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Engineering in Life Sciences

Wiley Online Library : Engineering in Life Sciences

Published: 2018-02-01T00:00:00-05:00


Enrichment of ANME-2 dominated anaerobic methanotrophy from cold seep sediment in an external ultrafiltration membrane bioreactor


Anaerobic oxidation of methane (AOM) coupled to sulfate reduction is a microbially mediated unique natural phenomenon with an ecological relevance in the global carbon balance and potential application in biotechnology. This study aimed to enrich an AOM performing microbial community with the main focus on anaerobic methanotrophic Archaea (ANME) present in sediments from the Ginsburg mud volcano (Gulf of Cadiz), a known site for AOM, in a membrane bioreactor (MBR) for 726 days at 22 (± 3) °C and an ambient pressure. The MBR was equipped with a cylindrical external ultrafiltration membrane, fed a defined medium containing artificial seawater and operated at a cross flow velocity of 0.02 m min−1. Sulfide production with simultaneous sulfate reduction was in equimolar ratio between days 480 and 585 of MBR operation, whereas methane consumption was in oscillating trend. At the end of the MBR operation (day 726), the enriched biomass was incubated with 13C labeled methane, 13C labeled inorganic carbon was produced and the AOM rate based on 13C-inorganic carbon was 1.2 μmol.gdw−1.d−1. Microbial analysis of the enriched biomass at 400 and 726 days of MBR operation showed that ANME-2 and Desulfosarcina type sulfate reducing bacteria were enriched in the MBR, which formed closely associated aggregates. The major relevance of this study is the enrichment of an AOM consortium in a MBR system which can assist to explore the ecophysiology of ANME and provides an opportunity to explore the potential application of AOM. This article is protected by copyright. All rights reserved

Supplement comprising of laccase and citric acid as an alternative for antibiotics– in vitro triggers of melanin production


An indiscriminate use of antibiotics in humans and animals has led to the widespread selection of antibiotic-resistance, thus constricting the use of antibiotics. A possible solution to counter this problem could be to develop alternatives that can boost the host immunity, thus reducing the quantity and frequency of antibiotic use. In this work, for the first time, citric acid and laccase were used as extracellular inducers of melanin production in yeast cells and human cell lines. It is proposed that the formulation of laccase and citric acid together could further promote melatonin-stimulated, melanocyte-derived melanin production. Melanization as a probe of immunity described in this study, is an easy and a rapid test compared to other immunity tests and it allows performing statistical analyses. The results showed the synergistic effect of citric acid and laccase on melanin production by yeast cells, with significant statistical differences compared to all other tested conditions (p: 0.0005-0.005). Laccase and citric acid together boosted melanin production after 8 days of incubation. An increase in melanin production by two human colon cells lines (Cacao-2/15 and HT-29) was observed on supplementation with both laccase and citric acid in the cell growth medium. Produced melanin showed antimicrobial properties similar to antibiotics. Therefore, a formulation with citric acid and laccase may prove to be an excellent alternative to reduce the antibiotic use in human and animal subjects. This article is protected by copyright. All rights reserved

Graphene oxide/silver nanohybrid: Optimization, antibacterial activity and its impregnation on bacterial cellulose as a potential wound dressing based On GO-Ag nanocmposite-coated BC


Recently, bacterial cellulose (BC) based wound dressing have raised significant interests in medical fields. However, to our best knowledge, it is apparent that the BC itself has no antibacterial activity. In this study, we optimized graphene oxide-silver (GO-Ag) nanohybrid synthesis using Response Surface Methodology and impregnate it to BC and carefully investigate their antibacterial activities against both the Gram-negative bacteria Escherichia coli and the Gram-positive bacteria Staphylococcus aureus. We discover that, compared to silver nanoparticles, GO-Ag nanohybrid with an optimal GO suspension's pH and [GO]/[AgNO3] ratio is much more effective and shows synergistically enhanced, strong antibacterial activities at rather low dose. The GO-Ag nanohybrid is more toxic to E. coli than that to S. aureus. The antibacterial and mechanical properties of BC/GO-Ag composite are further investigated. This article is protected by copyright. All rights reserved

Effects of osmotic pressure and pH on citric acid and erythritol production from waste cooking oil by Yarrowia lipolytica


Erythritol and citric acid could be produced from waste cooking oil (WCO) by Yarrowia lipolytica under different medium conditions, and osmotic pressure together with pH were considered to be the critical factors in this process. High osmotic pressure (2.76 osmol/L) combined with low pH (pH 3.0) promoted the highest yield of erythritol (21.8 g/L) accompanied by low-producing citric acid (2.5 g/L). By contrast, the highest citric acid biosynthesis (12.6 g/L) was detected under a pH of 6.0 and an osmotic pressure of 0.75 osmol/L, when only 4.0 g/L of erythritol was yielded. Moreover, lipase activities in these two media were also detected, and pH 3.0–OP 2.76 was supposed to be more beneficial to lipase activity. Biochemical pathways involved in the biosynthesis of erythritol and citric acid were subsequently investigated, and the products yielded from WCO were assumed to be correlated with the activities of transketolase, erythrose reductase, citrate synthase, and glycerol kinase. However, RT-PCR analysis revealed that mRNA levels of these enzymes did not significantly differ, confirming that metabolic flux regulations of erythritol and citric acid mostly took place at the post-transcriptional level. This article is protected by copyright. All rights reserved

Addition of aluminum oxide microparticles to Trichoderma viride My preculture enhances cellulase production and influences fungal morphology


Morphological engineering techniques have recently become popular, since they are used to increase the production of a variety of metabolites and enzymes when fungi are grown in submerged cultures. This study aimed to facilitate cellulase production by adding aluminum oxide to Trichoderma viride My precultures. The results showed that the highest cellulase activity was achieved when aluminum oxide at 10 g/L was used, and the activities of cellulase for filter paper and endoglucanase activity assays increased from 519.11 to 607.35 U/mL by 17.1%, and from 810.08 U/mL to 917.59 U/mL by 13.3%, compared with the control, respectively. Addition of aluminum oxide decreased the size of T. viride My pellets and increased the final pH. The changes in pellet diameter after the addition of different concentrations of aluminum oxide were fitted using a modified exponential decay model, which could precisely predict the pellet size by controlling aluminum oxide concentration. The optimum concentration of microparticles, and therefore pellet size, could significantly improve cellulase production, which is an encouraging step towards commercial cellulase production.

Biosurfactant production by Mucor circinelloides: Environmental applications and surface-active properties


Biosurfactants are structurally a diverse group of surface-active molecules widely used for various purposes in industry. In this study, among 120 fungal isolates, M-06 was selected as a superior biosurfactant producer, based on different standard methods, and was identified as Mucor circinelloides on the basis of its nucleotide sequence of the internal transcribed spacer (ITS) gene. M. circinelloides reduced the surface tension to 26 mN/m and its EI24 index was determined to be 66.6%. The produced biosurfactant exhibited a high degree of stability at a high temperature (121°C), salinity (40 g/L), and acidic pH (2–8). The fermentation broth's ability to recover oil from contaminated sand was 2 and 1.8 times higher than those of water and Tween 80, respectively. The ability of biosurfactant to emulsify crude oil in the sea and fresh water was 64.9 and 48% respectively. This strain could remove 87.6% of crude oil in the Minimal Salt Medium (MSM) crude oil as the sole carbon source. The results from a primary chemical characterization of crude biosurfactant suggest that it is of a glycolipid nature. The strain and its biosurfactant could be used as a potent candidate in bioremediation of oil-contaminated water, soil, and for oil recovery processes.

Effects of copper on expression of methane monooxygenases, trichloroethylene degradation, and community structure in methanotrophic consortia


Copper plays a key role in regulating the expression of enzymes that promote biodegradation of contaminants in methanotrophic consortia (MC). Here, we utilized MC isolated from landfill cover to investigate cometabolic degradation of trichloroethylene (TCE) at nine different copper (Cu2+) concentrations. The results demonstrated that an increase in Cu2+ concentration from 0 to 15 μM altered the specific first-order rate constant k1,TCE, the expression levels of methane monooxygenase (pmoA and mmoX) genes, and the specific activity of soluble methane monooxygenase (sMMO). High efficiency TCE degradation (95%) and the expression levels of methane monooxygenase (MMO) were detected at a Cu2+ concentration of 0.03 μM. Notably, sMMO-specific activity ranged from 74.41 nmol/(mgcell h) in 15 μM Cu2+ to 654.99 nmol/(mgcell h) in 0.03 μM Cu2+, which contrasts with cultures of pure methanotrophs in which sMMO activity is depressed at high Cu2+ concentrations, indicating a special regulatory role for Cu2+ in MC. The results of MiSeq pyrosequencing indicated that higher Cu2+ concentrations stimulated the growth of methanotrophic microorganisms in MC. These findings have important implications for the elucidation of copper-mediated regulatory mechanisms in MC.

The chemo enzymatic functionalization of chitosan zeolite particles provides antioxidant and antimicrobial properties


Silicate-based microporous materials like zeolites are nano enabled particles and used for various applications including pharmaceutical formulations. This study reports on the chemo-enzymatic functionalization of chitosan-zeolite particles (CTS-zeolites) with caffeic acid (CA) and glucose oxidase (GOX) to impart combined antioxidant and antimicrobial properties. CA was grafted on the chitosan moieties by using laccase generating stable particles (zeta potential –36.7 mV) of high antioxidant activity (44% DPPH inhibition). GOX was immobilized both on CTS-zeolites and on CA modified CTS-zeolites and creating a hydrogen peroxide generation system continuously and in-situ producing this oxidative and antimicrobial agent. The system prevented bacterial growth of E. coli and S. aureus over 24 h whereby a steady-state concentration of around 60 μM hydrogen peroxide in the culture medium was observed. CA and GOX functionalized CTS-zeolite particles additionally showed combinatorial antioxidant and antimicrobial properties providing a powerful bioactive system for medical applications. These particles proved their suitability for incorporation in bioactive formulations which could be used, inter alia, for topical wound treatments.

Assembly of graphene oxide-formate dehydrogenase composites by nickel-coordination with enhanced stability and reusability


Featuring unique planar structure, large surface area and biocompatibility, graphene oxide (GO) has been widely taken as an ideal scaffold for the immobilization of various enzymes. In this regard, nickel-coordinated graphene oxide composites (GO-Ni) were prepared as novel supporters for the immobilization of formate dehydrogenase. The catalytic activity, stability and morphology were studied. Compared with GO, the enzyme loading capacity of GO-Ni was enhanced by 5.2-fold, besides the immobilized enzyme GO-Ni-FDH exhibited better thermostability, storage stability and reuse stability than GO-FDH. GO-Ni-FDH retained 40.9% of its initial activity after 3 h at 60°C, and retained 31.4% of its initial relative activity after 20 days’ storage at 4°C. After eight times usages, GO-Ni-FDH maintained 63.8% of its initial activity. Mechanism insights of the multiple interactions of enzyme with the GO-Ni were studied, considering coordination bonds, hydrogen bonds, electrostatic forces, coordination bonds, and etc. A practical and simple immobilization strategy by metal ions coordination for multimeric dehydrogenase was developed.

The ejector loop reactor: Application for microbial fermentation and comparison with a stirred-tank bioreactor


Ejector loop reactors (ELR) are successfully used in industrial chemical processes for gas/liquid reactions. They achieve higher mass transfer rates compared to the stirred-tank reactor (STR) at comparable specific power input. Insufficient oxygen transport and shear stress induced growth inhibition are limiting parameters during microbial fermentation. Due to its better mass transfer characteristics, the ELR was expected to have beneficial effects on biomass and recombinant protein production. One concern, however, was whether the ELR's shear stress characteristics would have a negative effect. This study evaluated the suitability of using the Buss-Loop® Reactor (BLR), one of the most advanced ELR technologies, as a bioreactor. The well-studied STR was used as a reference. A lab scale BLR was adapted for microbial fermentation. Mass transfer rates and specific power inputs were within the same order of magnitude in the ELR and the reference STR. Maximum kLa values of 207 and 205 h−1 at power inputs of 6.9 and 9.7 W/L were measured in the ELR and STR, respectively. During batch fermentation of Escherichia coli K12 MG1655, maximum cell densities were higher in the ELR (OD600 of 22) than in the STR (OD600 of 18). Green fluorescence protein (GFP) production with pGS1 was comparable; however, more GFP was released into the media in the ELR. This indicates higher cell disruption compared to the STR. Despite this drawback of the first prototype, our work clearly demonstrates the potential of the ELR as a system for microbial fermentations.

Engineering aspects of immobilized lipases on esterification: A special emphasis of crowding, confinement and diffusion effects


Cross-linked enzyme crystal (CLEC) and sol-gel entrapped pseudomonas sp. lipase were investigated for the esterification of lauric acid with ethanol by considering the effects of reaction conditions on reaction rate. The activation energy for the reaction was estimated to be 1097.58 J/mol and 181.75 J/mol for sol-gel and CLEC entrapped lipase respectively. CLEC lipase exhibited a marginal internal diffusion effect on reaction rate over sol-gel lipases and found to be interesting. The overall reaction mechanism was found to conform to the Ping Pong Bi Bi mechanism. The higher efficiency of sol-gel lipases over CLEC lipases in esterification reaction is mainly due to the combined effects of crowding, confinement and diffusional limitations.

Enhanced production of the pharmaceutically important polyphenolic compounds in Vitex agnus castus L. shoot cultures by precursor feeding strategy


Agitated Vitex agnus castus L. shoot cultures were established to analyse the content of selected pharmaceutically important flavonoids and phenolic acids. Two variants (selected from nine ones) of MS medium were prepared: A (BAP 1 mg/L; NAA 0.5 mg/L; GA3 0.25 mg/L) and B (BAP 2 mg/L; NAA 0.5 mg/L). The biomass was harvested after 1, 2, 3,4, 5 and 6 weeks. Four-week cultures (variant A) were selected to perform the precursor feeding experiment. The L-phenylalanine dose of 1.6 g/L appears to be the most advantageous. Compared to the control cultures, the content of the individual compounds increased in a range from 1.4 to 17.3-fold (e.g. p-coumaric acid – 17.3 fold; casticin – 4.8-fold). The biomass from in vitro cultures is richer in neochlorogenic acid (16-fold), p-coumaric acid (5.3-fold), rutin (2.8-fold), caffeic acid (1.5-fold) and cinaroside (1.5-fold) than the leaves of its parent greenhouse-cultivated plants. Extracts contained 30 mg/100 g DW of casticin, but after the hydrolysis its amount increased up to 200 mg/100 g DW and twice exceeded the content in the greenhouse leaves. The results indicate that V. agnus castus agitated shoot cultures might be considered as a potential biotechnological source of some pharmaceutically important compounds, especially casticin, rutin, neochlorogenic and p-coumaric acids.

Rhamnolipid as new bio-agent for cleaning of ultrafiltration membrane fouled by whey


In this work, rhamnolipid biosurfactant as an eco-friendly and biodegradable cleaning agent was produced by Pseudomonas aeruginosa bacteria and was used to evaluate the chemical cleaning efficiency of whey fouled ultrafiltration membranes. Thin layer chromatography (TLC) and Fourier transform infrared spectroscopy (FTIR) confirmed the successful synthesis of rhamnolipid. The produced rhamnolipid was compared to chemical cleaners including sodium hydroxide (NaOH), sodium dodecyl sulfate (SDS) and Tween 20. Ultrafiltration membranes used for fouling and cleaning analysis were prepared using phase inversion via immersion precipitation technique. For studying the fouling mechanisms, Hermia's model adapted to cross-flow was used. From the fouling mechanism experiments, it was found that the complete blocking and cake formation were the dominant fouling mechanisms. The highest values of cleaning efficiency were achieved using rhamnolipid and NaOH as cleaning agents with the flux recovery of 100%, but with considering the low concentration of the rhamnolipid used in the cleaning solution compared to NaOH (0.3 versus 4 g/L for NaOH), its application is preferred.

Detection and identification of Staphylococcus aureus using magnetic particle enhanced surface plasmon spectroscopy


In this work, an approach for SPR spectroscopy using the liSPR system is examined that combines signal amplification by PCR and magnetic nanoparticles in one injection step. Therefore, the synthesis of PCR products was performed on the beads similar to a solid-phase PCR, termed PCR-on-a-bead. The functionality of this PCR was proven using an enzymatic assay. For validation the detection of oligonucleotides by SPR, an asymmetric PCR product was investigated. A signal increase upon binding of the PCR product to the specific probes was observed. In addition, surface regeneration of the chip was examined and reuse for at least two times ascertained. Amplification of the SPR signal by magnetic beads was verified but no signal was detected for PCR products immobilized on particles prior to injection.

Covalent immobilization of acetylcholinesterase on a novel polyacrylic acid-based nanofiber membrane


In this study, polyacrylic acid-based nanofiber (NF) membrane was prepared via electrospinning method. Acetylcholinesterase (AChE) from Electrophorus electricus was covalently immobilized onto polyacrylic acid-based NF membrane by demonstrating efficient enzyme immobilization, and immobilization capacity of polymer membranes was found to be 0.4 mg/g. The novel NF membrane was synthesized via thermally activated surface reconstruction, and activation with carbonyldiimidazole upon electrospinning. The morphology of the polyacrylic acid-based membrane was investigated by scanning electron microscopy, Fourier Transform Infrared Spectroscopy, and thermogravimetric analysis. The effect of temperature and pH on enzyme activity was investigated and maxima activities for free and immobilized enzyme were observed at 30 and 35°C, and pH 7.4 and 8.0, respectively. The effect of 1 mM Mn2+, Ni2+, Cu2+, Zn2+, Mg2+, Ca2+ ions on the stability of the immobilized AChE was also investigated. According to the Michaelis–Menten plot, AChE possessed a lower affinity to acetylthiocholine iodide after immobilization, and the Michaelis–Menten constant of immobilized and free AChE were found to be 0.5008 and 0.4733 mM, respectively. The immobilized AChE demonstrated satisfactory reusability, and even after 10 consecutive activity assay runs, AChE maintained ca. 87% of its initial activity. Free enzyme lost its activity completely within 60 days, while the immobilized enzyme retained approximately 70% of the initial activity under the same storage time. The favorable reusability of immobilized AChE enables the support to be employable to develop the AChE-based biosensors.

Jet aeration as alternative to overcome mass transfer limitation of stirred bioreactors


In industrial biotechnology increasing reactor volumes have the potential to reduce production costs. Whenever the achievable space time yield is determined by the mass transfer performance of the reactor, energy efficiency plays an important role to meet the requirements regarding low investment and operating costs. Based on theoretical calculations, compared to bubble column, airlift reactor, and aerated stirred tank, the jet loop reactor shows the potential for an enhanced energetic efficiency at high mass transfer rates. Interestingly, its technical application in standard biotechnological production processes has not yet been realized. Compared to a stirred tank reactor powered by Rushton turbines, maximum oxygen transfer rates about 200% higher were achieved in a jet loop reactor at identical power input in a fed batch fermentation process. Moreover, a model-based analysis of yield coefficients and growth kinetics showed that E. coli can be cultivated in jet loop reactors without significant differences in biomass growth. Based on an aerobic fermentation process, the assessment of energetic oxygen transfer efficiency [kgO2 kW−1 h−1] for a jet loop reactor yielded an improvement of almost 100%. The jet loop reactor could be operated at mass transfer rates 67% higher compared to a stirred tank. Thus, an increase of 40% in maximum space time yield [kg m−3 h−1] could be observed.

An environment-friendly approach to isolate and purify glucan from spent cells of recombinant Pichia pastoris and the bioactivity characterization of the purified glucan


While the methylotrophic yeast Pichia pastoris enables the industrial-scale biosynthesis of many recombinant products, large amount of nutrient-rich biomass emerged along this process. Polysaccharides, especially glucans that are abundant in the cell wall of P. pastoris, are yet to be better utilized owing to their various biological activities. However, the isolation and purification of cell wall glucan from P. pastoris has not been reported. In this study, we established an environment-friendly approach, including induced autolysis, hot-water treatment, ultrasonication, isopropanol extraction, and protease treatment, to isolate and purify glucan from the cell wall of P. pastoris. We achieved a purity of 85.3% and a yield of 11.7% for the purified glucan. Proteins, nucleic acids, lipids, and ash were efficiently removed during the purification. The activities of the purified glucan were investigated in mice fed with a high-fat diet. The purified glucan decreased the level of total cholesterol and triglycerides by 30.3 and 29.7%, respectively. This result suggested that the cell wall glucan of P. pastoris could be developed to a therapeutic agent for dyslipidemia. Our study proposed an environment-friendly and effective method to isolate and purify the glucan from P. pastoris, providing solid foundation for the high-value utilization of this yeast.

Biotechnological production of the angiotensin-converting enzyme inhibitory dipeptide isoleucine-tryptophan


Peptides with angiotensin-converting enzyme (ACE)-inhibitory and antihypertensive effects are suggested as innovative food additives to prevent or treat hypertension. Currently, these substances are isolated from food proteins following nonselective hydrolysis as a mixture of ACE-inhibitory peptides and other protein fragments. This study presents an innovative biotechnological method, based on recombinant DNA technology that was established to specifically produce the ACE-inhibitory dipeptide isoleucine-tryptophan. In a first step, a repetitive isoleucine-tryptophan construct fused to the maltose-binding protein was generated and expressed in Escherichia coli BL21 cells. The chromatographically purified recombinant fusion protein was enzymatically hydrolyzed using α-chymotrypsin to liberate the dipeptide isoleucine-tryptophan. The identity of the liberated isoleucine-tryptophan was confirmed by MS and derivatization of its N-terminus. The ACE-inhibitory effect of the recombinant dipeptide on soluble and membrane bound ACE was found to be indistinguishable from the inhibitory potential of the chemically produced commercially available dipeptide. The established experimental strategy represents a promising approach to the biotechnical production of sufficient amounts of recombinant peptide-based ACE-inhibitory and antihypertensive substances that are applicable as functional food additives to delay or even prevent hypertension.

High nitrate removal by starch-stabilized Fe0 nanoparticles in aqueous solution in a controlled system


This study was conducted to investigate biodenitrification efficiency with starch-stabilized nano zero valent iron (S-nZVI) as the additional electron donor in the presence of S2O3 in aqueous solutions, under anaerobic conditions. The main challenge for nZVI application is their tendency to agglomeration, thereby resulting in loss of reactivity that necessitates the use of stabilizers to improve their stability. In this study, S-nZVI was synthesized by chemical reduction method with starch as a stabilizer. The synthesized nanoparticles were characterized by TEM, XRD, and FTIR. Transmission electron microscopy (TEM) image shows S-nZVI has a size in the range of 5–27.5 nanometer. Temperature and S-nZVI concentration were the important factors affecting nitrate removal. Biodenitrification increased at 35°C and 500 mg/L of S-nZVI, in these conditions, biodenitrification efficiency increased from 40.45 to 78.84%. Experimental results suggested that biodenitrification increased by decreasing initial nitrate concentration. In the bioreactor biodenitrification rate was 94.07% in the presence of S-nZVI. This study indicated that, Fe2+ could be used as the only electron donor or as the additional electron donor in the presence of S2O3 to increase denitrification efficiency.

Role of oxygen supply in α, ω-dodecanedioic acid biosynthesis from n-dodecane by Candida viswanathii ipe-1: Effect of stirring speed and aeration


α, ω-Dodecanedioic acid (DC12) usually serves as a monomer of polyamides or some special nylons. During the biosynthesis, oxygenation cascaded in conversion of hydrophobic n-dodecane to DC12, while the oxidation of n-dodecane took place in the intracellular space. Therefore, it was important to investigate the role of oxygen supply on the cell growth and DC12 biosynthesis. It was found that stirring speed and aeration influenced the dissolved oxygen (DO) concentration which in turn affected cell growth as well as DC12 biosynthesis. However, the effect of culture redox potential (Orp) level on DC12 biosynthesis was more significant than that of DO level. For DC12 biosynthesis, the first step was to form the emulsion droplets through the interaction of n-dodecane and the cell. When the stirring speed was enhanced, slits in the surface layer of the emulsion droplets would be increased. Thus, the substances transportation by water through the slits would be intensified, leading to an enhanced DC12 production. Compared with the batch culture at a lower stirring speed (400 rpm) without culture redox potential (Orp) control, the DC12 concentration was increased by 5 times up to 201.3 g/L with Orp controlled above 0 mV at a higher stirring speed (800 rpm).

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes


The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO2 concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO2 in batch and fed-batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO2 values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO2. The presented results are especially interesting for large-scale and perfusion processes where increased pCO2 concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO2 accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO2 might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.

Influence of cavitation and high shear stress on HSA aggregation behavior


Neither the influence of high shear rates nor the impact of cavitation on protein aggregation is fully understood. The effect of cavitation bubble collapse-derived hydroxyl radicals on the aggregation behavior of human serum albumin (HSA) was investigated. Radicals were generated by pumping through a micro-orifice, ultra-sonication, or chemically by Fenton's reaction. The amount of radicals produced by the two mechanical methods (0.12 and 11.25 nmol/(L min)) was not enough to change the protein integrity. In contrast, Fenton's reaction resulted in 382 nmol/(L min) of radicals, inducing protein aggregation. However, the micro-orifice promoted the formation of soluble dimeric HSA aggregates. A validated computational fluid dynamic model of the orifice revealed a maximum and average shear rate on the order of 108 s−1 and 1.2 × 106 s−1, respectively. Although these values are among the highest ever reported in the literature, dimer formation did not occur when we used the same flow rate but suppressed cavitation. Therefore, aggregation is most likely caused by the increased surface area due to cavitation-mediated bubble growth, not by hydroxyl radical release or shear stress as often reported.

Protein adsorption onto monoliths: A surface energetics study


This part of work was done to explore the basic understanding of the adsorption chromatography by determining the interaction of selected model proteins (n = 5) to monolithic chromatographic materials, with varying densities of butyl and phenyl ligands. Surface energetics approach was applied to study the interaction behavior. The physicochemical properties of the proteins and monolithic chromatographic materials were explored by contact angle and zeta potential values. These values were used to study protein to monolith interaction under various operating conditions. Surface energetics approach allowed the calculation of interaction energy as a function of distance, i.e. energy minimum values. Calculations were performed at various conditions to analyze the effect of major operating parameters on the interaction strength. The interaction strength exposed the hydrophobic nature of the monoliths which increases with increasing ligand density. Further, interaction energy of proteins were higher with monolith with butyl ligand compared to monolith with phenyl ligand. For instance, lactoferrin interaction to monoliths with butyl represents more interaction, i.e. 24.38 kT as compared to monoliths with phenyl i.e. 23.28 kT, keeping lambda as 0.2 nm and salt concentration as 100 mM of ammonium sulphate. Hence, more energy and time will be consumed for elution of proteins immobilized to monoliths with butyl. Similarly, the effect of solid surface for proteins immobilization, effect of ligand density and effect of lambda showed some interesting insights on the interaction behavior. The knowledge generated from the present work will help in the basic understanding as well as development of an efficient, low cost downstream processing design and may mimic the real chromatographic experiments.

Promotion of phenolic compounds production in Salvia miltiorrhiza hairy roots by six strains of rhizosphere bacteria


Salvia miltiorrhiza Bunge is an important herb for the treatment of cerebrovascular and cardiovascular diseases with bioactive compounds (phenolic acids and tanshinones). Abundant studies showed that tanshinones could be stimulated by biotic and abiotic stresses, but limited information is available on biosynthesis of phenolic acids promoted by biotic stresses. The aim of the present work was to isolate and identify rhizosphere bacteria which stimulated phenolic compound in Salvia miltiorrhiza hairy roots and investigated the internal mechanism, providing a potential means to enhance content of pharmaceuticals in S. miltiorrhiza. The results showed that six bacteria, namely, HYR1, HYR26, SCR22, 14DSR23, DS6, and LNHR13, belonging to the genus Pseudomonas and Pantoea, significantly promoted the growth and content of major phenolic acids, RA and SAB. Bacteria LNHR13 was the most effective one, with the contents of RA and SAB reaching ∼2.5-fold (30.1 mg/g DW) and ∼2.3-fold (48.3 mg/g DW) as those of the control, respectively. Phytohormones and polysaccharides produced by bacteria showed potential responsibility for the growth and biosynthesis of secondary metabolites of S. miltiorrhiza. Meanwhile, we found that the more abundant the types and contents of phytohormones, the stronger their stimulating effect on the content of salvianolic acids.

Cover Picture: Engineering in Life Sciences 2'18


Issue Information


Experimental characterization and simulation of amino acid and peptide interactions with inorganic materials


Inspired by nature, many applications and new materials benefit from the interplay of inorganic materials and biomolecules. A fundamental understanding of complex organic–inorganic interactions would improve the controlled production of nanomaterials and biosensors to the development of biocompatible implants for the human body. Although widely exploited in applications, the interaction of amino acids and peptides with most inorganic surfaces is not fully understood. To date, precisely characterizing complex surfaces of inorganic materials and analyzing surface–biomolecule interactions remain challenging both experimentally and computationally. This article reviews several approaches to characterizing biomolecule–surface interactions and illustrates the advantages and disadvantages of the methods presented. First, we explain how the adsorption mechanism of amino acids/peptides to inorganic surfaces can be determined and how thermodynamic and kinetic process constants can be obtained. Second, we demonstrate how this data can be used to develop models for peptide–surface interactions. The understanding and simulation of such interactions constitute a basis for developing molecules with high affinity binding domains in proteins for bioprocess engineering and future biomedical technologies.

Stability of polymersomes with focus on their use as nanoreactors


The increased membrane stability of polymersomes compared to their liposomal counterparts is one of their most important advantages. Due to this benefit, polymer vesicles are intended to be used not only as carrier systems for drug delivery purposes but also as nanoreactors for biotechnological applications. Within this work, the stability of polymersomes made of the triblock copolymer poly(2-methyloxazoline)15-poly(dimethylsiloxane)68-poly(2-methyloxazoline)15 (PMOXA15-PDMS68-PMOXA15) toward mechanical stress, typically prevailing in stirred-tank reactors being the most often used reactor type in the biotechnological industry, was characterized. Dynamic light scattering and turbidity measurements showed that stirrer rotation causing a maximum local energy dissipation of up to 1.23 W/kg−1 did not result in any loss of vesicle quality or quantity. Nevertheless, most probably due to local membrane defects, 6.6% release of the previously encapsulated model dye calcein was recognized at 25°C within 48 h. Moreover, increased temperature, leading to decreased membrane viscosity and increased membrane fluidity, respectively, led to a higher molecule leakage. Besides, the stability of polymersomes in two-phase systems was investigated. Although alkanes and ionic liquids were shown not to lead to complete vesicle damage, no efficient calcein retention was achieved in either case.

Micro free-flow isoelectric focusing with integrated optical pH sensors


Recently, a new observation method for monitoring of pH gradients in microfluidic free-flow electrophoresis has emerged. It is based on the use of chip-integrated fluorescent or luminescent micro sensor layers. These are able to monitor pH gradients in miniaturized separations in real time and spatially resolved; this is particularly useful in isoelectric focusing. Here these multifunctional microdevices that feature continuous separation, monitoring, and in some instances other functionalities, are reviewed. The employed microfabrication procedures to produce these devices are discussed and the different pH sensor matrices that were integrated and their applications in the separation of different types of biomolecules. The procedures for obtaining spatially resolved information about the separated molecules and the pH at the same time and different detection modalities to achieve this such as deep UV fluorescence as well as time-resolved referenced pH sensing and the integration of a precolumn labeling step into these platforms are also highlighted.

Protein micropatterns printed on glass: Novel tools for protein-ligand binding assays in live cells


Micrometer-sized patterns of proteins on glass or silica surfaces are in widespread use as protein arrays for probing with ligands or recombinant proteins. More recently, they have been used to capture the surface proteins of mammalian cells seeded onto them, and to arrange these surface proteins into pattern structures. Binding of small molecule ligands or of other proteins, transmembrane or intracellular, to these captured surface proteins can then be quantified. However, reproducible production of protein micropatterns on surfaces can be technically difficult. In this review, we outline the wide potential and the current practical uses of printed protein micropatterns in a historical overview, and we detail some potential pitfalls and difficulties from our own experience, as well as ways to circumvent them.

A modified 384-well-device for versatile use in 3D cancer cell (co-)cultivation and screening for investigations of tumor biology in vitro


Pancreatic cancer exhibits a worst prognosis owed to an aggressive tumor progression i.a. driven by chemoresistance or tumor-stroma-interactions. The identification of candidate genes, which promote this progression, can lead to new therapeutic targets and might improve patient's outcome. The identification of these candidates in a plethora of genes requires suitable screening protocols. The aim of the present study was to establish a universally usable device which ensures versatile cultivation, screening and handling protocols of cancer cells with the 3D spheroid model, an approved model to study tumor biology. By surface modification and alternative handling of a commercial 384-well plate, a modified device enabling (i) 3D cultivation either by liquid overlay or by a modified hanging drop method for (ii) screening of substances as well as for tumor-stroma-interactions (iii) either with manual or automated handling was established. The here presented preliminary results of cell line dependent dose-response-relations and a stromal-induced spheroid-formation of the pancreatic cancer cells demonstrate the proof-of-principle of the versatile functionality of this device. By adapting the protocols to automation, a higher reproducibility and the ability for high-throughput analyses were ensured.

Cell-free production of pore forming toxins: Functional analysis of thermostable direct hemolysin from Vibrio parahaemolyticus


The pore forming characteristic of TDH1 and TDH2 variants of thermostable direct hemolysin (TDH), a major toxin involved in the pathogenesis of Vibrio parahaemolyticus, was studied on a planar lipid bilayer painted over individual picoliter cavities containing microelectrodes assembled in a multiarray. Both proteins formed pores upon insertion into the lipid bilayer which was shown as a shift in the conductance from the baseline current. TDH2 protein was able to produce stable currents and the currents were influenced by external factors like concentration, type of salt and voltage. The pore currents were influenced and showed a detectable response in the presence of polymers which makes them suitable for biotechnology applications.

A microfluidic device for the delivery of enzymes into cells by liposome fusion


Liposomes are versatile carriers of drugs or biomolecules and are ideally suited to transport molecules into cells. However, mechanistic studies to understand and improve the fusion of liposomes with cell membranes and endosomes are difficult. Here, we report a method that allows for stable coimmobilization of liposomes and living cells, thereby bringing the membranes into close contact, which is essential for membrane fusion. The small unilamellar liposomes are tethered to the surface by a linker so that no modification of the liposome membrane for cell binding is required. The cells are positioned above the liposomes by posts that are integrated into the microfluidic device, and a pH drop induces the fusion of the cell-liposome membranes. Both membrane fusion and release of molecules into the cytosol are visualized by fluorescence dequenching assays. Furthermore, we proved the efficient delivery of the enzyme β-galactosidase into the cells when a fusogenic liposome composition was used. The device could be used for fusion studies but is also a versatile means for cell transfection.