Biochemical Engineering Journal (v.105, #PA)


BEJ Keywords (II).

Preparing tauroursodeoxycholic acid (TUDCA) using a double-enzyme-coupled system by Qingzhi Ji; Jun Tan; Liancai Zhu; Deshuai Lou; Bochu Wang (1-9).
Tauroursodeoxycholic acid (TUDCA) has been used to treat many diseases effectively. TUDCA generation in vivo is a complex process involving five steps catalyzed by five enzymes in the enterohepatic circulation of bile salt. In this paper, we report a novel in vitro TUDCA preparation technology in which the epimerization of taurochenodeoxycholic acid (TCDCA) to TUDCA is directly catalyzed by immobilized 7α- and 7β-hydroxysteroid dehydrogenases (7α- and 7β-HSDH) in a double-enzyme-coupled system. One pair of oxidoreductases from Clostridium absonum DSM599, which was made up of 7α- and 7β-HSDH, was immobilized on a modified chitosan microsphere by separate immobilization and co-immobilization. The protein loading yields of 7α- and 7β-HSDH were 79.06% and 87.18%, and their activity yields were 57.23% and 53.97%, respectively. In the batch-wise reactions catalyzed by double-enzyme-coupled system, 72.76% of the TCDCA was transformed, and only 22.08% TUDCA was obtained on the basis of separate immobilization. The TUDCA yield was 62.49% under the catalytic effects of co-immobilized 7α- and 7β-HSDH microspheres, while it was only 41.23% based on the mixture of separate immobilized 7α- and 7β-HSDH microspheres. The enzyme catalytic technology described in this paper offers a potential application for synthesizing TUDCA and other high-value bile acid derivatives in vitro.
Keywords: Immobilized enzyme; Enzyme biocatalysis; Biotransformations; Enzyme activity; 7α-hydroxysteroid dehydrogenases; 7β-hydroxysteroid dehydrogenases; Bile acid;

Converting oils high in phospholipids to biodiesel using immobilized Aspergillus oryzae whole-cell biocatalysts expressing Fusarium heterosporum lipase by Jerome Amoah; Shih-Hsin Ho; Shinji Hama; Ayumi Yoshida; Akihito Nakanishi; Tomohisa Hasunuma; Chiaki Ogino; Akihiko Kondo (10-15).
Display OmittedThe presence of phospholipids in oil has been a major hurdle in the production of biodiesel using immobilized Aspergillus oryzae whole-cell biocatalysts. A density of phospholipids within the range of 10–30% could reduce both the rate of production and the final yield of biodiesel. Phospholipids in the oil leads to the formation of water-in-oil phospholipid-based reverse micelles. The water that activates the enzymatic process is observed to be trapped inside these reverse micelles. This has resulted in the inactivation of the reaction systems and has subsequently led to the deactivation of the immobilized lipase by the extended residence time of the added methanol. A reaction system involving gentle agitation and higher amount of water was found to reduce the reverse micelles formation. This simple technique improved the conversion efficiency by approximately 3-folds, producing a final biodiesel of more than 90%, using immobilized A. oryzae whole cells expressing Fusarium heterosporum lipase. This demonstrates that, the above technique could be successfully applied to the enzymatic biodiesel conversion of oils containing high amounts of phospholipids such as that from microalgae.
Keywords: Biodiesel production; Bioconversion; Whole-cell immobilized biocatalysts; Enzyme deactivation; Phospholipids; Reverse micelles;

Adipic acid has been widely used for producing thermoplastic polyurethane resins, nylon 6-6, adhesives, synthetic lubricants and plasticizers, and the global market for adipic acid is estimated at approximate $6.3 billion per year. Currently, 2.85 million tons of adipic acid are produced from petrochemical sources, mostly by oxidation of KA oil (cyclohexanol and cyclohexanone) catalyzed by nitric acid. The chemical synthesis of adipic acid is of serious consequences such as greenhouse gases and heavy pollution to the environment, etc. The researchers are struggling to establish more reliable, renewable and affordable adipic acid synthesis methods, and among which, biological synthesis of adipic acid is attracting the most attention. There are two biological routes for adipate synthesis: (1) biological accumulations of precursors of adipic acid such as d-glucaric acid and cis,cis-muconic acid, which can be further catalyzed to adipate; (2) synthesis of adipate directly from carbon source. With the emergence of metabolic engineering and synthetic biology, biological synthesis of adipic acid from renewable substrates is now feasible. Here, we review the new approaches and technologies to produce adipic acid biologically, with the direct synthesis of adipic acid from the renewable substrates of special interest.
Keywords: Adipic acid; Metabolite over production; Biosynthesis; Consolidated bioprocessing (CBP); Biocatalysis; Fermentation;

Display OmittedThe adsorption of Cr(VI) and Ni(II) applying amine-functionalized modified rice straw (MRS) as an adsorbent in single and binary systems was investigated. The MRS was characterized by Fourier transform infrared spectroscopy (FTIR), Thermo-gravimetric Analysis (TG), Brunauer–Emmett–Teller (BET) analysis, Scanning Electron Microscope (SEM) and the Energy Dispersive Spectrometer (EDS) analysis. The adsorption study was conducted systematically by varying adsorbent dosage, initial pH value, temperature and initial metal ion concentration. The results showed that the maximum adsorption capacity for Cr(VI) was 15.82 mg/g with MRS dosage 10 g/L at pH 2.0, temperature 318 K and initial metal ion concentration 200 mg/L, and 3.95 mg/g for Ni(II) at pH 7.0, temperature 318 K and initial metal ion concentration 80 mg/L with MRS dosage 10 g/L, respectively. The adsorption data were suitable for Langmuir isotherm model better both in single and binary systems. The pseudo-second-order model could better describe the adsorption process in single and binary systems. In binary systems, the existence of Ni(II) hindered the adsorption of Cr(VI) but the removal of Ni(II) was enhanced with the existence of Cr(VI) ions. Thermodynamic constant values (ΔG 0  < 0, ΔH 0  > 0, ΔS 0  > 0) illustrated that the adsorption of Cr(VI) and Ni(II) onto MRS were spontaneous and endothermic.
Keywords: Modified rice straw; Binary systems; Adsorption; Waste treatment; Kinetic parameters; Environmental preservation;

A new conductometric immunoassay based on biofunctionalized prussian blue-gold hybrid nanostructure (PBGN) was designed for sensitive determination of tissue polypeptide antigen (TPA). The immunosensor was prepared by immobilizing anti-TPA capture antibody on the interdigitated transducer, while the PBGN was used for bioconjugation of detection antibody and horseradish peroxidase (HRP). Upon target TPA introduction, the sandwiched immunocomplex was formed between capture antibody and detection antibody on the transducer. Accompanying the PBGN, the carried HRP and prussian blue catalyzed iodine ion into iodine relative to H2O2-KI system, thus resulting in the variations of local conductivity. Under optimal conditions, the immunosensor exhibited good conductometric responses toward target TPA, and allowed the detectable concentration as low as 0.28 pg mL−1. The precision, specificity and reproducibility were acceptable. Importantly, our strategy was also utilized for analysis of 13 human serum specimens, giving well matched results with those obtained from human TPA ELISA kit.
Keywords: Immunoassay; Biomedical; Biosensors; Enzyme biocatalysis; Prussian blue-gold hybrid nanostructures; Tissue polypeptide antigen;

An alternative anaerobic treatment process for treatment of heavy oil refinery wastewater containing polar organics by Yu Wang; Qinghong Wang; Min Li; Yingnan Yang; Wei He; Guangxu Yan; Shaohui Guo (44-51).
Heavy oil is an important part of energy sources, but the refining wastewater is difficult to treat by the conventional anaerobic process, which has low efficiency and poor ability to resist impact load. In this study, an up-flow anaerobic sludge bed (UASB) reactor was applied to treat heavy oil refinery wastewater containing large amounts of polar organics. Through a progressive increase of hydraulic conditions, the average removal efficiencies of COD and total oil reached 70% and 72%, respectively, at an organic loading rate (OLR) of 3.44 kg COD/m3  d. GC–MS analysis revealed that more biodegradable organic acids and alcohols were generated and macromolecular polar organics were degraded into small molecular intermediates after UASB treatment. The morphology observation of the sludge demonstrated that granular sludge with an average particle size of 1 mm was formed. Moreover, the predominant species and microbial community shift could reflect the performance of the reactor. The long-term operation of UASB exhibited excellent polar organic removal efficiency. The study demonstrated the potential of UASB as an alternative for high-efficiency anaerobic treatment of heavy oil refinery wastewater.
Keywords: Anaerobic processes; Heavy oil refinery wastewater; Polar organics; Bioreactors; Biodegradation; Waste-water treatment;

Display OmittedBiodiesel can be produced from vegetable oils using different catalysts including enzymes. This publication presents the development of a mathematical model for biodiesel production using the liquid lipase Callera Trans L (CTL) and analyzes the first block of reactions: esterification of free fatty acids (FFA) in biodiesel and hydrolysis of the latter. The relevant rate constants were evaluated by changing water, methanol, FFA and enzyme concentrations. The results were compared to the immobilized catalyst Novozym 435 (Nvz). The intriguing difference was observed for the apparent equilibrium constants of CTL (high K eq app) and Nvz (low K eq app). This thermodynamic “inconsistency” was explained by absence or presence of the catalyst carrier. Nvz carrier particles apparently help to disperse water, increasing its surface and hydrolytic activity in comparison to CTL. Another reactant, methanol, had a dual effect acting as (i) a substrate and (ii) a solvent of water in oil phase. The latter effect added to hydrolytic activity and decreased K eq app at increasing methanol (0–0.5 M). Inhibition and inactivation of CTL by methanol (<8% v/v) were insignificant. FFA acted as both substrate and reversible inhibitor of the enzyme suppressing its activity to approximately 25% at FFA >1.5 M.
Keywords: Esterification; Liquid lipase; Enzyme; Equilibrium; Methanol; Biodiesel;

The functional stabilities of ecosystems are still overlooked by pollution treatment specialists concerning the bioprocesses design and operation. Indeed, specialized wastes treatment bioreactors (SWTBR) used to remove pollution are not sustainable due to their greenhouse gases emission. TRIZ theory (Russian acronym for Theory of Solving Inventive Problem) was applied to propose an inventive sustainable bioreactor design thanks to its capacity to increase the level of abstraction for creative problems resolution. The SWRBR was designed after applying the three fundamental concepts of TRIZ and identification of the principal contradictions in SWTBR. The proposed coupling between the essential ecosystems characteristics especially, the trophic reactions and the matter recycling was used to solve the identified contradictions in SWTBR and to design SWRBR. The dense microbial communities especially associated with animals through gastrointestinal tract and plants through rhizosphere constitute excellent models to design sustainable wastes recycling bioreactors (SWRBR). The designed SWRBR avoids the unbalance of the geochemical cycles, and can help process engineers systematically find creative solutions for sustainable waste treatment.
Keywords: Ecosystem preservation; Waste-water treatment; Bioprocess design; Bioreactors; Engineered microbial ecosystems; TRIZ method;

Display OmittedAlthough a successive dearylation is recognized as a triphenyltin biodegradation pathway, the cleavage pattern of the various chemical bonds of phenyltins is still not clear. Moreover, the correlation among phenyltins biosorption, degradation, molecular properties and metabolic impacts is far from fully understood. Therefore, phenyltins treatment of Bacillus thuringiensis was conducted. After degradation for 7 d in the presence of 50 mg L−1 of the surfactant sucrose fatty acid ester, the degradation efficiency of 1 mg L−1 triphenyltin reached its peak value of 89%. The surfactant altered the topological structure of the cellular peptide chains, accelerated triphenyltin binding and transport, increased cellular viability, Na+/K+- and Ca2+/Mg2+-ATPase activities, increased PO4 3–and Na+ assimilation, and decreased K+ and Mg2+ release, resulting in the enhancement of triphenyltin degradation. However, surfactant did not change the successive dephenylation pathway, which was primarily determined by the bond energy of each Sn―C bond of triphenyltin
Keywords: Biodegradation; Biosorption; Biotransformation; Enzyme activity; Organotin; Ion;

Anoxic–aerobic sequencing batch reactor (SBR) system was operated for 180 days (under ambient temperature, 20 days SRT, 24 h HRT, influent COD/nitrate: 4 and COD/phosphate: 137) to treat a high strength wastewater (1000 mg/L nitrate and 4000 mg/L COD). The unique aspect was elimination of anaerobic cycle due to availability of nitrate (NO3) and oxygen as electron acceptors in anoxic and aerobic phases respectively. Simultaneous removal of nitrate (98%), phosphate (86%), and COD (72%) was achieved in anoxic phase. The subsequent aerobic phase experienced 26% of residual COD removal along with phosphate release (∼3.4 mg/L), reducing the overall P-removal to 76%. A long anoxic phase (18 h) could sustain denitrifying dephosphatation with less MLSS generation. Pyrosequencing data were analyzed through Ribosomal database project (RDP) and DECIPHER while diversity of sampling was analyzed using Chao1 and Shannon index. Rarefaction curve reflected adequacy of sampling for total species diversity study. Overall analysis revealed Proteobacteria, Alphaproteobacteria, Rhodobacterales, Rhodobacteraceae and Paracoccous as the prominent phylum, class, order, family, and genus respectively.Surplus electron donor and acceptor in anoxic phase (feasting) were advantageous for enrichment of DNPAOs over OHOs while nitrate exhaustion in the aerobic phase provided adequate fasting condition to maintain DNPAOs dominance. Low specific denitrification rate values in comparison to other heterotrophs, also supported enrichment of denitrifying phosphate accumulating organisms (DNPAOs) in the anoxic–aerobic sequencing batch reactor. Diverse micro flora ensured robustness and performance stability in high strength wastewater.
Keywords: SBR; Nitrate (NO3); Phosphate and COD removal; Anoxic; Aerobic; Microbial diversity;

Butanol inhibition is one of the major obstacles limiting the economic viability of acetone–butanol–ethanol (ABE) fermentation. In this study, a butanol-tolerant mutant (Clostridium beijerinckii BT14) was generated by atmospheric and room temperature plasmas (ARTP). This mutant showed significant advantage over its parent strain in terms of butanol tolerance. Compared to its parent strain, batch fermentation by this mutant produced 25% higher butanol and 33% higher ABE solvents due to its efficient generation of intracellular NADH and high NADH-dependent butanol dehydrogenase activity. Furthermore, C. beijerinckii BT14 was applied to fed-batch fermentation with pervaporation (PV). As a results, C. beijerinckii BT14 grew to a high cell density and this process generated highly concentrated ABE solution with a high solvent productivity of 0.98 g/(L h) and glucose consumption rate of 2.64 g/(L h). Thus, this work provides an appropriate strategy to develop an efficient process for ABE production in the PV coupled fermentation.
Keywords: Biosynthesis; Butanol tolerance; Atmospheric and room-temperature plasmas; Fed-batch culture; Pervaporation; Process integration;

Cyanophycin from recombinant Escherichia coli is composed of aspartic acid as a backbone with arginine and lysine as the side chains. Cyanophycin exists in insoluble and soluble forms based on its solubility in aqueous solution. This study aims to assess the physical properties and cellular biocompatibility of cyanophycin prepared with recombinant E. coli. The decomposition temperature of cyanophycin was around 230 °C for both forms of cyanophycin, as measured by thermogravimetric analysis. Soluble cyanophycin showed no toxicity to Chinese Hamster Ovary (CHO) cells at a concentration of 5 mg/mL as revealed by the thiazolyl blue tetrazolium bromide method. When the insoluble cyanophycin formed thin films, the films exhibited a structure of stacking lamellae. CHO cells grown on the films had a higher relative cell density, or 107–142% that of those grown on tissue culture polystyrene (TCPS), 48 h after seeding. After the removal of serum-containing medium, the CHO cells maintained cell morphology for up to 72 h in Dulbecco’s modified Eagle medium without serum, and the relative cell density was 150–170% that of the cells grown on TCPS 48 h after serum removal, indicating that the cyanophycin substratum could provide sustained cell growth. When RAW 246.7 cells were grown on the films of insoluble cyanophycin for 96 h, nitric oxide concentration released from the macrophages was below 2 mM/mg protein, suggesting that a minimal immune response was elicited. The results showed that cyanophycin might have the potential to serve as a biocompatible, degradable material in biomedical applications, such as tissue engineering and drug delivery.
Keywords: Tissue cell culture; Biomedical; Protein; Biomimetics; Macrophage activation;

Cyclodextrin glycosyltransferase (CGTase) from Bacillus sp. C26 was highly stable when it was immobilized in mixed alginate–gelatin gel beads. Enzymatic synthesis of β-cyclodextrin (β-CD) from starch by immobilized CGTase was performed in a continuous stirred-tank reactor (CSTR) and a packed-bed reactor (PBR). A comparative study on reaction kinetics found that the apparent maximum production rate (V max,app) for reaction in CSTR (0.5742 g L−1  h−1) was higher than that in PBR (0.33 g L−1  h−1) likely due to the higher mass transfer rate in CSTR. The lower apparent Michaelis–Menten constant (K m,app) in PBR (31.88 g L−1) compared to that in CSTR (58.3 g L−1) indicate that the enzyme in PBR required lower amount of substrate to become half-saturated. The appropriate dilution rate for operation in both reactors was the same at 0.75 h−1. To effectively produce β-CD, an integrated CSTR-PBR was developed. Through this integrated system, the final β-CD concentration and yield were improved from 6.10 g L−1 and 15.3% in a single CSTR up to 10.6 g L−1 and 26.5%, respectively. Moreover, the immobilized CGTase had good operational stability for 96 h of continuous use. This study has shown that the integrated CSTR-PBR was effective for continuous production of β-CD from starch and may greatly contribute to developing industrialized production of β-CD.
Keywords: Cyclodextrin glycosyltransferase; β-Cyclodextrin; Enzyme bioreactors; Immobilised enzymes; Kinetic parameters; Packed bed bioreactors;

Display OmittedThe current study describes the research on treatment of arsenic (As(III) or As(V)) containing industrial wastewater in a bio-column reactor. Bacillus arsenicus MTCC 4380 has been immobilized on Sawdust/MnFe2O4 composite bed in the bio-column reactor. The significant influence of bed height (20–100 cm), flow rate (251.2–837.33 mL/h), empty bed contact time (8–20 h), and initial arsenic (either As(III) or As(V)) concentration (2000–5000 mg/L) on the arsenic (either As(III) or As(V)) removal was investigated. The breakthrough curve for the bed height specified that a longer bed column extended the life span of the column with a maximum capacity of 87.573 and 88.990 mg/g for the As(III) and As(V) column, respectively. The impact of co-existing ions on biosorption/bioaccumulation of arsenic was investigated in column mode. The column was regenerated by eluting As(III) or As(V) using 0.05 M NaOH after biosorption/bioaccumulation studies. The desorption of As(III) or As(V) in each three cycles was about 51–64% and 53–65%, respectively. The critical bed depth, graphically determined using BDST model for As(III) and As(V) removal were 17.778 and 18.095 cm at 1% breakthrough, respectively. The working lifetime of MTZ (Tc), HMTZ, RMTZ were estimated to be 66 h, 80.291 cm, 1.409 cm/h and 9 h, 80.516 cm 1.413 cm/h at 100 cm bed depth for the removal of As(III) and As(V) by immobilized bacterial cells, respectively.
Keywords: Arsenic; SD/MnFe2O4 composite (MSD); Bioremediation; Fixed-bed bioreactors; Immobilised cells; Mass transfer;

Effects of a higher hydraulic shear force on denitrification granulation in upflow anoxic sludge blanket reactors by Yaoqi Xue; Jianbo Guo; Jing Lian; Yuanyuan Zhang; Chao Zhang; Yue Zhao (136-143).
The objectives of this study were to culture a more stable denitrification granular sludge and to investigate the effects of hydraulic shear force on the stability of the granular sludge. The stability consists of shear stability and removal stability, which are characterized by the shear sensitivity (Kss) and by the relative standard deviation of the specific nitrogen removal rate for three consecutive days (RSD-N3), respectively. Two upflow granular sludge blanket (USB) reactors under different hydraulic shear conditions were used to culture granular sludge. The Kss of the mature granular sludge in USBH (G  = 24.7 s−1) and USBL (G  = 14.5 s−1) were 0.000024 and 0.0051, respectively. The USBH only required 56 days to obtain mature granular sludge, whereas the USBL required 70 days. These results indicated that higher hydraulic shear tended to shorten the granulation time and enhance the shear stability of the granular sludge. The RSD-N3 of the USBL during the maturation period was only 3.68%, which is approximately 32.84% of the value for the USBH, indicating better removal stability for the nitrogen with the USBL. SEM indicated bacillus bacteria were the largest component of the granular microbial community, and metagenomics using high-throughput sequencing identified Methyloversatilis and Azospira as the dominant microorganisms. These findings are important for the development of technologies in this field and have extensive applications in the denitrification of granular sludge.
Keywords: Waste-water treatment; Bioreactors; Biodegradation; Immobilised cells; Hydraulic shear force; Kss;

The functional improvement of endoglucanase (EG), a key cellulose-hydrolyzing biocatalyst, is imperative for the practical use of cellulosic materials such as lignocellulose, stove and straws. Here, we employed a bio-inspired silica-encapsulation method to improve the stability and reusability of EG. We introduced a new silica-forming peptide (SFP) from Ectocarpus siliculosus at the C-terminus of EG to generate a recombinant fusion protein, EG-SFP, with auto-silicifying ability. We obtained an EG-SFP-encapsulated silica matrix (EG-SFP@Silica) via the EG-SFP-mediated auto-silicification process under ambient conditions. The immobilization efficiency was 90%. The introduction of SFP did not significantly affect the functionality of EG, and moreover, EG-SFP@Silica demonstrated higher thermostability by 5 °C than free EG-SFP or EG. In addition, EG-SFP@Silica retained 90% of its initial residual activity with up to 18 uses. These results provide a platform for the development of a practical enzymatic hydrolysis process for cellulosic materials.
Keywords: Cellulase; Immobilised enzymes; Immobilization; Polypeptides; Biosilicification; Silica forming peptide;

Kinetics of Pseudomonas veronii 2E biofilm development under different nutritional conditions for a proper bioreactor design by María Alejandra Daniel; Matías R. Barrionuevo; Santiago R. Doyle; Diana L. Vullo (150-158).
Display OmittedBiofilm-mediated bioreactors represent a proficient alternative, as extracellular materials of bacterial biofilms enhance metal immobilization by biosorption. Bacterial attachment or detachment is related to environmental conditions. The aim of this work is to study the nutritional dependence of Pseudomonas veronii 2E biofilm development to be applied in a future biofilm-reactor for wastewater treatments. For such purpose, biofilm establishment kinetics was explored over glass coverslips changing medium composition and nutrient concentration and in Fe(II) presence. After crystal violet cell staining, biofilm was visualized using direct microscopic observation; sample scanning-image analysis implemented in MATLAB and ethanol extraction-Absorbance 590 nm measurement. Biofilm structure developed with mineral basal medium appeared as a monolayer, but in complex basal media cell aggregates were appreciated. Optimal attachment was achieved with complex basal medium-5 g/L glucose at 55 h. The crystal violet concentration complemented with the image capturing supported these results. Biofilm establishment was enhanced by biosurfactant production in P. veronii 2E. The obtention of mutants deficient in biosurfactant secretion confirmed the influence of these compounds on cell motility and hence on cell attachment. The information of the biofilm structure defined under such conditions is the useful first step for the future bioreactor design.
Keywords: Wastewater biotreatment; Metal bioremediation; Biofilm reactor; Pseudomonas veronii; Biofilm stability; Biosorption;

Microbial production of 2,3-butanediol through a two-stage pH and agitation strategy in 150 l bioreactor by Anchal Priya; Prem Dureja; Pooja Talukdar; Rohit Rathi; Banwari Lal; Priyangshu M Sarma (159-167).
Display OmittedThis study was focused on scaling up of 2,3-butanediol (2,3-BD) production, via fermentation in a 150 l bioreactor using the newly isolated strain Enterobacter cloacae TERI BD 18. Operational parameters were customized for the strain E. cloacae using a fed- batch strategy, which enhanced the glucose to 2,3-BD conversion yield (0.44 g/g). Furthermore, dual stage pH and agitation control regime was considered where in the initial 10 h the pH value was 7.5 and the agitation rate used was 200 rpm favoring bacterial growth, while these values were subsequently changed to a pH value of 6.5 and an agitation rate of 150 rpm leading to enhanced accumulation of 2,3-BD. Production of 85 g/l of 2,3-BD was achieved with ethanol and acetoin as the only by-products. Total productivity of 1.73 g/l/h with a yield of 0.48 g of 2,3-BD per g of glucose was achieved in the 150 l bioreactor. Additionally, the total fermentation time was reduced to 50 h with this strategy. The integrated approach of fed-batch strategy together with dual regime in pH and agitation control favors 2,3-BD production in the pilot scale bioreactor system employed in this study.
Keywords: Glucose; Microbial growth; Fermentation; Fed-batch culture; 150 l bioreactor; 2,3-Butanediol (2,3-BD);

Optimizing storage conditions to prevent cold denaturation of trypsin for sequencing and to prolong its shelf life by Brankica Rašković; Saša Vatić; Boban Anđelković; Vladimir Blagojević; Natalija Polović (168-176).
Display OmittedTrypsin is a serine protease with widespread applications, including protein sequencing and trypsin mass fingerprinting. In the present study, the storage of trypsin in acidic conditions significantly affected the recovery of activity (40%) after 7 freeze–thaw cycles. Further, trypsin lost parts of its native secondary structure elements, which resulted in a 10% increase in β-sheet content (band maximum detected at a frequency of 1634 cm−1 in the Fourier transform infrared (FT-IR) spectrum) indicative of freezing-induced denaturation of the protein. The cold storage of trypsin in ammonium bicarbonate (pH 8.2) with the addition of cryoprotectants, such as glycerol or lysine, led to protein stabilization (complete secondary structure content preservation was detected by FT-IR), higher activity recovery (>90%) and modest autolysis (<10%). High activity recovery (>90%) was also detected with the addition of propylene glycol and polyethylene glycol, saccharides and arginine. Nevertheless, trypsin stored at pH 8.2 with the addition of glycerol or lysine was as efficient as untreated trypsin in the trypsin mass fingerprinting analysis of BSA, suggesting that the cold storage of trypsin in slightly alkaline conditions with the addition of cryoprotectants could prolong its shelf life.
Keywords: Enzyme activity; Cold stability; Protein recovery; Protein denaturation; Proteolysis; Proteomics;

In this work, a new approach for smooth control profiles parameterization (requiring a small number of parameters) is presented and especially recommended for bioprocess applications because of the smoothness of the profiles obtained, which are not only continuous time functions but also differentiable along the whole control interval. The importance of smooth profiles relays on the fact that abrupt changes in the cells’ environment may affect the metabolism of the cell, usually leading to a decrease in the process productivity. The parameterization proposed in this work is based on sinusoidal functions, which not only describe smooth functions but also are flexible and naturally constrained. Two very well-known bioprocess case studies have been successfully addressed by using the new parameterization approach.
Keywords: Optimization; Smooth control profiles; Substrate shock; Ethanol; Fed-batch culture; Penicillin;

Insights into the operational characteristics of a multi-habitat membrane bioreactor: Internal variation and membrane fouling by Bing Tang; Xuan Chen; Bing Qiu; Zi Zhang; Liying Bin; Shaosong Huang; Fenglian Fu (189-196).
Display OmittedThis work investigated the operational characteristics of a multi-habitat membrane bioreactor to reveal its internal variation and membrane fouling. During the 100d operating period, a combined anaerobic and aerobic zone gradually formed in a single membrane bioreactor. In the anaerobic zone, DO decreased to nearly zero, while in the aerobic zone, DO value still remained at above 3.1 mg/L, which created beneficial conditions for the removal of nitrogen (TN removal = 87.1%) and phosphorus (TP removal = 90.2%), although the phosphorus removal efficiency decreased in the later stage of operation (TP removal = 44.6%). The increase of torque in the bioreactor accorded with the Boltzmann model (r 2  = 0.97891), and the growth trend between torque and apparent viscosity of the fluid was highly consistent, which implied that an on-line torque approach may play an important role in the timely detection of rheological information about the mixed liquor suspended solids. As operational time increased, the size of sludge particles in the aeration zone tended to decrease, and the permanent membrane resistance gradually increased. These relationships revealed the reason for accelerated membrane fouling. Scanning electron microscopy revealed that the membrane fouling was mainly caused by a compact fouling layer that formed on the surface of membrane module.
Keywords: Membrane bioreactors; Waste-water treatment; Non-Newtonian fluids; Viscosity; Rheological behavior; Torque approach;

Detailed study of efficient ethanol production from elmwood by alkali pretreatment by ​Mahboubeh S. Noori; Keikhosro Karimi (197-204).
Display OmittedAn alkaline pretreatment was performed on hardwood elm to improve enzymatic hydrolysis and ethanol production. The pretreatment was conducted with 8% (w/v) NaOH solution at 0, 25, and 80 °C for 2 h, and the best results were obtained by the pretreatment at 0 °C. The glucose yield from untreated wood was only 8.0% and improved to 71.5% after the pretreatment at 0 °C, whereas the corresponding ethanol yield was improved from 11.1% to 45.7%. In order to decrease ineffective adsorption of cellulase enzyme on lignin and enhance the enzymatic hydrolysis and fermentation yields, a non-ionic surfactant, Tween-20, was used in the hydrolysis process. The addition of 2.5 g L−1 Tween-20 further favorably modified the yields of enzymatic hydrolysis and ethanol production to 79.8% and 57.3%, respectively. Changes in the wood’s structural properties by the pretreatment were followed in detail by swelling and buffering capacity measurements as well as SEM and FTIR analyses. Furthermore, the adsorption and desorption of cellulase during the enzymatic hydrolysis were investigated, and a consistent relation was observed between adsorbed and desorbed enzymes and enzymatic hydrolysis yield.
Keywords: Alkaline pretreatment; Adsorption; Bioconversion; Elmwood; Ethanol; Fermentation;

Lactic acid is a platform chemical useful for the production of polymers, oxychemicals, solvents, and for biological nutrient removal in wastewater streams. Food waste offers a renewable feedstock to produce lactic acid, but the co-digestion with sludge has not been suitably studied. In this study, response surface methodology was used to identify the pH, temperature, loading rate, and retention time for co-digestion of foodwaste and primary sludge that optimized lactic acid production. The optimum conditions occur at pH 5.5 and temperature 41 °C. A loading rate of 150 g L−1 volatile solids food waste maximizes lactate yield while 250 g L−1 volatile solids maximizes lactate concentration, resulting in 48 g L−1 and 58 g L−1 lactate respectively. Optical purity and ammonium concentration were evaluated to inform end uses. This research indicates that the co-digestion can achieve 97% of theoretical yield while requiring less pH adjustment and retention time than experiments that did not co-digest with primary sludge.
Keywords: Waste treatment; Bioconversion; Anaerobic processes; Lactic acid; Food waste; Biological nutrient removal;

Biosynthesis of FeS nanoparticles from contaminant degradation in one single system by Xiang Xiao; Wen-Wen Zhu; Hang Yuan; Wen-Wei Li; Qian Li; Han-Qing Yu (214-219).
Display OmittedBiogenetic nanomaterials have attracted growing interests in recent years attributed to their “green” synthesis nature, but expensive precursors are typically needed. On the other hand, release of hazardous intermediates during contaminant biodegradation/conversion is usually encountered in wastewater treatment processes. This study reports an effective coupling of both processes in one simple system to overcome the individual limitations. By using Shewanella oneidensis MR-1 as the inoculum, the Fe2+ ions released from naphthol green B (NGB) bioreduction and H2S from thiosulfate reduction were utilized in-situ to generate ferrous sulfide (FeS) nanoparticles, with an average size of ∼30 nm. In addition, we discovered for the first time that FeS nanoparticles could be synthesized both extracellarly and intracellularly by this strain, and identified the essential role of the Mtr respiratory pathway in the biosynthesis process. This study deepens our understanding of the bioconversion behaviors of metal-complex dyes, and may provide implications for development of sustainable nanomaterial fabrication processes.
Keywords: FeS nanoparticle; Shewanella oneidensis MR-1; Biosynthesis; Anaerobic process; Biodegradation; Waste-water treatment;

This paper presents the determination of reaction kinetic parameters and the kinetic analysis of a chemostat cultivation performed using a multiphase microreactor (mMR). Saccharomyces cerevisiae was cultivated aerobically in continuous chemostat mode using an mMR. Steady-state biomass, substrate and ethanol concentrations were determined at dilution rates between 0.14 ≤  D  ≤ 0.42 h−1 with a glucose-feed concentration of 10 g L−1. Modelling the aerobic chemostat culture was based on stationary balance equations. Maximal specific growth rate and Monod constant were determined using different linearization methods. The aerobic yeast metabolism was considered using two validity ranges of the model: (a) with purely oxidative metabolism in which glucose was converted into biomass or was used for maintenance metabolism, and no ethanol was generated; (b) with oxido-reductive metabolism employing an active Crabtree effect, in which ethanol was generated at the expense of biomass production. The different yield coefficients could be determined using the plots of the specific substrate consumption rate qS  = f(D) and the specific product formation rate qP  = f(D), respectively. Using this model, the reaction kinetic parameters were determined from the stationary concentrations of the biomass, glucose and ethanol that were determined during aerobic cultivation in the mMR. Finally, the kinetic parameters were compared with those reported in the literature that had been obtained using laboratory-scale reactors.
Keywords: Chemostat; Biokinetics; Kinetic Parameters; Microbioreactor; Scale-Down; Yeast;

Accelerated vascularization of tissue engineering constructs in vivo by preincubated co-culture of aortic fragments and osteoblasts by Paul Schumann; Andreas Kampmann; Gisa Sauer; Daniel Lindhorst; Constantin von See; Marcus Stoetzer; Frank Tavassol; Nils-Claudius Gellrich; Martin Rücker; Harald Essig (230-241).
There is an urgent critical need for the development of clinically relevant tissue-engineered large bone substitutes that can promote early vascularization after transplantation. To promote rapid blood vessel growth in the engineered tissue, we preincubated aortic fragments, as well as, co-cultures of aortic fragments and osteoblast-like cells in matrigel-filled PLGA scaffolds before implantation into the dorsal skinfold chambers of balb/c mice. Despite an acceptable and low inflammatory response, preincubated aortic fragments accelerate early angiogenesis of tissue-engineered constructs; the angiogenesis was found to occur faster than that observed in previous studies. Thus, the time-period for achieving a denser microvascular network could be reduced to half. In addition, co-culture with osteoblasts enhances this angiogenic effect significantly (against preincubated aortic fragments alone). During the preincubation period, aortic fragments begin to form a network of vessel-like structures additionally supported by osteoblast-like cells. After transplantation, further development of a dense microvasculature continues rapidly. Therefore, preincubation of aortic fragments, especially in co-culture with osteoblast-like cells, in 3D extracellular matrices supports the rapid vascularization of tissue-engineered constructs. This method is a promising approach to establish a dense microvascular network in these constructs.
Keywords: Biodegradation; Tissue cell culture; Animal cell culture; Oxygen transfer; Angiogenesis; Intravital flourescence microscopy;

Direct channeling is a well-known process in which intermediates are funneled between enzyme active sites through a physical tunnel and can be a potential way to enhance the biocatalytic efficiency for cascading bioreactions. However, the exact mechanism of the substrate channeling remains unclear. In this work, we used mathematical models to describe the mass transfer in the physical tunnels and to gain further understanding of direct proximity channeling. Simulation with a diffusion-reaction model showed that the reduction of the diffusion distance of intermediates could not cause proximity channeling. A second kinetic model, which considered the physical tunnel as a small sphere capable of preventing diffusion of the intermediate into the bulk, was then constructed. It was used to show that the maximum channeling degree in branched pathways depends on the strength of the side reactions, suggesting that proximity channeling in a physical tunnel is more suitable for a pathway with strong side reactions. On the other hand, for a linear pathway, proximity channeling is more beneficial when the constituting enzymes have relatively low activities and expression levels. Our kinetic model provides a theoretical basis for engineering proximity channeling between sequentially acting enzymes in microbial cell factories and enzyme engineering.
Keywords: Proximity channeling; Modelling; Diffusion-reaction; Enzymes; Kinetic parameters; Mass transfer;

Mechano growth factor-C24E, a potential promoting biochemical factor for ligament tissue engineering by Yang Song; Can Yu; Chunli Wang; Xingshuang Ma; Kang Xu; Juila Li Zhong; Yonggang Lv; K.L. Paul Sung; Li Yang (249-263).
Display OmittedLigaments play an important role in stabilizing and balancing knee joint movement. Anterior cruciate ligament (ACL) injuries remain a challenge in clinical and experimental settings due to the poor healing potential of ACL injuries. This study investigates whether the short peptide Mechano growth factor-C24E (MGF-C24E) is an efficient biochemical factor to promote repair of ACL injuries. We found that MGF-C24E-treated (5–30 ng/ml) synovial fibroblasts (SFs) subjected to the static stretching (12% strain) were experienced significant lower oxidative stress, endoplasmic reticulum stress, and the activity of matrix metalloproteinases (MMPs) by SFs was also decreased. In addition, when ACL fibroblasts (ACLFs) were injured and then cultured in SFs culture medium after the SFs had been treated with MGF-C24E, the ACLFs exhibited increased proliferation, migration, collagen synthesis and enhanced mechanical recovery. These results indicate that MGF-C24E can interact with injured SFs and affect the injury microenvironment of the knee to promote ACLFs mechanical injury repair.
Keywords: Ligament tissue engineering; Mechano growth factor; Biomedical; Polypeptides; Tissue cell culture; Physiology;

Facile bio-synthesis of gold nanoparticles by using extract of Hibiscus sabdariffa and evaluation of its cytotoxicity against U87 glioblastoma cells under hyperglycemic condition by Pratik Mishra; Sambit Ray; Sayantan Sinha; Bhaskar Das; Md. Imran Khan; Susant K. Behera; Soon-Il Yun; Suraj K. Tripathy; Amrita Mishra (264-272).
Display OmittedIn the present paper, a facile synthesis of gold nanoparticles is reported with leaf and stem extract of Hibiscus sabdariffa. Structural features of as synthesized nanoparticles are characterized by UV–vis spectroscopy, XRD, FTIR, and XPS. Morphology of the above synthesized gold nanoparticles is investigated by electron microscopy. The stability of the nanoparticles is studied in different concentrations of glucose which suggested their possible application under hyperglycemic condition. As synthesized nanoparticles has shown selective toxicity towards U87 glioblastoma multiforme cell line under normal and hyperglycemic condition, indicating their potential to be used in the development of value-added products in the biochemical industries. The possible mode of activity of the above nanoparticles has been studied by in vitro molecular techniques.
Keywords: Biomedical; Biosynthesis; Cytotoxicity; DNA; Nanoparticles; Optimisation;

In situ synthesized rGO–Fe3O4 nanocomposites as enzyme immobilization support for achieving high activity recovery and easy recycling by Dong Yang; Xueyan Wang; Jiafu Shi; Xiaoli Wang; Shaohua Zhang; Pingping Han; Zhongyi Jiang (273-280).
Display OmittedHerein, the reduced graphene oxide–Fe3O4 (rGO–Fe3O4) nanocomposites are synthesized by the simultaneous reduction of graphene oxide (GO) and in situ deposition of Fe3O4 nanoparticles (ca. 20 nm) enabled by Fe2+ ions. The rGO–Fe3O4 nanocomposites integrate the magnetic property of Fe3O4 nanoparticles and the large specific surface area of rGO nanosheets. Catalase (CAT), as a commonly used enzyme, can be efficiently immobilized on the rGO–Fe3O4 nanocomposites through physical adsorption. The CAT loading capacity is as high as 312.5 ± 12.6 mg g−1, while the activity recovery of CAT can be high up to nearly 98%. The strong magnetic response of immobilized CAT ensures its easy separation from the reaction system when an external magnetic field is applied. Owing to the hydrophobic and hydrogen bonding interactions between enzyme and the support, the immobilized CAT exhibits zero leaching, desirable stability and excellent reusability.
Keywords: rGO–Fe3O4 nanocomposites; Immobilised enzyme; Enzyme activity; Easy recycling; Kinetic parameters; Biocatalysis;

Promotion effects of ultrasound on sludge biodegradation by thermophilic bacteria Geobacillus stearothermophilus TP-12 by Wan-Qian Guo; He-Shan Zheng; Shuo Li; Shih-Hsin Ho; Shan-Shan Yang; Xiao-Chi Feng; Jo-Shu Chang; Xiang-Jing Wang; Nan-Qi Ren (281-287).
Sludge biodegradation using thermophilic bacteria is a promising method for sludge treatment. In order to further enhance the efficiency of sludge reduction and hydrolysis, low-frequency ultrasound was used to promote this process. We isolated a thermophilic strain that is effective in secreting extracellular protease to hydrolyze sludge. Then the key ultrasound parameters were selected using the response surface methodology method. After 12 h treatment using thermophilic bacteria with short-time ultrasound promotion, volatile suspended solids (VSS) reduction ratio was achieved 32.8%, which is 41.4% higher than that without ultrasound promotion. Meanwhile the contents of soluble chemical oxygen demand (SCOD), protein and carbohydrate were increased by 20.2%, 16.8% and 15.9%, respectively. The composition of dissolved organic matter of sludge products evaluated by excitation-emission matrix spectroscopy demonstrated the promotion effect and eliminated the possibility of the direct sludge degradation caused by ultrasound treatment. Low-frequency ultrasound could effectively promote the thermophilic bacteria hydrolysis to achieve higher sludge biodegradation ratio without directly degrading the raw sludge. The promoted process of sludge biodegradation can further reduce the environmental risk and make sludge to be more readily usable.
Keywords: Sludge; Waste treatment; Biodegradation; Thermophiles; Protease; Low-frequency ultrasound;

Cultivation of yeast in diffusion-based microfluidic device by A.F. Oliveira; V.B. Pelegati; H.F. Carvalho; C.L. Cesar; R.G. Bastos; L.G. de la Torre (288-295).
The capacity to create a diffusive chemical concentration gradient in microfluidic systems has the potential to improve the study of microbial processes. These tools allow the evaluation of microbial cell performance under different and controlled conditions. Diffusion-based gradient generators, in particular, have the capacity to maintain spatiotemporally constant gradient concentrations necessary to evaluate cell behavior in a precise environment. This work uses a known microfluidic device capable of generating a diffusive glucose concentration gradient to evaluate for the first time the behavior of Saccharomyces cerevisiae ATCC 7754 inside a microchannel. The cell growth along the microfluidic microchambers was observed and the kinetic parameters determined, with values statistically similar to those of conventional batch cultivation. Monod kinetic parameters could also be determined in the microfluidic device using small substrate concentrations. These results show the potential of this microbioreactor to investigate yeast growth with microliter samples and to evaluate experiments in triplicate performed and in parallel. The diffusive concentration gradient in a microfluidic device allowed the acquisition of results in a more practical way when compared to conventional techniques.
Keywords: Microfluidics; Gradient concentration; Bioprocess monitoring; Microbial growth; Kinetic parameters; Yeast;

Performance and microbial community analysis in alkaline two-stage enhanced anaerobic sludge digestion system by Yogananda Maspolim; Chenghong Guo; Keke Xiao; Yan Zhou; Wun Jern Ng (296-305).
This study investigated an alkaline two-stage enhanced anaerobic sludge digestion system, which simultaneously combined biological and chemical mode of degradation. The alkaline enhanced mesophilic stage-1 was operated at pH 8 with 3 days hydraulic retention time (HRT), while the mesophilic stage-2 was without pH adjustment at 17 days HRT. The system achieved higher chemical oxygen demand (COD) removal, volatile solids (VS) reduction, and methane yield than the conventional 20 days HRT single-stage system. Further enhancement was obtained by moving the stage-1 from 35 to 55 °C, but it did not yield better energy balance with the 3 + 17 days HRT configuration implemented in this study. 454 pyrosequencing revealed the acclimation of specialized communities in the alkaline two-stage system. Methanosarcina, Methanobrevibacter and Methanothermobacter could survive at pH 8 in the alkaline enhanced stage-1 and contributed to regulating the potentially inhibitory volatile fatty acids (VFA) or hydrogen levels under the enhanced sludge solubilization and acidogenesis condition. Various fermentative populations, distinct to those in the single-stage system, were also enriched in the stage-1s. These populations could grow at pH 8, were transferred into the stage-2, and ensured continuity of the biochemical reactions under mild alkaline condition, leading to the enhanced sludge digestion process.
Keywords: Anaerobic processes; Bioreactors; Biogas; Waste treatment; Two-stage anaerobic digestion; Microbial community;

Completely autotrophic nitrogen-removal over nitrite (CANON) has been considered as a promising nitrogen-removal technology in treating high ammonia and high temperature wastewater. In this study, the exact effect of different temperatures on process performance and microbial features based on two lab-scale CANON reactors was investigated. Results showed extraordinary nitrogen removal performance with mean total nitrogen (TN) removal efficiency of 80.01% and 66.90% under high TN removal loading of 2.09 kg m−3  d−1 and 1.02 kg m−3  d−1 at 30 °C and ambient temperature (16–23 °C) respectively. Biodiversity analysis indicated Nitrosomonas-related aerobic ammonia oxidizing bacteria (AerAOB) and Candidatus Brocadia-like anaerobic ammonia oxidizing bacteria (AnAOB) were predominant functional microorganisms that coexisted without distinguishable niche on the volcanic carriers in both two CANON reactors. Temperature could noticeably impact functional bacterial population: the population of AerAOB and AnAOB decreased to one to two orders of magnitude whereas Nitrospira and Nitrobacter increased at ambient temperature. Based on the bacterial experimental results, some feasible strategies have been discussed to improve the bioactivity and increase the function bacterial population, aiming at enhancing nitrogen-removal capacity at ambient temperature.
Keywords: CANON; Microbial growth; Biofilms; Bioreactors; Waste-water treatment; Community structure;

An approach for estimating the maximum specific growth rate of Gluconobacter japonicus in strawberry purée without cell concentration data by A.M. Cañete-Rodríguez; I.M. Santos-Dueñas; J.E. Jiménez-Hornero; M.J. Torija-Martínez; A. Mas; I. García-García (314-320).
The estimation of the maximum specific growth rate (μ max) for non-readily culturable bacteria, growing on complex media containing suspended solids, is a difficult task considering the important problems in obtaining reliable measures of cell concentration. An example of this situation can be a culture of Gluconobacter japonicus growing in strawberry purée for producing gluconic acid. Based on the dependency between energy requirements of the genus Gluconobacter and substrate uptake as well as its constant relationship between gluconic acid production and total substrate uptake, the total substrate concentration profile during the exponential growth phase could be used for estimating μ max without cell concentration measures. In this case, the high selectivity of the strain for glucose in comparison to fructose resulted in no fructose consumption during the batch; so, just using the glucose concentrations data during the exponential phase allow us to obtain an estimation of μ max. Additionally, a rough estimation of the apparent and stoichiometric yields of cell on glucose is also possible.
Keywords: Maximum specific growth rate; Gluconobacter japonicus CECT 8443; Growth kinetics; Biokinetics; Biotransformations; Batch processing;