Showing papers in "Engineering in Life Sciences in 2013"

Enzyme stabilization by nano/microsized hybrid materials

Abstract: Immobilization is a key technology for successful realization of enzyme-based industrial processes, particularly for production of green and sustainable energy or chemicals from biomass-derived catalytic conversion. Different methods to immobilize enzymes are critically reviewed. In principle, enzymes are immobilized via three major routes (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross-linking (carrier free). As a result, immobilizing enzymes on certain supports can enhance storage and operational stability. In addition, recent breakthroughs in nano and hybrid technology have made various materials more affordable hosts for enzyme immobilization. This review discusses different approaches to improve enzyme stability in various materials such as nanoparticles, nanofibers, mesoporous materials, sol–gel silica, and alginate-based microspheres. The advantages of stabilized enzyme systems are from its simple separation and ease recovery for reuse, while maintaining activity and selectivity. This review also considers the latest studies conducted on different enzymes immobilized on various support materials with immense potential for biosensor, antibiotic production, food industry, biodiesel production, and bioremediation, because stabilized enzyme systems are expected to be environmental friendly, inexpensive, and easy to use for enzyme-based industrial applications.

Strategies for recovery and purification of poly[(R)‐3‐hydroxyalkanoates] (PHA) biopolyesters from surrounding biomass

Abstract: To provide competitive alternatives to classical plastics, production of poly[(R)-3-hydroxyalkanoate] (PHA) biopolyesters has to become more economical. Downstream processing for PHA recovery from biomass plays a vital role in the PHA manufacturing process with respect to cost performance, material quality, and eco-balance. Several factors impact the selection of the adequate PHA recovery method: the microbial production strain, type and composition of PHA, PHA load in biomass, required product purity, availability of chemicals for PHA recovery, and impact on physical properties of PHA. In this review, we compare classical and novel strategies for PHA recovery from microbial biomass. Approaches for reducing solvent and energy inputs, focusing on obtaining endotoxin-poor PHA for medical application, are presented, as well as recent developments in efficient disruption of PHA-rich biomass. In addition, particularities of extremophiles and genetically modified microorganisms with properties facilitating the release and separation of PHA granules are discussed.

Microbial degradation of phenol and phenolic derivatives

Abstract: Phenol and its derivatives are one of the largest groups of environmental pollutants due to their presence in many industrial effluents and broad application as antibacterial and antifungal agents. A number of microbial species possess enzyme systems that are applicable for the decomposition of various aliphatic and aromatic toxic compounds. Intensive efforts to screen species with high-degradation activity are needed to study their capabilities of degrading phenol and phenolic derivatives. Mostofthecurrentresearchhasbeendirectedattheisolationandstudyofmicrobial species of potential ecological significance. In this review, some of the best achievementsindegradingphenoliccompoundsbybacteriaandyeastsarepresented,which draws attention to the high efficiency of strains of Pseudomonas, Candida tropicalis, Trichosporon cutaneum, etc. The unique ability of fungi to maintain their degradation potential under conditions unfavorable for other microorganisms is outstanding. Mathematical models of the microbial biodegradation dynamics of single and mixed aromatic compounds, which direct to the benefit of the processes studied in optimization of modern environmental biotechnology are also presented.

Consistent development of bioprocesses from microliter cultures to the industrial scale

Abstract: Bioprocess development today is slow and expensive compared to chemical process development. A drastic paradigm shift is necessary and possible by the consistent application of engineering strategies that are typically used in the process development phase already in the early product development. Aside from providing a consistent pathway, strategies such as statistical-based design of experiments, fed-batch, minibioreactors, new on-line sensors, process modeling, and control tools in combination with automation of manual steps offer a higher success rate and the opportunity to find the optimum parameters and operation point. This also directly benefits the early phases of biomolecular screening and initial production of small amounts of the target molecule. The paper reviews the bioprocess developmental phases from a business perspective and the available systems and technologies.

Bioprocessing of differentiated plant in vitro systems

Abstract: Plant cells contain a wide range of interesting secondary metabolites, which are used as natural pigments and flavoring agents in foods and cosmetics as well as phyto-pharmaceutical products. However, conventional industrial extraction from whole plants or parts of them is limited due to environmental and geographical issues. The production of secondary metabolites from in vitro cultures can be considered as alternative to classical technologies and allows a year-round cultivation in the bioreactor under optimal conditions with constant high-level quality and quantity. Compared to plant cell suspensions, differentiated plant in vitro systems offer the advantage that they are genetically stable. Moreover, the separation of the biomass from culture medium after fermentation is much easier. Nevertheless, several investigations in the literature described that differentiated plant in vitro systems are instable concerning the yield of the target metabolites, especially in submerged cultivations. Other major problems are associated with the challenges of cultivation conditions and bioreactor design as well as upscaling of the process. This article reviews bioreactor designs for cultivation of differentiated plant in vitro systems, secondary metabolite production in different bioreactor systems as well as aspects of process control, management, and modeling and gives perspectives for future cultivation methods.

Hairy root cultures: A suitable biological system for studying secondary metabolic pathways in plants

Abstract: Hairy roots, a plant disease caused by Agrobacterium rhizogenes, show distinctive features such as high growth rate, unlimited branching, and biochemical and genetic stability. Hairy roots resemble normal roots in terms of differentiated morphology and biosynthetic machinery, producing similar secondary metabolites compared to wild-type roots. As a result, hairy roots have been a topic of intense research for the past three decades, fueling innumerable attempts to develop in vitro hairy root cultures for a large number of plants for the commercial-scale production of secondary metabolites. The same characteristics have now led to further applications, such as using hairy root cultures as experimental systems for secondary metabolic pathway elucidation studies. Although the trend is relatively new, it has already gained momentum. This review summarizes these developments. The following discussion focuses on the rationale and advantages of using hairy root cultures for secondary metabolic pathway elucidation studies, the methods used, and the results that have been obtained so far.

Recent advances and future prospects for increased butanol production by acetone‐butanol‐ethanol fermentation

Abstract: Presently, several researchers are increasingly focusing on producing butanol as the next-generation fuel by acetone-butanol-ethanol (ABE) fermentation. Butanol has many superior characteristics compared to other biofuels, such as ethanol. However, its production by ABE fermentation faces the challenges of low productivity and yield because of product inhibition and heterofermentation, respectively, and thereby, high costs. Until date, molecular biological techniques and fermentation engineering methods have been applied for high butanol production. Although glucose remains the substrate of choice since traditional research, it is now necessary to substitute glucose derived from edible starch to other substrates from low-cost feedstock, such as agricultural residue. In addition, ABE-producing clostridia cannot directly produce butanol from lignocelluloses. Therefore, recent research is focusing on pretreatment and enzymatic saccharification of the complex molecules derived from agricultural residue for use as feedstock in butanol production. This article reviews traditional research, including the metabolism patterns and characteristics of ABE-producing clostridia. Furthermore, this article describes developments in ABE fermentation with respect to the establishment of highly efficient butanol production processes, such as batch, fed-batch, and continuous cultures, with the introduction of butanol removal, as well as butanol production from lignocellulosic biomasses or alternative substrates to sugars.

Synthesis of membrane proteins in eukaryotic cell‐free systems

Abstract: Cell-free protein synthesis (CFPS) is a valuable method for the fast expression of difficult-to-express proteins as well as posttranslationally modified proteins. Since cell-free systems circumvent possible cytotoxic effects caused by protein overexpression in living cells, they significantly enlarge the scale and variety of proteins that can be characterized. We demonstrate the high potential of eukaryotic CFPS to express various types of membrane proteins covering a broad range of structurally and functionally diverse proteins. Our eukaryotic cell-free translation systems are capable to provide high molecular weight membrane proteins, fluorescent-labeled membrane proteins, as well as posttranslationally modified proteins for further downstream analysis.

Hydrogen and methane production from desugared molasses using a two‐stage thermophilic anaerobic process

Abstract: Hydrogen and methane production from desugared molasses by a two-stage thermophilic anaerobic process was investigated in a series of two up-flow anaerobic sludge blanket (UASB) reactors. The first reactor that was dominated with hydrogen-producing bacteria of Thermoanaerobacterium thermosaccharolyticum and Thermoanaerobacterium aciditolerans could generate a high hydrogen production rate of 5600 mL H2/day/L, corresponding to a yield of 132 mL H2/g volatile solid (VS). The effluent from the hydrogen reactor was further converted to methane in the second reactor with the optimal production rate of 3380 mL CH4/day/L, corresponding to a yield of 239 mL CH4/g VS. Aceticlastic Methanosarcina mazei was the dominant methanogen in the methanogenesis stage. This work demonstrates that biohydrogen production can be very efficiently coupled with a subsequent step of methane production using desugared molasses. Furthermore, the mixed gas with a volumetric content of 16.5% H2, 38.7% CO2, and 44.8% CH4, containing approximately 15% energy by hydrogen is viable to be bio-hythane.

Biosynthesis “debugged”: Novel bioproduction strategies

Abstract: The production of chemicals from biogenic resources is widely performed using microorganisms. These often do not meet the requirements of technical processes due to the narrow limits of microbial physiology. As a consequence, alternatives are sought. Cell-free enzymatic reaction cascades were recently proposed as next-generation bioproduction systems. Here, we address problems of microbial systems, show potential advantages of cell-free concepts and give an overview over the state of the technology. Furthermore, we address current challenges of the cell-free strategy and give an outlook on future developments.

Dynamic process conditions in bioprocess development

Abstract: In this review, we summarise recent studies that purposefully employed dynamic conditions, such as shifts, pulses, ramps and oscillations, for fast physiological strain characterisation and bioprocess development. We show the broad applicability of dynamic conditions and the various objectives that can thereby be investigated in a short time. Dynamic processes reveal information about the analysed system faster than traditional strategies, like continuous cultivations, as process parameters can directly be linked to platform and product parameters. Furthermore, we demonstrate that dynamic operations can result in increased productivity and high product quality, making this strategy a valuable tool for bioprocess development. With this review, we would like to encourage bioprocess engineers to an increased use of dynamic conditions in bioprocess development.

Transglutaminase-modified wool keratin film and its potential application in tissue engineering

Abstract: Transglutaminase (TGase) catalyzes the cross-linking of many proteins and has been widely used to improve the properties of certain protein-based materials. Keratin is considered as a promising biomaterial candidate following traditional chemical modification. In this study, the effect of TGase on the properties of a wool keratin film was investigated. The TGase-modified film was applied to drug release and cell proliferation. Treatment with TGase (30 U/g keratin) for 18 h at 40°C increased the tensile strength of the film from 5.18 ± 0.15 MPa to 6.22 ± 0.11 MPa and decreased the elongation at break from 83.47 ± 1.79% to 72.12 ± 3.02%. The stability of the film in PBS and in artificial gastric juice was also improved. A rougher surface and a more compact cross-section were observed by scanning electron microscopy photographs of the TGase-treated film. SDS-PAGE analysis confirmed that higher molecular weight proteins were formed in the TGase-modified keratin solution and film. The results of the drug release assay using diclofenac indicated that both films with and without TGase treatment led to a high initial release in PBS, which was more constant in artificial gastric juice. The enzyme treatment led to a lower drug release rate from the film. Cell culture experiments suggested that the TGase-mediated cross-linked keratin film shows a good biocompatibility and that it can be used for tissue engineering applications.

Process analytical technology (PAT) tools for the cultivation step in biopharmaceutical production

Abstract: The process analytical technology (PAT) initiative is now 10 years old. This has resulted in the development of many tools and software packages dedicated to PAT application on pharmaceutical processes. However, most applications are restricted to small molecule drugs, mainly for the relatively simple process steps like drying or tableting where only a limited number of parameters need to be controlled. A big challenge for PAT still lies in applications for biopharmaceuticals and then especially in the cultivation process step, where the quality of a biopharmaceutical product is largely determined. This review gives an overview of the currently available tools for monitoring and controlling the biopharmaceutical cultivation step and of the main challenges for the most common cell platforms (i.e. Escherichia coli, yeast, and mammalian cells) used in biopharmaceutical manufacturing. The real challenge is to understand how intracellular mechanisms (from synthesis to excretion) influence the quality of biopharmaceuticals and how these mechanisms can be monitored and controlled to yield the desired end product quality. Modern “omics” tools and advanced process analyzers have opened up the way for PAT applications for the biopharmaceutical cultivation process step.

Carbonic anhydrase: Its biocatalytic mechanisms and functional properties for efficient CO2 capture process development

Abstract: The accumulation of carbon dioxide (CO2) is the main cause of global climate change and it is very important to develop technologies that can reduce atmospheric CO2 level. Although several physical or chemical CO2 capture processes are being developed, the efficiency of capture/sequestrationshould be increased. Carbonic anhydrase (CA, EC 4.2.1.1), a metalloenzyme, has been considered as an important biocatalyst for CO2 capture system development because CA has the highest efficiency of CO2 conversion via hydration (CO2 + H2O HCO3− + H+, kcat: ∼106 s−1). In this review, biocatalytic mechanism and functional properties of CA are reviewed in terms of its distinctive CO2 converting ability. In addition, recent applications of several CAs are presented in CO2 capture process development. This review would present guidelines for scientists and researchers to understand CA functions and develop interest in the practical applications of CA, in particular, as biocatalytic agent for more efficient CO2 capture process development.

Bacterial FadR and synthetic promoters function as modular fatty acid sensor- regulators in Saccharomyces cerevisiae

Abstract: Fatty acid derivatives have ideal properties for use as drop-in biofuels. An effective strategy in engineering microbial cells to maximize productivity and yield involves dynamic control of protein production in response to concentrations of key intermediates. In Saccharomyces cerevisiae, the activities of the native transcription factors responsive to fatty acids are repressed in the presence of a glucose carbon source. In order to develop a modular fatty acid regulation system in S. cerevisiae, we constructed fatty acid/fatty acyl-CoA biosensors in S. cerevisiae using bacterial FadR transcriptional repressors and yeast synthetic promoters containing DNA-binding operators. We demonstrated the functionality of FadR repressors in S. cerevisiae, and tuned the sensing system by varying the promoter strength upstream to the FadR-coding sequence by varying the number of operator sites in the synthetic promoter and by using FadR from two bacterial sources (Escherichia coli and Vibrio cholerae) with different ligand sensitivities. We envision that our fatty acid/fatty acyl-CoA biosensors can be used for regulation of protein expression based on the availability of fatty acid intermediates, which will assist in balancing of cellular metabolism during fatty acid derivatives production in yeast.

Biosynthesis of (R)‐epichlorohydrin at high substrate concentration by kinetic resolution of racemic epichlorohydrin with a recombinant epoxide hydrolase

Abstract: The substrate concentration and yield were shown to be very low in the production of (R)-epichlorohydrin by hydrolysis of racemic epichlorohydrin using epoxide hydrolases in previous studies. In this work, we synthesized an epoxide hydrolase gene from Agrobacterium radiobacter and expressed it in Escherichia coli by the PCR assembly method. The recombinant A. radiobacter epoxide hydrolase (ArEH) was applied in the preparation of (R)-epichlorohydrin and, a yield of 42.7% with ≥99% enantiomeric excess (ee) from 25.6 mM racemic epichlorohydrin was obtained. However, the ee of (R)-epichlorohydrin was not able to reach 99% due to substrate and product inhibition when the substrate concentration was over 320 mM. Inhibition studies revealed that (S)-3-chloro-1,2-propanediol displayed non-competitive inhibition in the conversion of (S)-epichlorohydrin but non-significant inhibition for (R)-epichlorohydrin. Moreover, ArEH was successfully applied in the preparation of (R)-epichlorohydrin at high substrate concentration by eliminating the substrate inhibition. The substrate concentration increased to 448 mM by intermittent feeding of the substrate and to 512 mM by using a two-phase reaction system, with a high yield (>27%) and ee (>98%) of (R)-epichlorohydrin. This is the first report of high-yield production of (R)-epichlorohydrin at high substrate concentration, laying the foundations for its application on the industrial scale.

Mimicking the biological world: Methods for the 3D structuring of artificial cellular environments

Abstract: Combining modern methods in microsystem technology with the latest advancements in the life sciences, namely those in tissue engineering and advanced cell culturing, is promoting the development of a promising toolbox for modeling biological systems. The core problem to solve using this toolbox is the design of 3D artificial cellular environments, both in fluidic systems and on solid substrates. The construction of 3D biological fluidic environments involves the use of microfluidic devices where fluid direction and behavior can be tightly regulated in a geometrically constrained environment for advanced cell cultivation. This is used in modern cultivation devices, such as bioreactors and multicompartment systems, including systems with integrated multielectrode arrays in both 2D and 3D. The construction of 3D cell cultures on substrates involves various fabrication techniques that use different polymers and biopolymers processed by micromachining, chemical pattern guided cell cultivation, photopolymerization, and organ printing methods. These methods together have the potential to create an artificial system with the complete hierarchical, geometrical, and functional organization found in an actual biological system. In this review, we describe representative developments in this research area and the fusion of formerly unrelated disciplines that are generating new beneficial applications in life sciences.

Potential bioavailability and chemical forms of Co and Ni in the biogas process—An evaluation based on sequential and acid volatile sulfide extractions

Abstract: Several previous studies reported stimulatory effects on biogas process performance after trace metal supplementation. However, the regulation of the bioavailability in relation to chemical speciation, e.g. the role of sulfide is not fully understood. The objective of the present study was to determine the effect of sulfide on chemical speciation and bioavailability of Co and Ni in lab-scale semicontinuous stirred biogas tank reactors treating stillage. The chemical forms and potential bioavailability of Co and Ni were studied by sequential extraction, analysis of acid-volatile sulfide (AVS), and simultaneously extracted metals. The results demonstrated that Ni was completely associated to the organic matter/sulfide fraction and AVS, suggesting low potential bioavailability. Cobalt was predominantly associated to organic matter/sulfide and AVS, but also to more soluble fractions, which are considered to be more bioavailable. Process data showed that both Co and Ni were available for microbial uptake. Although the actual bioavailability of Co could be explained by association to more bioavailable chemical fractions, the complete association of Ni with organic matter/sulfides and AVS implies that Ni was taken up despite its expected low bioavailability. It was concluded that extensive Co- and Ni-sulfide precipitation did not inhibit microbial uptake of Co and Ni in the reactors.

Selection and optimization of a salting-out extraction system for recovery of biobutanol from fermentation broth.

Abstract: High separation cost of biobutanol from the fermentation broth has become a bottleneck in acetone-butanol-ethanol (ABE) industrial production due to low ABE concentration. In this study, a suitable salting-out extraction (SOE) system was selected and optimized for the recovery of biobutanol from the fermentation broth. From the different SOE systems investigated, the acetone/K2HPO4 system appeared to be more favorable. To examine the potential of this SOE system, the partition coefficient combined with concentration fold of butanol in synthetic solutions was optimized by the response surface methodology. The optimum conditions were found to be 17.51% w/w acetone, 21.44% w/w dipotassium hydrogen phosphate at ABE concentration of 17.3 g/L (butanol = 9.5 g/L). Under these conditions, a favorable partition coefficient, extraction yield, and concentration fold of butanol were determined as 70, 99.08%, and 1.48, respectively. The optimum extraction conditions were then used to direct the recovery of biobutanol from a real fermentation broth. The partition coefficient, extraction yield, and concentration fold of butanol reached 63.13, 98.10%, and 1.53, respectively; while the removal ratio of cells and proteins were 99.12 and 95.70%, respectively. The SOE system of acetone/K2HPO4 appeared to be effective and feasible for the downstream processing of butanol from fermentation broth.

Leucojum aestivum plants propagated in in vitro bioreactor culture and on solid media containing cytokinins

Abstract: Cytokinins are growth regulators that stimulate cell division and control morphogenesis in plants, however their role in regulating secondary metabolism is not well studied. The influence of various cytokinins (benzyladenine, zeatin, kinetin, meta-topolin, thidiazuron) and culture systems (solid and temporary immersion RITA (R) system) on the quality Leucojum aestivum plant regenerated from somatic embryos was investigated. The largest number of regenerated plants (181.6 and 168.8) was obtained from the embryos cultivated on media enriched with meta-topolin and benzyladenine. Thidiazuron and meta-topolin led to the highest number of normally developed plants (94.8 and 90.6). The random amplified polymorphic DNA analysis of in vitro and in vivo plants showed four clusters of similarity. The highest biomass (growth index: 2.49) was obtained with the temporary immersion RITA (R) system. Alkaloid extracts were analyzed by LC-MS, leading to the quantification of galanthamine and lycorine both in plant materials and in liquid media. The highest contents of galanthamine (0.05% dry weight) were observed in plants cultivated in the presence of thidiazuron in bioreactor system. Galanthamine was accumulated (highest content 0.05% dry weight) in plants cultivated in the presence of thidiazuron in bioreactor system whereas lycorine was synthetized mainly in plants cultivated on solid media.

Growth kinetics of a Helianthus annuus and a Salvia fruticosa suspension cell line: Shake flask cultivations with online monitoring system

Abstract: Plants produce a variety of secondary metabolites to defend themselves against herbivores or to attract pollinating insects. Plant cell biotechnology offers excellent opportunities in order to use such secondary plant metabolites to produce goods with consistent quality and quantity throughout the year, and therefore to act independently from biotic and abiotic environmental factors. This article presents results of an extensive study of plant cell in vitro cultivation in a modern shake flask system with noninvasive online respiration activity monitoring unit. Comprehensive screening experiments confirm the successful transfer of a model culture (sunflower suspension) into the shake flask monitoring device and the suitability of this respiration activity monitoring unit as qualified tool for screening of plant in vitro cultures (sunflower and sage suspension). The authors demonstrate deviations between online and offline data due to varying water evaporation from different culture flask types. The influence of evaporation on growth-specific parameters thereby rises with increasing cultivation time. Furthermore, possibilities to minimize the impact of evaporation, either by adjusting the inlet air moisture or by measuring the evaporation in combination with an appropriate correction of the measured growth values are shown.

Micro reaction technology for valorization of biomolecules using enzymes and metal catalysts.

Abstract: The methodology of processing biomass for the production of fuels and chemicals has been well established; however, these processes are often not economical due to the high cost of materials and low selectivity. In order to more efficiently use the raw materials, and to decrease processing (and developing) time, it is necessary to consider novel synthesis routes and/or novel process technologies for biomass valorization. The use of micro-/millireactor technology for this purpose is still under investigation. This review introduces relevant processes for the conversion of biomolecules into the industrially valuable products developed (or under development) in a microreactor. The processes with the enzyme- and metal-based catalysts are considered and carbohydrates, amino acids, and their derivatives as starting materials for processing in microreactors are reviewed as well. Also, we discuss main motivations, achievements, and current challenges when using microreactor technology, and the status of a holistic process design approach in relevant fields. We suggest that although there are individual examples reported on sustainable process design including microreactor technology, the systematic approach is still missing.

Two-step salting-out extraction of 1,3-propanediol and lactic acid from the fermentation broth of Klebsiella pneumoniae on biodiesel-derived crude glycerol.

Abstract: Crude glycerol is a primary by-product in the biodiesel production process and its large surplus blocks the development of the biodiesel industry. In this study, crude glycerol was used as a substrate to co-produce 1,3-propanediol (1,3-PD) and lactic acid through fermentation of Klebsiella pneumoniae. The final concentrations of 1,3-PD and lactic acid were 62.6 and 33.4 g/L, respectively, and the total mass conversion yield was 55.7%. The two-step salting-out extraction method was adopted for the separation of 1,3-PD and lactic acid from the fermentation broth. In the first step of extraction, the maximal partition coefficient and recovery of 1,3-PD reached 9.81 and 92.4% under the optimal conditions of 30% isopropanol and 30% potassium carbonate. Subsequently, 28% ethanol was added into the salt phase to perform the second salting-out extraction at pH 6.5. The partition coefficient and recovery of lactic acid reached 1.27 and 73.8%, respectively. All cells and most of the proteins (98.5%) were finally removed. The results suggest that crude glycerol can be used as a promising feedstock to co-produce 1,3-PD and lactic acid from fermentation broths, and this could not only reduce the production cost but also promote the further development of the biodiesel industry.

Microbial canthaxanthin: Perspectives on biochemistry and biotechnological production

Abstract: Canthaxanthin (CX) as a high-value carotenoid is widely used in medical, pharmaceutical, cosmetic, chemical, and food industries. In this review, recent developments in the biotechnological production of CX associated with comprehensive insights on CX biosynthesis pathways to improve CX production are critically scrutinized. This article explains the role of carotenogenic genes in the precursor's synthesis of functional CX ingredients. It also explores the possibilities for productivity enhancement of microbial CX in upstream and downstream processes. Moreover, several promising directions for future studies are introduced.

Formation of ethyl acetate by Kluyveromyces marxianus on whey: Influence of aeration and inhibition of yeast growth by ethyl acetate

Abstract: The ability of the yeast Kluyveromyces marxianus to convert lactose into ethyl acetate offers good opportunities for the economical reuse of whey. The formation of ethyl acetate as a bulk product depends on aerobic conditions. Aeration of the bioreactor results in discharge of the volatile ester with the exhaust gas that allows its process-integrated recovery. The influence of aeration (varied from 10 to 50 L/h) was investigated during batch cultivation of K. marxianus DSM 5422 in 0.6 L whey-borne medium using a stirred reactor. With lower aeration rates, the ester accumulated in the bioreactor and reached higher concentrations in the culture medium and the off gas. A high ester concentration in the gas phase is considered beneficial for ester recovery from the gas, while a high ester concentration in the medium inhibited yeast growth and slowed down the process. To further investigate this effect, the inhibition of growth by ethyl acetate was studied in a sealed cultivation system. Here, increasing ester concentrations caused a nearly linear decrease of the growth rate with complete inhibition at concentrations greater than 17 g/L ethyl acetate. Both the cultivation process and the growth rate depending on ethyl acetate were described by mathematical models. The simulated processes agreed well with the measured data.

Consortium diversity of a sulfate‐reducing biofilm developed at acidic pH influent conditions in a down‐flow fluidized bed reactor

Abstract: Sulfate reduction is an appropriate approach for the treatment of effluents with sulfate and dissolved metals. In sulfate-reducing reactors, acetate may largely contribute to the residual organic matter, because not all sulfate reducers are able to couple the oxidation of acetate to the reduction of sulfate, limiting the treatment efficiency. In this study, we investigated the diversity of a bacterial community in the biofilm of a laboratory scale down-flow fluidized bed reactor, which was developed under sulfidogenic conditions at an influent pH between 4 and 6. The sequence analysis of the microbial community showed that the 16S rRNA gene sequence of almost 50% of the clones had a high similarity with Anaerolineaceae. At second place, 33% of the 16S rRNA phylotypes were affiliated with the sulfate-reducing bacteria Desulfobacca acetoxidans and Desulfatirhabdium butyrativorans, suggesting that acetotrophic sulfate reduction was occurring in the system. The remaining bacterial phylotypes were related to fermenting bacteria found at the advanced stage of reactor operation. The results indicate that the acetotrophic sulfate-reducing bacteria were able to remain within the biofilm, which is a significant result because few natural consortia harbor complete oxidizing sulfate-reducers, improving the acetate removal via sulfate reduction in the reactor.

High‐throughput process development methods for chromatography and precipitation of proteins: Advantages and precautions

Abstract: Although high-throughput process development methods using 96-well microplate (MP) systems are promising and attractive, they still have several unclear problems in the practical applications. Several case studies for the purification process development by using manually operated 96-well MP systems are presented. In the first example, the MPs with ion-exchange monolith disks or ion-exchange membranes were employed for linear gradient elution and breakthrough curve experiments. The data such as the peak salt concentration and dynamic binding capacity values obtained with the MP system were in good agreement with those by the LC system. In the second example, batch adsorption experiments with a 96-well filter MP were performed for a protein A resin (particle)–antibody system in order to obtain the adsorption isotherms. The static binding capacities calculated from the isotherms were similar to those calculated from the LC system. In the last example, protein precipitation experiments by PEG were carried out for determining the solubility curve. It was found that the presence of dimer and aggregates affect the solubility curve measurement significantly. The proposed methods suggest powerful practical applications of high-throughput process development for chromatography and precipitation process when the experiments are carefully designed and carried out.

Detection of dairy fouling: Combining ultrasonic measurements and classification methods

Abstract: Fouling and cleaning in heat exchangers are severe and costly (up to 0.3% of gross national product) issues in dairy and food processing. Therefore, reducing cleaning time and cost is urgently needed. In this study, two classification methods [artificial neural network (ANN) and support vector machine (SVM)] for detecting protein and mineral fouling presence and absence based on ultrasonic measurements were presented and compared. ANN is based on a multilayer perceptron feed forward neural network, whereas SVM is based on clustering between fouling and no fouling using a hyperplane. When both fouling types (1239 datasets) were combined, ANN showed an accuracy of 71.9% while SVM displayed an accuracy of 97.6%. Separate fouling detection of mineral/protein fouling by ANN/SVM was comparable: dependent on fouling type detection accuracies of 100% (protein fouling, ANN and SVM), and 98.2% (SVM), and 93.5% (ANN) for mineral fouling was reached. It was shown that it was possible to detect fouling presence and absence offline in a static setup using ultrasonic measurements in combination with a classification method. This study proved the applicability of combining classification methods and fouling measurements to take a step toward reducing cleaning costs and time.

Multi-photon structuring of native polymers: A case study for structuring natural proteins

Abstract: The trend of mimicking the real biological world has created an intensive search for methods that are able to engineer 3D-structured biological environments using nano- and micro-system technologies. Recently published methods show the design of 3D structures by multi-photon induced polymerization of artificial polymers, such as chemically modified natural polymers. However, limitations of this approach are the long processing time and the fact that no native polymers have been used up to date. In this communication, a case study of multi-photon structuring of unmodified, native proteins (e.g. collagen and fibrinogen) and liquids, such as natural human blood, or cell culture medium supplements, such as fetal calf serum, is presented. Based on a computer-assisted process, the structures are polymerized precisely. Even adhesion and gluing of cells with this technique are possible. These encouraging results open new avenues for further inquiry, which are discussed in the paper.

Pretreatment of raw glycerol with activated carbon for 1,3-propanediol production by Clostridium butyricum

Abstract: National "863" Project [2006AA020103]; National Natural Science Foundation of China [21076172]; University of Science and Technology in Fujian province [2010H6023]