Modelling and design of a high productivity ethanol process using Zymomonas mobilis — I

Growth kinetics and ethanol production of Zymomonas mobilis in a bioreactor with cell recycle were modelled. High specific growth rates can be used to control excessive biomass accumulation in the system. Predicted peak productivity with a cell concentration of 80 g l⁻¹, a dilution rate of 6.5 h⁻¹, and a feed glucose concentration of 120 g l⁻¹ is 350 g l⁻¹ h⁻¹. The design of a special recycle reactor using a filter which should permit the operating conditions required for the validation of the model is proposed.     

Transfer and expression of a Bacillus licheniformis α-amylase gene in Zymomonas mobilis

The gene from Bacillus licheniformis coding for a thermostable -amylase was subcloned into the broad-host-range plasmid pKT210 in Escherichia coli. The recombinant plasmid pGNB6 was transferred into Zymomonas mobilis ATCC 31821 by conjugation. Plasmid pGNB6 was stably maintained in E. coli and unstable in Z. mobilis. The amylase gene was expressed in Z. mobilis at a lower level (25%) than in E. coli and regulation of enzyme biosynthesis was different in the host cells. Almost all the -amylase activity was recovered in the culture medium of Z. mobilis. This enzyme localization seemed to be the result of protein secretion rather than cell lysis. Integration of the amylase gene into a cryptic plasmid of Z. mobilis was observed. The amylase gene was still expressed, although at a lower level, and the -amylase activity, associated with a protein of molecular mass 62,000 daltons, was immunologically identical in Z. mobilis, E. coli and B. licheniformis.     

Production of glucose–fructose oxidoreductase and ethanol by Zymomonas mobilis ATCC 29191 in medium containing corn steep liquor as a source of vitamins

Different concentrations of corn steep liquor (CSL) were tested in the cultivation of Zymomonas mobilis. Cell growth, ethanol production, and the formation of glucose-fructose oxidoreductase (GFOR) and glucono-delta-lactonase (GL), the enzymes responsible for the bio-production of gluconic acid and sorbitol, were examined. The cell yields using 25 g CSL l(-1) and 40 g CSL l(-1) (Y(X,S) approximately 0.031 g g(-1)) were close to that obtained with 5 g yeast extract (YE) l(-1). With 5 g CSL l(-1) and 15 g CSL l(-1), the nutritional limitation led to smaller Y(X/S). Using 100 g CSL l(-1) produced an inhibitory effect on cell growth. Similar ethanol yields (92-95%) were calculated for each concentration of CSL and also for YE medium. The highest specific GFOR/GL activities (13.2-13.5 U g(-1) dry cell) were reached with 25 g CSL l(-1) and 40 g CSL l(-1), values comparable to that achieved with 5 g YE l(-1). The results confirm that CSL is an effective and cheap supplement for Z. mobilis medium, increasing the economic potential of a large-scale bio-production of sorbitol and gluconic acid by untreated Z. mobilis cells. The economic feasibility of the process is discussed.     

Continuous production of ethanol in a two-stage fermentation process using a protein — Phospholipid complex as a protective agent

A two-stage continuous fermentation process, using a continuous stirred tank fermenter (CSTF) and a tower fermenter (TF) connected in series, has been studied for ethanol production from d-glucose. The addition of a protein-phospholipid complex as a protective agent (PA) in the TF led to a three-fold increase in ethanol productivity and a 23.63% increase in final ethanol concentration in the tower effluent. The results are consistent with our previous findings on a cascade operation of CSTFs, namely, that the addition of PA to the tower increases cell tolerance towards ethanol at ethanol concentrations up to 70 gl^?1. Both studies indicate that beyond this experimentally determined critical ethanol concentration, ethanol production is significantly inhibited. Mathematical modelling of the behaviour of a single flocculated yeast floc suggested that, for yeast flocs up to 1 mm diameter, neither internal nor external diffusion of substrate is limiting. Therefore, a simplified mathematical method was developed for the analysis of the TF system. By plotting the calculated values from the derived performance equation and the experimental values of substrate conversion vs. residence time, good agreement was obtained between these two for the addition of PA. However, a small deviation was observed for the PA-free system.     

Growth and lipid production of Umbelopsis isabellina on a solid substrate—Mechanistic modeling and validation

Microbial lipids are an interesting feedstock for biodiesel. Their production from agricultural waste streams by fungi cultivated in solid-state fermentation may be attractive, but the yield of this process is still quite low. In this article, a mechanistic model is presented that describes growth, lipid production and lipid turnover in a culture of Umbelopsis isabellina on κ-carrageenan plates containing the monomers glucose and alanine as C-source and N-source, respectively, and improves the understanding of the complex solid-state system. The model includes reaction kinetics and diffusion of glucose, alanine and oxygen. It is validated empirically and describes the different phases of the culture very well: exponential growth, linear growth because of oxygen limitation, accumulation of lipids and carbohydrates after local N-depletion and turnover of lipids after local C-depletion. Extending the model with an unidentified extracellular product improved the fit of the model to the data. The model shows that oxygen limitation is extremely important in solid-state cultures using monomers, and explains the difference in production rate with submerged cultures. However, the results also show that the specific lipid production rate in solid-state cultures is much lower than in submerged cultures, which results in a low lipid yield.     

Prospective validation of the EuroSCORE II risk model in a single Dutch cardiac surgery centre

Objective

The EuroSCORE I was one of the most frequently used pre-operative risk models in cardiac surgery. In 2011 it was replaced by its successor the EuroSCORE II. This study aims to validate the EuroSCORE II and to compare its performance with the EuroSCORE I in a Dutch hospital.

Methods

The EuroSCORE II was prospectively validated in 2,296 consecutive cardiac surgery patients between 1 April 2012 and 1 January 2014. Receiver operating characteristic curves on in-hospital mortality were plotted for EuroSCORE I and EuroSCORE II, and the area under the curve was calculated to assess discriminative power. Calibration was assessed by comparing observed versus expected mortality. Additionally, analyses were performed in which we stratified for type of surgery and for elective versus emergency surgery.

Results

The observed mortality was 2.4% (55 patients). The discriminative power of the EuroSCORE II surpassed that of the EuroSCORE I (area under the curve EuroSCORE II 0.871, 95% confidence interval (CI) 0.832–0.911; area under the curve additive EuroSCORE I 0.840, CI 0.798–0.882; area under the curve logistic EuroSCORE I 0.761, CI 0.695–0.828). Both the additive and the logistic EuroSCORE I overestimated mortality (predictive mortality additive EuroSCORE I median 5.0%, inter-quartile range 3.0–8.0%; logistic EuroSCORE I 10.7%, inter-quartile range 5.8–13.9), while the EuroSCORE II underestimated mortality (median 1.6%, inter-quartile range 1.0–3.5). In most stratified analyses the EuroSCORE II performed better.

Conclusion

Our results show that the EuroSCORE II produces a valid risk prediction and outperforms the EuroSCORE I in elective cardiac surgery patients.

     

Enhancement of β-glucosidase stability and cellobiose-usage using surface-engineered recombinant Saccharomyces cerevisiae in ethanol production

To enhance the use of cellobiose by a recombinant Sachharomyces cerevisiae, the expressed -glucosidase that hydrolyzes cellobiose was stabilized using a surface-display system. The C-terminal half of -agglutinin was used as surface-display motif for the expression of -glucosidase in the cell wall. The surface-displayed -glucosidase had a half-life time (t _1/2) of 100 h in acidic culture broth conditions, while secreted -glucosidase had a t _1/2 of 60 h. With such stabilization of -glucosidase, the surface-engineered S. cerevisiae utilized 7.5 g cellobiose l^–1 over 60 h, while S. cerevisiae secreting -glucosidase into culture broth used 5.8 g cellobiose l^–1 over the same period.     

Computational analysis reveals the coupling between bistability and the sign of a feedback loop in a TGF-β1 activation model

Background

Bistable behaviors are prevalent in cell signaling and can be modeled by ordinary differential equations (ODEs) with kinetic parameters. A bistable switch has recently been found to regulate the activation of transforming growth factor-β1 (TGF-β1) in the context of liver fibrosis, and an ordinary differential equation (ODE) model was published showing that the net activation of TGF-β1 depends on the balance between two antagonistic sub-pathways.

Results

Through modeling the effects of perturbations that affect both sub-pathways, we revealed that bistability is coupled with the signs of feedback loops in the model. We extended the model to include calcium and Krüppel-like factor 2 (KLF2), both regulators of Thrombospondin-1 (TSP1) and Plasmin (PLS). Increased levels of extracellular calcium, which alters the TSP1-PLS balance, would cause high levels of TGF-β1, resembling a fibrotic state. KLF2, which suppresses production of TSP1 and plasminogen activator inhibitor-1 (PAI1), would eradicate bistability and preclude the fibrotic steady-state. Finally, the loop PLS???TGF-β1???PAI1 had previously been reported as negative feedback, but the model suggested a stronger indirect effect of PLS down-regulating PAI1 to produce positive (double-negative) feedback in a fibrotic state. Further simulations showed that activation of KLF2 was able to restore negative feedback in the PLS???TGF-β1???PAI1 loop.

Conclusions

Using the TGF-β1 activation model as a case study, we showed that external factors such as calcium or KLF2 can induce or eradicate bistability, accompanied by a switch in the sign of a feedback loop (PLS???TGF-β1???PAI1) in the model. The coupling between bistability and positive/negative feedback suggests an alternative way of characterizing a dynamical system and its biological implications.

     

Performance of an anaerobic baffled reactor and hybrid constructed wetland treating high-strength wastewater in Nepal—A model for DEWATS

Centralized wastewater treatment systems require sophisticated technologies and skilled manpower for their operation and maintenance (O&M). These systems have huge construction as well as O&M costs. Therefore, a Decentralized Wastewater Treatment System (DEWATS) rather than a centralized system might be especially beneficial in developing countries. A model for DEWATS is developed in Nepal with Anaerobic Baffled Reactor (ABR) and hybrid Constructed Wetland (CW). The DEWATS treats high-strength wastewater from 80 households (400 PE). This paper summarizes the performance of the DEWATS from July 2006 to August 2007 in the removal efficiencies of TSS, BOD₅, COD, NH₄–N, TP and FC. The ABR is very effective in the removal of organic pollutants and could achieve TSS removal up to 91%, BOD₅ up to 78% and COD up to 77%. The average removal efficiencies of the DEWATS is 96% TSS, 90% BOD₅, 90% COD, 70% NH₄–N, 26% TP and 98% FC.     

Commercial production of β-glucuronidase (GUS): a model system for the production of proteins in plants

We have generated transgenic maize seed containing -glucuronidase(GUS) for commercial production. While many other investigators have demonstrated the expression of GUS as a scoreable marker, this is one of the first cases where a detailed characterization of the transgenic plants and the protein were performed which are necessary to use this as a commercial source of GUS. The recombinant -glucuronidase was expressed at levels up to 0.7% of water-soluble protein from populations of dry seed, representing one of the highest levels of heterologous proteins reported for maize. Southern blot analysis revealed that one copy of the gene was present in the transformant with the highest level of expression. In seeds, the majority of recombinant protein was present in the embryo, and subcellular localization indicated that the protein was dispersed throughout the cytoplasm. The purified recombinant -glucuronidase (GUS) was compared to native -glucuronidase using SDS-PAGE and western blot analysis. The molecular mass of both the recombinant and native enzymes was 68 000 Da. N-terminal amino acid sequence of the recombinant protein was similar to the sequence predicted from the cloned Escherichia coli gene except that the initial methionine was cleaved from the recombinant GUS. The recombinant and native GUS proteins had isoelectric points (pI) from 4.8 to 5.0. The purified proteins were stable for 30 min at 25, 37, and 50 ° C. Kinetic analysis of the recombinant and native GUS enzymes using 4-methylumbelliferyl glucuronide (MUG) as the substrate was performed. Scatchard analysis of these data demonstrated that the recombinant enzyme had a K_m of 0.20 mM and a V_max of 0.29 mM MUG per hour, and the native enzyme had a K_m and V_max of 0.21 mM and 0.22 mM/h respectively. Using D-saccharic acid 1,4-lactone, which is an inhibitor of -glucuronidase, the K_i of the native and recombinant enzymes was determined to be 0.13 mM. Thus, these data demonstrate that recombinant GUS is functionally equivalent to native GUS. We have demonstrated the expression of high levels of GUS can be maintained in stable germlines and have used an efficient recovery system where the final protein product, GUS, has been successfully purified. We describe one of the first model systems for the commercial production of a foreign protein which relies on plants as the bioreactor.     

Biocatalytic ketone reduction—a powerful tool for the production of chiral alcohols—part II: whole-cell reductions

The tricarboxylic acid (TCA) cycle is an energy-producing pathway for aerobic organisms. However, it is widely accepted that the phylogenetic origin of the TCA cycle is the reductive TCA cycle, which is a non-Calvin-type carbon-dioxide-fixing pathway. Most of the enzymes responsible for the oxidative and reductive TCA cycles are common to the two pathways, the difference being the direction in which the reactions operate. Because the reductive TCA cycle operates in an energetically unfavorable direction, some specific mechanisms are required for the reductive TCA-cycle-utilizing organisms. Recently, the molecular mechanism for the “citrate cleavage reaction” and the “reductive carboxylating reaction from 2-oxoglutarate to isocitrate” in Hydrogenobacter thermophilus have been demonstrated. Both of these reactions comprise two distinct consecutive reactions, each catalyzed by two novel enzymes. Sequence analyses of the newly discovered enzymes revealed phylogenetic and functional relationships between other TCA-cycle-related enzymes. The occurrence of novel enzymes involved in the citrate-cleaving reaction seems to be limited to the family Aquificaceae. In contrast, the key enzyme in the reductive carboxylation of 2-oxoglutarate appears to be more widely distributed in extant organisms. The four newly discovered enzymes have a number of potential biotechnological applications.     

Bioconversion of lutein using a microbial mixture—maximizing the production of tobacco aroma compounds by manipulation of culture medium

The generation of aroma compounds by carotenoid cleavage in the 9–10 position was studied, due to the importance of these compounds in the flavor and fragrance industry. The bioconversion of the carotenoid lutein to C₁₃ norisoprenoids utilizing a microbial mixture composed of Trichosporon asahii and Paenibacillus amylolyticus was carried out by a fermentation process. Applying an experimental design methodology, the effects of nutritional factors on the production of aroma compounds present in the tobacco profile were studied. After an assessment of the significance of each nutritional factor, the levels of the variables yielding the maximum response were calculated. Glucose, tryptone, and yeast extract exerted a strong negative effect over the objective function, with glucose being the strongest. Lutein possessed a positive effect over the tobacco aroma production, while sodium chloride and trace elements showed no influence over the process. The yield attained after culture medium manipulation was almost ten-fold higher, compared with the base medium; and the aroma mixture was characterized as: 7,8-dihydro--ionol (95.2%), 7,8-dihydro--ionone (3.7%), and -ionone (1.1%).     

Biodiesel production—current state of the art and challenges

Biodiesel is a clean-burning fuel produced from grease, vegetable oils, or animal fats. Biodiesel is produced by transesterification of oils with short-chain alcohols or by the esterification of fatty acids. The transesterification reaction consists of transforming triglycerides into fatty acid alkyl esters, in the presence of an alcohol, such as methanol or ethanol, and a catalyst, such as an alkali or acid, with glycerol as a byproduct. Because of diminishing petroleum reserves and the deleterious environmental consequences of exhaust gases from petroleum diesel, biodiesel has attracted attention during the past few years as a renewable and environmentally friendly fuel. Since biodiesel is made entirely from vegetable oil or animal fats, it is renewable and biodegradable. The majority of biodiesel today is produced by alkali-catalyzed transesterification with methanol, which results in a relatively short reaction time. However, the vegetable oil and alcohol must be substantially anhydrous and have a low free fatty acid content, because the presence of water or free fatty acid or both promotes soap formation. In this article, we examine different biodiesel sources (edible and nonedible), virgin oil versus waste oil, algae-based biodiesel that is gaining increasing importance, role of different catalysts including enzyme catalysts, and the current state-of-the-art in biodiesel production. JIMB 2008: BioEnergy—special issue.     

Imperfect Batesian mimicry—the effects of the frequency and the distastefulness of the model

Batesian mimicry is the resemblance between unpalatable models and palatable mimics. The widely accepted idea is that the frequency and the unprofitability of the model are crucial for the introduction of a Batesian mimic into the prey population. However, experimental evidence is limited and furthermore, previous studies have considered mainly perfect mimicry (automimicry). We investigated imperfect Batesian mimicry by varying the frequency of an aposematic model at two levels of distastefulness. The predator encountered prey in a random order, one prey item at a time. The prey were thus presented realistically in a sequential way. Great tits (Parus major) were used as predators. This experiment, with a novel signal, supports the idea that Batesian mimics gain most when the models outnumber them. The mortalities of the mimics as well as the models were significantly dependent on the frequency of the model. Both prey types survived better the fewer mimics there were confusing the predator. There were also indications that the degree of distastefulness of the model had an effect on the survival of the Batesian mimic: the models survived significantly better the more distasteful they were. The experiment supports the most classical predictions in the theories of the origin and maintenance of Batesian mimicry.     

The structure and function of the chloroplast photosynthetic membrane — a model for the domain organization

Recent work on the domain organization of the thylakoid is reviewed and a model for the thylakoid of higher plants is presented. According to this model the thylakoid membrane is divided into three main domains: the stroma lamellae, the grana margins and the grana core (partitions). These have different biochemical compositions and have specialized functions. Linear electron transport occurs in the grana while cyclic electron transport is restricted to the stroma lamellae. This model is based on the following results and considerations. (1) There is no good candidate for a long-range mobile redox carrier between PS II in the grana and PS I in the stroma lamellae. The lateral diffusion of plastoquinone and plastocyanin is severely restricted by macromolecular crowding in the membrane and the lumen respectively. (2) There is an excess of 14±18% chlorophyll associated with PS I over that of PS II. This excess is assumed to be localized in the stroma lamellae where PS I drives cyclic electron transport. (3) For several plant species, the stroma lamellae account for 20±3% of the thylakoid membrane and the grana (including the appressed regions, margins and end membranes) for the remaining 80%. The amount of stroma lamellae (20%) corresponds to the excess (14–18%) of chlorophyll associated with PS I. (4) The model predicts a quantum requirement of about 10 quanta per oxygen molecule evolved, which is in good agreement with experimentally observed values. (5) There are at least two pools of each of the following components: PS I, PS II, cytochrome bf complex, plastocyanin, ATP synthase and plastoquinone. One pool is in the grana and the other in the stroma compartments. So far, it has been demonstrated that the PS I, PS II and cytochrome bf complexes each differ in their respective pools.Abbreviations PS I and PS II Photosystem I and II - P 700 reaction center of PS I - LHC II light-harvesting complex II     

Effect of the aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor: mathematical modeling

The effect of aeration rate and agitation speed on β-carotene production and morphology of Blakeslea trispora in a stirred tank reactor was investigated. B. trispora formed hyphae, zygophores and zygospores during the fermentation. The zygospores were the morphological form responsible for β-carotene production. Both aeration and agitation significantly affected β-carotene concentration, productivity, biomass and the volumetric mass transfer coefficient (K_La). The highest β-carotene concentration (1.5 kg m⁻³) and the highest productivity (0.08 kg m⁻³ per day) were obtained at low impeller speed (150 rpm) and high aeration rate (1.5 vvm). Also, maximum productivity (0.08 kg m⁻³ per day) and biomass dry weight (26.4 kg m⁻³) were achieved at high agitation speed (500 rpm) and moderate aeration rate (1.0 vvm). Conversely, the highest value of K_La (0.33 s⁻¹) was observed at high agitation speed (500 rpm) and high aeration rate (1.5 vvm). The experiments were arranged according to a central composite statistical design. Response surface methodology was used to describe the effect of impeller speed and aeration rate on the most important fermentation parameters. In all cases, the fit of the model was found to be good. All fermentation parameters (except biomass concentration) were strongly affected by the interactions among the operation variables. β-Carotene concentration and productivity were significantly influenced by the aeration, agitation, and by the positive or negative quadratic effect of the aeration rate. Biomass concentration was principally related to the aeration rate, agitation speed, and the positive or negative quadratic effect of the impeller speed and aeration rate, respectively. Finally, the volumetric mass transfer coefficient was characterized by the significant effect of the agitation speed, while the aeration rate had a small effect on K_La.     

Development and validation of a new method to measure walking speed in free-living environments using the actibelt® platform

Walking speed is a fundamental indicator for human well-being. In a clinical setting, walking speed is typically measured by means of walking tests using different protocols. However, walking speed obtained in this way is unlikely to be representative of the conditions in a free-living environment. Recently, mobile accelerometry has opened up the possibility to extract walking speed from long-time observations in free-living individuals, but the validity of these measurements needs to be determined. In this investigation, we have developed algorithms for walking speed prediction based on 3D accelerometry data (actibelt?) and created a framework using a standardized data set with gold standard annotations to facilitate the validation and comparison of these algorithms. For this purpose 17 healthy subjects operated a newly developed mobile gold standard while walking/running on an indoor track. Subsequently, the validity of 12 candidate algorithms for walking speed prediction ranging from well-known simple approaches like combining step length with frequency to more sophisticated algorithms such as linear and non-linear models was assessed using statistical measures. As a result, a novel algorithm employing support vector regression was found to perform best with a concordance correlation coefficient of 0.93 (95%CI 0.92-0.94) and a coverage probability CP1 of 0.46 (95%CI 0.12-0.70) for a deviation of 0.1 m/s (CP2 0.78, CP3 0.94) when compared to the mobile gold standard while walking indoors. A smaller outdoor experiment confirmed those results with even better coverage probability. We conclude that walking speed thus obtained has the potential to help establish walking speed in free-living environments as a patient-oriented outcome measure.