Industrial Bioprocesses Research Articles

Enhanced thermostability of mesophilic endoglucanase Z with a high catalytic activity at active temperatures

This is the first study for therrmostable mutants of mesophilic endoglucanase EngZ from Clostridium cellulovorans using by site-directed mutagenesis. K94R, S365P and their double mutant K94R/S365P had a wide range of active temperatures (30⿿60°C). In addition, the optimal temperature of K94R/S365P was increased by 7.5°C. K94R/S365P retained 78.3% relative activity at 70°C, while the wild type retained only 5.8%. Especially, K94R/S365P remained 45.1-fold higher activity than the wild type at 70°C. In addition, K94R/S365P was 3.1-fold higher activity than the wild type at 42.5°C, which is the optimal temperature of the wild type. K94R/S365P showed also stimulated in 2.5-fold lower concentration of CaCl2 and delayed aggregation temperature in the presence of CaCl2 compared to the wild type. In pH stability, K94R/S365P was not influenced, but the optimum pH was transferred from pH 7 to pH 6. In long-term hydrolysis, K94R/S365P reduced the newly released reducing sugar yields after 12h reaction; however, the yields consistently increased until 72h. Finally, the total reducing sugar of K94R/S365P was 5.0-fold higher than the wild type at 50°C, pH6. EngZ (K94R/S365P) can support information to develop thermostability of GH9 endoglucanase with a high catalytic efficiency as the potential industrial bioprocess candidate.

Enhanced production of glycyrrhetic acid 3-O-mono-β-d-glucuronide by fed-batch fermentation using pH and dissolved oxygen as feedback parameters

Glycyrrhetic acid 3-O-mono-β-d-glucuronide (GAMG), the major functional ingredient in licorice, has widespread applications in food, pharmacy and cosmetics industry. The production of GAMG through Penicillium purpurogenum Li-3 cultivation was for the first time performed through both batch and fed-batch processes in bioreactors. In batch process, under optimal conditions (pH5.0, temperature 32°C, agitation speed 100r·min−1), 3.55g·L−1 GAMG was obtained in a 2.5L fermentor. To further enhance GAMG production, a fine fed-batch process was developed by using pH and DO as feedback parameters. Starting from 48h, 100ml 90g·L−1 substrate Glycyrrhizin (GL) was fed each time when pH increased to above 5.0 and DO was increased to above 80%. This strategy can significantly enhance GAMG production: the achieved GL conversion was 95.34% with GAMG yield of 95.15%, and GAMG concentration was 16.62g·L−1 which was 5 times higher than that of batch. Then, a two-step separation strategy was established to separate GAMG from fermentation broth by crude extraction of 15ml column packed with D101 resin followed by fine purification with preparative C18 chromatography. The obtained GAMG purity was 95.79%. This study provides a new insight into the industrial bioprocess of high-level GAMG production.

Kinetics and modeling for extraction of chrysin from Oroxylum indicum seeds

Research on extraction of chrysin is crucial for theoretical purposes and for food industrial bioprocesses. Optimization and kinetics of chrysin extraction from seeds of Oroxylum indicum (L.) Vent. were analyzed using agitated solid-liquid extractions with ethanol and water mixtures. The influence of extraction process parameters was investigated. Optimized conditions for chrysin extraction were a 0.2 mole fraction of ethanol as an extraction solvent, a temperature of 318 K, an agitation speed 1,400 rpm, and a solid to solvent ratio of 1:30. The extraction kinetic behavior of chrysin followed first order kinetics. The kinetic expression developed by Spiro and Siddique was used and the model was in agreement with experimental results. The diffusion coefficient ranged from 1.38×10−11 to 19.43×10−11 m2·s−1 and the activation energy for extraction kinetics was 21.85 kJ·mol−1.

Industrial Views to Microbe-Metal Interactions in Sub-Arctic Conditions

This paper covers industrial views and challenges related to microbe-metal interactions in the sub-arctic conditions in Finland. The first issue is to operate bioleaching and bio-precipitation processes in cold and rainy environments where microbial activities tend to be low and solutions get diluted. On the other hand, industrial challenges in cold climates are related to the need to hinder the activity of microbe-metal interactions in certain applications, such as closed mines and nuclear waste repositories. Our case examples show the potential of industrial bioprocess utilization in cold climates, but also emphasize their special characteristics and challenges.

Purification and characterization of a new thermoalkaliphilic pectate lyase from Actinomadura keratinilytica Cpt20

This study was carried out to investigate the purification and biochemical characterization of a new extracellular alkaliphilic and thermostable pectate lyase (Pel-20) isolated from Actinomadura keratinilytica strain Cpt20. Pure protein was obtained after sequential chromatographies on a fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC) columns. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 34125.11-Da. The enzyme had an NH2-terminal sequence of GFATNQGGTTGGAGGTLS, thus, sharing high homology with actinomycetes pectate lyase family. The results showed that this enzyme was completely inhibited by EDTA, which supports its belonging to the pectate lyase superfamily. It showed optimum activity at pH 10.5 and 70°C. The thermoactivity and thermostability of Pel-20 were enhanced in the presence of 1mM Ca2+. Its half-life times at 70, 80, 90, and 100°C were 18, 12, 7, and 2h, respectively. Its kinetic parameters, Km and Vmax values were 0.45mM and 21,700U/mg, respectively. Low-esterified pectin was the optimum substrate for the Pel-20. However, higher-esterified pectin was also weakly cleaved. Overall, the alkaliphilicity and thermostability properties of Pel-20 make it a potential candidate for future application in industrial bioprocesses.

Systems strategies for developing industrial microbial strains.

Industrial strain development requires system-wide engineering and optimization of cellular metabolism while considering industrially relevant fermentation and recovery processes. It can be conceptualized as several strategies, which may be implemented in an iterative fashion and in different orders. The key challenges have been the time-, cost- and labor-intensive processes of strain development owing to the difficulties in understanding complex interactions among the metabolic, gene regulatory and signaling networks at the cell level, which are collectively represented as overall system performance under industrial fermentation conditions. These challenges can be overcome by taking systems approaches through the use of state-of-the-art tools of systems biology, synthetic biology and evolutionary engineering in the context of industrial bioprocess. Major systems metabolic engineering achievements in recent years include microbial production of amino acids (L-valine, L-threonine, L-lysine and L-arginine), bulk chemicals (1,4-butanediol, 1,4-diaminobutane, 1,5-diaminopentane, 1,3-propanediol, butanol, isobutanol and succinic acid) and drugs (artemisinin).

Increased Microbial Butanol Tolerance by Exogenous Membrane Insertion Molecules.

Butanol is an ideal biofuel, although poor titers lead to high recovery costs by distillation. Fluidization of microbial membranes by butanol is one of the major factors limiting titers in butanol-producing bioprocesses. Starting with the hypothesis that certain membrane insertion molecules would stabilize the lipid bilayer in the presence of butanol, we applied a combination of in vivo and in vitro techniques within an in silico framework to describe a new approach to achieve solvent tolerance in bacteria. Single-molecule tracking of a model supported bilayer showed that COE1-5C, a five-ringed oligo-polyphenylenevinylene conjugated oligoelectrolyte (COE), reduced the diffusion rate of phospholipids in a microbially derived lipid bilayer to a greater extent than three-ringed and four-ringed COEs. Furthermore, COE1-5C treatment increased the specific growth rate of E. coli K12 relative to a control at inhibitory butanol concentrations. Consequently, to confer butanol tolerance to microbes by exogenous means is complementary to genetic modification of strains in industrial bioprocesses, extends the physiological range of microbes to match favorable bioprocess conditions, and is amenable with complex and undefined microbial consortia for biobutanol production. Molecular dynamics simulations indicated that the π-conjugated aromatic backbone of COE1-5C likely acts as a hydrophobic tether for glycerophospholipid acyl chains by enhancing bilayer integrity in the presence of high butanol concentrations, which thereby counters membrane fluidization. COE1-5C-mitigated E. coli K12 membrane depolarization by butanol is consistent with the hypothesis that improved growth rates in the presence of butanol are a consequence of improved bilayer stability.

Identification of molecular species of polyol oils produced from soybean oil by Pseudomonas aeruginosa E03-12 NRRL B-59991

Abstract The objective of this study is to identify the chemical species of the polyol oil products produced from soybean oil by Pseudomonas aeruginosa E03-12 NRRL B-59991. We reported earlier the polyol products produced from soybean oil by Acinetobacter haemolyticus A01-35 (NRRL B-59985) ( Hou and Lin, 2013 ). The polyol oil produced by strain A01-35 were a mixture of 57 molecular species of DAG containing tri-, di-, mono-hydroxy FA and normal FA. In this study, tricaprylin was selected as internal standard for HPLC quantitative estimation of products. A CombiFlash chromatographic method was established for separation of the polyol oil products. The molecular species of the polyol oils produced from soybean oil by strain E03-12 were identified with HPLC/MS. We identified 41 derivatives of DAG, among them 32 molecular species containing one hydroxy FA and one normal FA, 8 molecular species containing two hydroxy FA without normal FA, and one molecular specie containing two normal FA without hydroxylated FA. The hydroxy FA included mono-, di- and tri-hydroxy FA. Eight molecular species of DAG containing one trihydroxy FA and 14 molecular species of DAG containing one dihydroxy FA. We have also identified 64 molecular species of TAG, among them 13 molecular species containing two hydroxy FA, 42 molecular species containing one hydroxy FA and 9 molecular species containing no hydroxylated FA. This is different from our previous findings with A. haemolyticus A01-35 which produced only DAG polyol oils. E03-12 is a better strain for developing into an industrial bioprocess.

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

The Application of an On-Line Optical Sensor to Measure Biomass of a Filamentous Bioprocess

Monitoring of all critical process parameters in bioprocess engineering is essential. Sensors have been previously developed for specific parameters such as on-line temperature, pH or stirring control and data logging. However, biomass monitoring needs further development. All current non-invasive technology, such as Near Infra-Red, is limited on biomass measurement of animal and insect cells. Biomass monitoring of industrial bioprocesses of filamentous microorganisms still requires sample removal from the vessel, which could potentially compromise sterility. This study has focused on the application of a non-invasive optical sensor in the on-line monitoring of the biomass of the filamentous microorganism Streptomyces coelicolor A3 (2). Raw output data from the biomass monitor were directly compared to data from the sensors measuring dissolved oxygen levels and off gas evolution and the results successfully demonstrate that the optical sensor is sensitive in identifying different levels of biomass. Therefore, it is possible to use the simple output data to provide real time information on biomass levels of filamentous microorganisms, a very powerful tool in bioprocess engineering.

On the use of electrochemical multi-sensors in biologically charged media

Abstract. For the investigation and characterisation of liquid media with microorganisms, electrochemical sensors are typically used. Usually the microorganisms are part of the process or cannot be excluded for different reasons. This paper describes the application of various electrodes, which are partly miniaturised and combined with multi-sensor systems for several applications in processes containing microorganisms. The application in industrial bioprocesses like beer brewing and biogas production, and in paper manufacturing, is described. The performance of the multi-sensor systems, and thus their suitability for a contribution to improved process monitoring, is evaluated. The multi-sensor systems represent an interesting tool to enhance monitoring capacities at installed systems without the necessity for huge port installations and offer the possibility to monitor the spatial distribution of gradients. The developed systems presented here allow location-independent measurements in process plants with a variable positioning of the sensors in the industrial reactors.

Utilization of papaya waste and oil production by Chlorella protothecoides

Microalgae derived oils have outstanding potential for use in biodiesel production. Chlorella protothecoides has been shown to accumulate lipids up to 60% of its cellular dry weight with glucose supplementation under heterotrophic growth conditions. To reduce production costs, alternative carbon feedstocks have been evaluated and show promise as low-cost alternatives. Regional agricultural residues, such as off-quality fruits and vegetables that cannot be used as human food or animal feed, as well as inedible plant byproducts, represent an abundant and underutilized resource as a feedstock for supporting microbial production of biofuels. Here, we present the finding that C. protothecoides isolates are capable of robust cell growth and oil production in growth medium comprised of pH-adjusted puree of culled, waste papaya fruit without any additional growth supplements. Optimization of culture medium and growth conditions was used for lab-scale strain characterization and demonstration of the potential for scale-up to an industrial bioprocess. The most rapid cell doubling time observed was 6.6h and the maximum oil production rate observed was 1.9g/l/day.

Lactate and glucose concomitant consumption as a self-regulated pH detoxification mechanism in HEK293 cell cultures.

One of the most important limitations of mammalian cell-based processes is the secretion and accumulation of lactate as a by-product of their metabolism. Among the cell lines commonly used in industrial bioprocesses, HEK293 has been gaining importance over the last years. Up recently, HEK293 cells were known to consume lactate in late stages of cell culture usually when glucose and/or glutamine were depleted from media. Remarkably, in both scenarios, no significant cell growth was reported. However, we have observed a different metabolic behavior regarding lactate production and consumption in HEK293 cultures. HEK293 cells were able to co-metabolize glucose and lactate simultaneously, even in exponentially growing cell cultures. Our deep study of the effects of environmental conditions on lactate metabolism revealed that pH was the key to trigger the metabolic shift from lactate production to lactate and glucose concomitant consumption. Remarkably, this shift could be triggered at will when pH was set at 6.8. Even more interesting was the fact that lowering pH to 6.6 and supplementing media with exogenous lactate resulted in co-consumption of glucose and lactate from the beginning of cell culture, without affecting cell growth or protein productivity. On the contrary, cell growth was clearly hampered at this low pH if extracellular lactate was lacking. From our results, we hypothesize that HEK293 cells metabolize extracellular lactate as a strategy for pH detoxification, by means of co-transporting extracellular protons together with lactate into the cytosol. This novel hypothesis for unraveling lactate metabolism in HEK293 cells could open a door to re-direct genetic engineering strategies in order to obtain more efficient cell lines and also to further develop animal cell technology applications.

English

Saliva containing amylase was collected from an individual 5 min after a carbohydrate meal and filtered using a dialysis bag to remove starch particles. The filtered saliva was immobilized on calcium alginate beads. The effect of experimental parameters like pH, temperature and substrate concentration on the activity of the immobilized post-carbohydrate meal salivary α-amylase was determined. The immobilized salivary α-amylase had an optimum activity at temperature 40°C and pH 7.0. The activation energy (Ea) as obtained from the Arrhenius plot was 31.4 kJ/Mol. The kinetic parameters Km and Vmax of the immobilized α-amylase were found to be 1.6 mg/ml and 16.4 μmol/min, respectively; this was compared to that of free salivary α-amylase (Km = 0.0048 mg/ml) and α-amylases from fungi and bacteria sources. Immobilization tends to increase the Km of the immobilized enzyme, indicating a low affinity for substrate, however the enzymes Km was lower than that of some microbial α-amylases. The results obtained from the characterization of immobilized post-carbohydrate meal salivary α-amylase in this study show that immobilization had no significant effect on the enzyme and compared to kinetic parameters of microbial α-amylase, immobilized salivary α-amylase may not be of significant benefit as alternative source of α-amylase in the industrial bioprocesses.   Key words: Enzyme activity, carbohydrate, immobilized enzyme, industrial bioprocess, kinetics, salivary α-amylase.

Partitioning in aqueous two-phase systems: Analysis of strengths, weaknesses, opportunities and threats.

For half a century aqueous two-phase systems (ATPSs) have been applied for the extraction and purification of biomolecules. In spite of their simplicity, selectivity, and relatively low cost they have not been significantly employed for industrial scale bioprocessing. Recently their ability to be readily scaled and interface easily in single-use, flexible biomanufacturing has led to industrial re-evaluation of ATPSs. The purpose of this review is to perform a SWOT analysis that includes a discussion of: (i) strengths of ATPS partitioning as an effective and simple platform for biomolecule purification; (ii) weaknesses of ATPS partitioning in regard to intrinsic problems and possible solutions; (iii) opportunities related to biotechnological challenges that ATPS partitioning may solve; and (iv) threats related to alternative techniques that may compete with ATPS in performance, economic benefits, scale up and reliability. This approach provides insight into the current status of ATPS as a bioprocessing technique and it can be concluded that most of the perceived weakness towards industrial implementation have now been largely overcome, thus paving the way for opportunities in fermentation feed clarification, integration in multi-stage operations and in single-step purification processes.

Control of ATP concentration in Escherichia coli using synthetic small regulatory RNAs for enhanced S-adenosylmethionine production.

ATP is the limiting precursor and driving force for S-adenosylmethionine (SAM) biosynthesis in Escherichia coli. In contrast to traditional optimization of fermentation processes, the synthetic sRNA-based repression strategy, which was developed as a highly efficient gene knockdown approach, has been applied for the regulation of the intracellular ATP concentration in order to enhance SAM production. In this work, proB, glnA and argB, all involved in the synthesis of ATP-dependent by-products in the S-adenosylmethionine production were selected as candidates for repression. The results show that the S-adenosylmethionine titer and yield in the recombinant strain were doubled compared with the control. The best-performing strain, Anti-argB, produced the highest SAM titer (1.21 mg L(-1)), and strain Anti-glnA gave the highest yield (0.13 mg g(-1), 12 h). Both the concentration of ATP and the ratio of ATP to ADP were shown to have a positive effect on the S-adenosylmethionine synthesis. Overall, the synthetic sRNA-based downregulation strategy has a high potential for cofactor regulation and will be useful for industrial ATP-driven bioprocesses.

A mid-ranging control strategy for non-stationary processes and its application to dissolved oxygen control in a bioprocess

In this study a modified mid-ranging strategy is proposed where the controller for the secondary manipulated variable uses its own output as its setpoint, possibly with an offset and/or re-scaling. This modification allows the manipulated variables to increase in unison so that the mid-ranging advantage of utilizing the fast dynamics of the primary controller to regulate the process can be achieved also in non-stationary processes, while not adding complexity to the controller. The proposed control strategy has been implemented in pilot-scale (500l) industrial bioprocesses where it is used to control the dissolved oxygen level by manipulating agitator speed and aeration rate. The controller is demonstrated to perform well in these, outperforming a reference controller which has previously been shown to give satisfactory control performance. It is also shown in similar experiments that the strategy can easily be adapted to control dissolved oxygen in bioprocesses where the feed rate is controlled using an extremum-seeking controller. The proposed strategy is generally applicable to non-stationary processes where a mid-ranging approach is suitable.

Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

Development of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere.

Multiplicity of steady states in glycolysis and shift of metabolic state in cultured mammalian cells.

Cultured mammalian cells exhibit elevated glycolysis flux and high lactate production. In the industrial bioprocesses for biotherapeutic protein production, glucose is supplemented to the culture medium to sustain continued cell growth resulting in the accumulation of lactate to high levels. In such fed-batch cultures, sometimes a metabolic shift from a state of high glycolysis flux and high lactate production to a state of low glycolysis flux and low lactate production or even lactate consumption is observed. While in other cases with very similar culture conditions, the same cell line and medium, cells continue to produce lactate. A metabolic shift to lactate consumption has been correlated to the productivity of the process. Cultures that exhibited the metabolic shift to lactate consumption had higher titers than those which didn’t. However, the cues that trigger the metabolic shift to lactate consumption state (or low lactate production state) are yet to be identified. Metabolic control of cells is tightly linked to growth control through signaling pathways such as the AKT pathway. We have previously shown that the glycolysis of proliferating cells can exhibit bistability with well-segregated high flux and low flux states. Low lactate production (or lactate consumption) is possible only at a low glycolysis flux state. In this study, we use mathematical modeling to demonstrate that lactate inhibition together with AKT regulation on glycolysis enzymes can profoundly influence the bistable behavior, resulting in a complex steady-state topology. The transition from the high flux state to the low flux state can only occur in certain regions of the steady state topology, and therefore the metabolic fate of the cells depends on their metabolic trajectory encountering the region that allows such a metabolic state switch. Insights from such switch behavior present us with new means to control the metabolism of mammalian cells in fed-batch cultures.

Identification of genetic variations associated with epsilon-poly-lysine biosynthesis in Streptomyces albulus ZPM by genome sequencing

The biosynthesis of the antibiotic epsilon-poly-lysine (ε-PL) in Streptomyces albulus is performed by polylysine synthase (pls); however, the regulatory mechanism of this process is still unknown. Here, we first obtained the complete genome sequence of S. albulus ZPM, which consists of 9,784,577 bp and has a GC content of 72.2%. The genome houses 44 gene clusters for secondary metabolite biosynthesis, in which 20 gene clusters are involved in the biosynthesis of polyketides and nonribosomally synthesized peptides. High-throughput sequencing was further performed, and genetic variants were identified from pooled libraries consisting of the 30 highest-yield mutants or 30 lowest-yield mutants. More than 350 genetic variants associated with ε-PL yield have been identified. One hundred sixty-two affected proteins, from important metabolic enzymes to novel transcriptional regulators, were identified as being related to ε-PL synthesis. HrdD, one of the affected genes, is a sigma factor that shows the most sensitive response to pH change and contains a non-synonymous mutation (A132V) in mutant strains with lower ε-PL yields. Electrophoretic mobility shift assays showed that the pls gene is likely regulated by transcriptional activator HrdD. The data obtained in this study will facilitate future studies on ε-PL yield improvement and industrial bioprocess optimization.