Yongmanitchai; Kohsuke Honda; Takao Ohashi; Toshiomi Yoshida; Kazuhito Fujiyama; Savitree Limtong (208-218).
Lipid production from crude glycerol by Rhodosporidium fluviale DMKU-RK253 was optimized using response surface methodology (RSM). The optimal medium (100 mL in 500 mL Erlenmeyer flask) consisting of 70 g/L crude glycerol, 0.55 g/L (NH4)2SO4, 1 g/L monosodium glutamate, 2 g/L MgSO4·7H2O, 0.4 g/L KH2PO4 (C/N ratio of 140) and optimal cultivation conditions, namely pH 5.5, 25 °C and 200 rpm shaking speed (129 h−1 of kLa) were obtained. Batch cultivation in a 5 L jar fermenter performed with the optimal nutrients and conditions resulted in a high lipid content of 63.8% of dry biomass with a lipid quantity of 8.99 g/L and a lipid yield of 0.16 g/g. To enhance lipid production, a two-stage cultivation process with three different strategies viz. nutrient and temperature shift cultivation, temperature shift cultivation and temperature shift cultivation with high cell density was performed. The temperature shift cultivation (30 °C to 25 °C) with the high cell density strategy provided a high lipid quantity of 15.98 g/L, a biomass of 22.93 g/L (69.5% lipid content and lipid yield of 0.21 g/g) and small amount of lipid droplets were observed in the cultivation medium.
Keywords: Biosynthesis; Glycerol; Yeast; Optimisation; Two-stage cultivation; Microbial lipid;

A single-layer structured microbial sensor for fast detection of biochemical oxygen demand by Yijin Li; Jizhou Sun; Jinfen Wang; Chao Bian; Jianhua Tong; Yang Li; Shanhong Xia (219-225).
Display OmittedIn this paper, a novel biochemical oxygen demand (BOD) sensor based on a single layer of immobilized microorganisms on electrode for fast detection is investigated. Instead of the previous two-layer structure of oxygen permselective membrane and microorganism membrane, a single layer structure using electrodes as the support for microorganism immobilization has been studied. Amino functional groups on the surface of microorganisms have been bonded with carboxyl modified electrodes. Without extra supports for immobilization, mass transfer resistances are reduced, which facilitates fast current response. Bacillus subtilis cells with high biodegradation ability are obtained after 12 h cultivation. The immobilization process has been investigated by CV and EIS. B. subtilis cultivation time, response time and linear range have been optimized. The proposed sensor can reach equilibrium in 5 min which has the advantage for real-time BOD determination in the future. The linear range is 5–30 mg/l with correlation coefficient of 0.978 and limit of detection of 1.65 mg/l.
Keywords: Biosensors; Immobilized cells; Dissolved oxygen; Fast detection; Biochemical oxygen demand (BOD);

Analysis of exopolysaccharide production patterns of Cordyceps militaris under various light-emitting diodes by Chung-Hua Kho; Shu-Chen Kan; Chih-Yuan Chang; Heng-Yi Cheng; Chia-Chi Lin; Pin-Chiuan Chiou; Chwen-Jen Shieh; Yung-Chuan Liu (226-232).
Light-emitting diodes (LEDs) with varying light wavelengths (blue, green, yellow, red and white) were used to study the effects of light source on the biomass and exopolysaccharide (EPS) production of Cordyceps militaris. In a solid-state culture, green and red light sources gave the best hyphal growth. Logistic rate equations were used to calculate the kinetic behavior of the biomass growth and EPS production of a submerged culture under various light sources. According to the results, a maximal biomass concentration of 17.06 g/L was obtained using red light, with a specific growth rate of 1.47 day−1. In contrast, the highest EPS production of 2404.2 mg/L was obtained under blue light, giving a growth- and non-growth-associated product formation coefficient of 17.32 mg/g and 10.83 mg/g/day, respectively. According to the model fitting, a mixed-growth-associated pattern emerged when the culture was illuminated with LEDs, which is quite different from the non-growth-associated mode under dark conditions. The extraordinarily high EPS production of C. militaris under these conditions offers a good source of polysaccharides for further academic and medical applications.
Keywords: Cordyceps militaris; Fermentation; Filamentous fungi; Light-emitting diodes (LEDs); Modeling; Submerged culture;

This study was conducted to investigate the nitrogen removal efficiency and N2O emission characteristics of a novel thermophilic aerobic denitrifying bacterium, Chelatococcus daeguensis TAD1, under different C/N ratios and pH values in a batch reactor. Nitrogen removal efficiency and N2O emissions were dramatically influenced by C/N ratio and pH. Moreover, multifactor analysis of variance suggested that these two factors also had significant interaction effects on N2O emissions. The optimum C/N ratio was determined to be 8 (where pH was set to 7), at which a very high nitrogen removal efficiency (>99%) was achieved. Under these conditions, N2O emissions were only 34.43 μg/L and the N2O emission factor was 0.046%, which could offer a promising new microbial resource for N removal and reduction of greenhouse gas emissions during wastewater treatment.
Keywords: N2O emissions; Thermophilic; Aerobic denitrification; C/N ratio; pH;

Microbial community analysis of anaerobic granules in phenol-degrading UASB by next generation sequencing by Jeong-Geol Na; Mo-Kwon Lee; Yeo-Myeong Yun; Chungman Moon; Mi-Sun Kim; Dong-Hoon Kim (241-248).
Display OmittedThe objective of this study was to investigate microbial communities in a continuous anaerobic phenol-degrading system using a next generation sequencing tool. The anaerobic granules adapted to phenol were first obtained by repeated-batch operation, which were then inoculated in an up-flow anaerobic sludge blanket reactor (UASB) operated at various organic loading rates (OLRs). Lag periods for both phenol degradation and CH4 production decreased as batch fermentation was repeated, indicating a progressive adaptation of the granules to phenol. In the UASB operation, the highest OLR handled was 6 kg COD/m3/d, in which the attained biogas production rate, phenol degradation, and CH4 contents were 2.1 m3/m3/d, 79.0%, and 75.3%, respectively. Syntrophorhabdus and Clostridium were found to be the dominant bacteria, whose sum occupied around 60% of total bacterial sequences. In particular, there was a significant increase in Syntrophorhabdus (39.2% of total bacterial sequences), known to degrade phenol to benzoate and subsequently to acetate and hydrogen in syntrophic association with a hydrogenotrophic methanogen. In terms of archaea, Methanosaeta (42.1% of total archaeal sequences), and Methanobacterium (24.5% of total archaeal sequences) became dominant as operation continued, which were negligible in the inoculum.
Keywords: Phenol; Biogas; Microbial community analysis; Waste-Water treatment; Bioreactors; Anaerobic processes;

Effect of volumetric oxygen transfer coefficient (kLa) on ethanol production performance by Scheffersomyces stipitis on hemicellulosic sugarcane bagasse hydrolysate by Débora Danielle Virgínio Silva; Kelly Johana Dussán; Valentina Hernández; Silvio Silvério da Silva; Carlos Ariel Cardona; Maria das Graças de Almeida Felipe (249-257).
Experimental evaluation of the effect of the agitation speed and aeration rate (measured by kLa) and energy required for ethanol production using sugarcane bagasse hemicellulosic hydrolysate (SBHH) by Scheffersomyces stipitis were studied. Fermentation and purification stages were simulated using the software Aspen Plus with experimental data to understand the overall energy performance of the process. In all experiments, fermentative parameters and the thermal energy required in the ethanol production process were strongly influenced by kLa values. The optimum initial kLa to achieve the maximal ethanol concentration (15.03 g L−1) and the minimal thermal energy required (1.85 × 105  KW per kg ethanol), were found at 8.0 h−1 (450 rpm and 0.6 vvm). Under this condition, the ethanol yield and productivity were 0.37 g g−1 and 0.30 g L−1  h−1, respectively. The current study highlights the ethanol production improvement from hemicellulose hydrolysate by S. stipitis and will contribute to developing a more efficient strategies for fermentation of both cellulose and hemicellulose hydrolysates.
Keywords: Ethanol; Fermentation; Dissolved oxygen; Yeast Scheffersomyces stipitis; Hemicellulosic hydrolysate; Sugarcane bagasse;

Metabolomics analysis of phytohormone gibberellin improving lipid and DHA accumulation in Aurantiochytrium sp. by Xin-Jun Yu; Jie Sun; Ya-Qi Sun; Jian-Yong Zheng; Zhao Wang (258-268).
Display OmittedIn this study, the phytohormone gibberellin was evaluated for its effect on lipid and docosahexaenoic acid (DHA) accumulation in Aurantiochytrium sp.YLH70. It was found that 4 mg/mL of gibberellin increased biomass, lipid and DHA yield of Aurantiochytrium sp.YLH70 by 14.4%, 43.6% and 79.1%, respectively. Moreover, a GC–MS metabolomics method combined with a multivariate analysis was applied to reveal the metabolic mechanisms responsible for the increased lipid and DHA accumulation. The principal component analysis (PCA) revealed that metabolomics profiles from all groups were discriminated. 38 metabolites identified by the partial least-squares-discriminant analysis (PLS-DA) were responsible for responding to gibberellin treatment. Metabolic pathway analysis showed that gibberellin accelerated the rate of utilization of glucose, and metabolites in fatty acids biosynthesis and mevalonate pathway were increased, while metabolites in glycolysis and TCA cycle were decreased in Aurantiochytrium sp.YLH70. Moreover, the anti-stress mechanism in Aurantiochytrium sp.YLH70 might be induced by gibberellin.
Keywords: Gibberellin acid; Aurantiochytrium sp; Metabolomics; Anti-stress; Gas chromatography–mass spectrometry (GC–MS); Docosahexaenoic acid (DHA);

Microbial removal of carboxylic acids from 1,3-propanediol in glycerol anaerobic digestion effluent by PHAs-producing consortium by Chaozhi Pan; Giin-Yu Amy Tan; Liya Ge; Chia-Lung Chen; Jing-Yuan Wang (269-276).
Display OmittedAnaerobic fermentation of glycerol to 1,3-propanediol (1,3-PDO) is conceived as an economic feasible pathway to handle with increasing crude glycerol from biodiesel industry. However, glycerol anaerobic digestion effluent (ADE) consists of carboxylic acids and 1,3-PDO, imposing difficulties for separation. The objective of this study was, therefore, to investigate microbial removal of carboxylic acids from 1,3-PDO in glycerol ADE by polyhydroxyalkanoates (PHAs) producing consortium. Growth tests on carbon sources showed Corynebacterium hydrocarbooxydans had preference for butyrate while Bacillus megaterium for acetate and glycerol. Consequently, their consortium had a higher cell density and a faster substrate utilization rate than single strain grown in glycerol ADE. Acidic pH at 6.0 and 5.2 strongly inhibited cell growth and activity, while C:N ratio (w/w) at 8:1 could balance nitrogen demand for cell growth and PHA synthesis. Kinetic study further revealed over 80% of fed 1,3-PDO was preserved after depletion of carboxylic acids. Correspondingly, total organic carbon (TOC) contribution from 1,3-PDO rose from initial 55.8% to 84%. Produced PHAs comprised 3-hydroxybutyrate (3-HB) units. The results showed this study as the first attempt to provide a win-win solution to remove carboxylic acids from 1,3-PDO in glycerol ADE and converted them into PHAs as a secondary value-added product.
Keywords: Glycerol anaerobic digestion effluent; Polyhydroxyalkanoates; 1,3-Propanediol; Microbial removal; Microbial consortium;

A high-throughput screening method for identifying lycopene-overproducing E. coli strain based on an antioxidant capacity assay by Xian Xu; Weiyue Jin; Ling Jiang; Qing Xu; Shuang Li; Zhidong Zhang; He Huang (277-284).
A straightforward, high-throughput screening method for identifying recombinant Escherichia coli strains that yield high levels of lycopene was developed based on the antioxidative properties of lycopene. Twenty-four recombinant E. coli strains were constructed using plasmids with various orderings of lycopene biosynthesis genes, Shine-Dalgarno regions, and aligned spacing regions. The high-throughput screening was based on antioxidant capacity measurements; we assessed the 2,2-diphenyl-1-picrylhydrazyl method, reducing activity method, and hydroxyl free radical scavenging method; the hydroxyl free radical scavenging method was determined to be the most suitable for use with our lycopene-overproducing system. The accuracy of this method was comparable to that of assay by high-performance liquid chromatography. The strain E. coli IEB11, which had the strongest antioxidant capacity and the highest lycopene-accumulating capacity, was selected and investigated for its ability to produce lycopene at different temperatures, glycerol concentrations, and fermentation times. In the optimal conditions in a 5-L fermenter, lycopene content of 1288 mg/L and a mean productivity of 53.7 mg/L/h were obtained after 24 h.
Keywords: Biosynthesis; Recombinant DNA; Fermentation; Optimization; High-throughput screening; Lycopene;

Actinobacillus succinogenes: Advances on succinic acid production and prospects for development of integrated biorefineries by Chrysanthi Pateraki; Maria Patsalou; Anestis Vlysidis; Nikolaos Kopsahelis; Colin Webb; Apostolis A. Koutinas; Michalis Koutinas (285-303).
Actinobacillus succinogenes is a wild-type bacterial strain, isolated from bovine rumen, known as one of the most efficient natural producers of succinic acid. Herein, the factors contributing to the fermentative production of succinic acid by A. succinogenes are reviewed with particular focus on raw materials, culture conditions, significance of carbon dioxide availability and downstream separation and purification. The metabolic potential of this strain is evaluated through discussion of the pathways involved in succinic acid production, genome analysis as well as the development of A. succinogenes mutants. The review also addresses the importance of by-product formation during fermentation that constitutes an important aspect regulating succinic acid production by A. succinogenes. The prospect of integrating succinic acid production in future biorefineries is assessed.
Keywords: Actinobacillus succinogenes; Bioconversion; Product inhibition; Downstream processing; Integrated biorefineries; Renewable resources;