Comparative metabolomic analysis on industrial continuous and batch ethanol fermentation processes by GC-TOF-MS

The intracellular metabolic profile characterization of Saccharomyces cerevisiae throughout industrial ethanol fermentation was investigated using gas chromatography coupled to time-of-flight mass spectrometry. A total of 143 and 128 intracellular metabolites in S. cerevisiae were detected and quantified in continuous and batch fermentations, respectively. The two fermentation processes were both clearly distinguished into three main phases by principal components analysis. Furthermore, the levels of some metabolites involved in central carbon metabolism varied significantly throughout both processes. Glycerol and phosphoric acid were principally responsible for discriminating seed, main and final phases of continuous fermentation, while lactic acid and glycerol contributed mostly to telling different phases of batch fermentation. In addition, the levels of some amino acids such as glycine varied significantly during both processes. These findings provide new insights into the metabolomic characteristics during industrial ethanol fermentation processes. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Acceleration of ethanolic fermentation by zeolites

Summary Ethanolic fermentation of glucose by Saccharomyces cerevisiae was accelerated in presence of Silicalite only when the yeast was highly flocculent, the zeolite lowering floc size. With the sucrose and molasses, however, fermentation was accelerated even when the yeasts have low flocculence, the zeolite enhancing invertase activity of the yeast cells.     

Two-phase partitioning bioreactors in fermentation technology

The two-phase partitioning bioreactor concept appears to have a great potential in enhancing the productivity of many bioprocesses. The proper selection of an organic solvent is the key to successful application of this approach in industrial practice. The integration of fermentation and a primary product separation step has a positive impact on the productivity of many fermentation processes. The controlled substrate delivery from the organic to the aqueous phase opens a new area of application of this strategy to biodegradation of xenobiotics. In this review, the most recent advances in the application of two-liquid phase partitioning bioreactors for product or substrate partitioning are discussed. Modeling and performance optimization studies related to those bioreactor systems are also reviewed.     

Non-linear control of continuous bioreactors

Control of bioreactors has achieved importance in the recent years. This may be due to the fact that they are difficult to control which may be attributed to its nonlinear dynamic behavior. The model parameters of the bioreactor also vary in an unpredictable manner. The complexity of the biochemical processes inhibits the accurate modeling and also the lack of suitable sensors make the process state difficult to characterize. Considerable emphasis has been placed on the control of fed-batch fermentors because of their prevalence in industries. However, when production of biomass is to be optimized, continuous operation is desirable. Several procedures are available for the nonlinear control of processes, viz., differential geometric approach, internal model control approach, reference synthesis technique, predictive control design, etc., but the major disadvantage of these approaches is the computational time required to perform the prediction optimization. In this study, a nonlinear controller based on a polynomial discrete time model (NARMAX) is evaluated for its performance on a fermentor. It can be shown that a nonlinear self-tuning controller based on NARMAX model can be extended to the control of fermentors. The response is smooth for both load and setpoint changes even when process parameters are assumed to be zero and uncertainty in parameters are present and in the presence of controller constraints. The control action can be made more or less robust by changing the design parameters appropriately. Therefore, nonlinear self-tuning controller is suitable for control of industrial processes.     

Apparent viscosity for non-Newtonian fermentation media in bioreactors

The apparent viscosity of non-Newtonian fermentation media is examined. The present state of this subject is discussed. The energy dissipation rate concept is used for a new evaluation of the apparent viscosity in bioreactors, i.e. stirred tank and bubble column bioreactors. The proposed definition of the apparent viscosity is compared with the definitions available in the literature.List of Symbols A _d m ² downcomer cross-sectional area - A _r m ² riser cross-sectional area - a m^–1 specific surface area - C constant in eq. (13) - D m column diameter - D _I m impeller diameter - g m s^–2 gravitational acceleration - h J m^–2 s^–1 K^–1 heat transfer coefficient - K Pa s ⁿ consistency index in a power-law model - k constant in eq. (3) - k _L m s ^–1 liquid-phase mass transfer coefficient - N s^–1 impeller speed - n flow index in a power-law model - P W power input - Re Reynolds number ND _I ^/2 /(/) - U _sg m s ^–1 superficial gas velocity - (U _sg ) _r m s^–1 superficial gas velocity based on riser - V-m³ liquid volume - v ₀ m s^–1 friction velocity Greek Symbols s^–1 shear rate - _c s^–1 characteristic shear rate - W kg^–1 energy dissipation rate per unit mass - W kg^–1 characteristic energy dissipation rate per unit mass - Pa s viscosity - _app Pa s apparent viscosity - kg m^–3 density - Pa shear stress     

Feed component inhibition in ethanolic fermentation by Saccharomyces cerevisiae

Inhibition by secondary feed components can limit productivity and restrict process options for the production of ethanol by fermentation. New fermentation processes (such as vacuum or extractive fermentation), while selectively removing ethanol, can concentrate nonmetabolized feed components in the remaining broth. Stillage recycle to reduce stillage waste treatment results in the buildup of nonmetabolized feed components. Continuous culture experiments are presented establishing an inhibition order: CaCl(2), (NH(4))(2)xSO(4) > NaCl, NH(4)Cl > KH(2)PO(4) > xylose, MgCl(2) > MgSO(4) > KCl. Reduction of the water activity alone is not an adequate predictor of the variation in inhibitory concentration among the different components tested. As a general trend, specific ethanol productivity increases and cell production decreases as inhibitors are added at higher concentration. We postulate that these results can be interpreted in terms of an increase in energy requirements for cell maintenance under hypertonic (stressed) conditions. Ion and carbohydrate transport and specific toxic effects are reviewed as they relate to the postulated inhibition mechanism. Glycerol production increases under hypertonic conditions and glycerol is postulated to function as a nontoxic osmoregulator. Calcium was the most inhibitory component tested, causing an 80%decline in cell mass production at 0.23 mol Ca(2+)/L and calcium is present at substantial concentration in many carbohydrate sources. For a typical final cane molasses feed, stillage recycle must be limited to less than onethird of the feed rate; otherwise inhibitory effects will be observed.     

Theoretical and methodological studies of continuous microbial bioreactors

This article reviews most of the author's studies on process development and reactor design for continuous microbial reactions. (1) Enzyme reactions of growing and non-growing microbial cells immobilized in agar gel beads were analyzed pertaining to the effects of external and internal diffusion of substrate on reaction kinetics. (2) Experimental correlations of production rates of beta-fructosidase and acid phosphatase with dilution rate of continuous culture were simulated based on an operon model for enzyme regulation. (3) Population dynamics of an amylase-producing bacteria and their mutant were discussed in relation to enzyme productivity in a continuous culture of spore-forming bacteria. (4) Plasmid mobilization in a mixed population of donor, recipient, and helper cells was investigated in a continuous culture as a model study of accidental release of a genetically modified plasmid into a natural environment. (5) A production rate increase of up to 100-fold was achieved by cell-recycle culturing of continuous acetic acid fermentation using a filter module with a hollow fiber membrane. (6) The feasibility of a continuous surface culture for the biooxidation of organic substances was ascribed to an enhanced oxygen absorption rate in the presence of a microbial film on a liquid surface. (7) Simultaneous separation of inhibitory products using an electrodialysis module during some organic acid fermentations was effective for increasing production in a continuous culture.     

Competitive yeast fermentation with selective flocculation and recycle

Continuous fermentations were carried out involving competition between two strains of Saccharomyces cerevisiae. One of the strains has a lower specific growth rate and is very flocculent, whereas the fastergrowing strain is nonflocculent. The product stream from the chemostat was fed into an inclined settler where the flocculent strain was partially separated from the nonflocculent strain as a result of the higher sedimentation rate of the flocculent cells. The underflow from the inclined settler, which was concentrated and enriched with flocculent cells, was recycled to the chemostat. When no recycle was used, the fastergrowing, nonflocculent yeast rapidly overtook the culture. With selective recycle, however, the experiments demonstrated that the slower-growing flocculent yeast could be maintained as the dominant species. A theoretical development is also presented in order to describe the competition between two strains in the bioreactor-settler system. The concept of selective recycle via selective flocculation and sedimentation offers a possible means of maintaining unstable recombinant microorganisms in continuous fermentations.     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

合成气厌氧发酵生物反应器的研究进展

对合成气厌氧发酵生物反应器的研究进展进行综述,包括生物反应器操作原理、类型、构造、应用和对发酵过程的影响等,并对其未来的发展作出展望。     

Dynamic modeling and simulation of continuous airlift bioreactors

For dynamic behaviors of continuous airlift bioreactors, a mathematical model based on a tanks-in-series model with backflow has been developed. The equations describing the dynamics of airlift bioreactors are material balances for micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and heat balances. Non-ideal mixing of liquid and gas phases is taken into account using a tanks-in-series model with backflow. The batch operation, startup operation and the consequence of plant failure were simulated and the effects of design and operating parameters for an airlift bioreactor on its dynamic behaviors were discussed. The concentration profiles of micro-organism, substrate, dissolved oxygen and oxygen in gas-phase and the temperature profile in an airlift bioreactors and their dynamics were obtained. The computational results indicate that the transients of a chemostat in the case of bubble column bioreactor are slower compared with those in the case of airlift bioreactor. The proposed simulator is more precise as compared with models published previously in the literature and therefore provides more reliable and rational examination of continuous airlift bioreactor performance.     

Penicillin fermentation: mechanisms and models for industrial-scale bioreactors

Even after many years of research and industrial practice, the production of penicillin G in fed-batch fermentation by Penicillium crysogenum continues to attract research interest. There are many reasons: the commercial and therapeutic importance of penicillin and its derivatives, the complexity of cell growth, and the impact of engineering variables, the last of which are significant in large bioreactors but are not yet fully understood. Extensive research has generated new information on the mechanisms of cellular reactions and morphological features of the mycelia and their role in the synthesis of the product. Given a choice of mechanisms, models of different degrees of complexity, for both cellular differentiation and bioreactor performance, have been proposed. The more complex models require and provide more information. They are also more difficult to evaluate and apply in automatic control systems for production-scale bioreactors. The present review considers the evolution of recent knowledge and models from this perspective.     

Static magnetic fields enhancement of Saccharomyces cerevisae ethanolic fermentation

Magnetic effects induced in ethanolic fermentation by Saccharomyces cerevisiae strain DAUFPE-1012 were studied during a 24 h exposure to 220 mT steady magnetic fields (SMF) at 23 +/- 1 degrees C, produced by NdFeB rod magnets. The magnets were attached diametrically opposed (N to S) to a cylindrical tube reactor. The biomass growth in the reactor culture media (yeast extract + glucose 2%) during 24 h was monitored by measurements of optical density, which was correlated to cell dry weight. Ethanol concentration and glucose level were measured every 2 h. The pH of the culture media was maintained between 4 and 5. As a result, biomass (g/L) increased 2.5-fold and ethanol concentration 3.4-fold in magnetized cultures (n = 8) as compared with SMF nonexposed cultures (n = 8). Glucose consumption was higher in magnetized cultures, which correlated to the ethanol yield.     

On-line measurement of Brewer's yeast flocculation during fermentation

Summary The separation of yeast at the and of a beer fermentation depends on the ability of the yeast to become flocculent, form floes and sediment to the bottom of the fermenter. To monitor these processes an on-line method has been developed. With the instrument, a Photometric Dispersion Analyser, it is possible to determine flocculation on-line at the same conditions as in the fermenter.     

Microbial community analysis of two field-scale sulfate-reducing bioreactors treating mine drainage

The microbial communities of two field-scale pilot sulfate-reducing bioreactors treating acid mine drainage (AMD), Luttrell and Peerless Jenny King (PJK), were compared using biomolecular tools and multivariate statistical analyses. The two bioreactors were well suited for this study because their geographic locations and substrate compositions were similar while the characteristics of influent AMD, configuration and degree of exposure to oxygen were distinct. The two bioreactor communities were found to be functionally similar, including cellulose degraders, fermenters and sulfate-reducing bacteria (SRB). Significant differences were found between the two bioreactors in phylogenetic comparisons of cloned 16S rRNA genes and adenosine 5'-phosphosulfate reductase ( apsA ) genes. The apsA gene clones from the Luttrell bioreactor were dominated by uncultured SRB most closely related to Desulfovibrio spp., while those of the PJK bioreactor were dominated by Thiobacillus spp. The fraction of the SRB genus Desulfovibrio was also higher at Luttrell than at PJK as determined by quantitative real-time polymerase chain reaction analysis. Oxygen exposure at PJK is hypothesized to be the primary cause of these differences. This study is the first rigorous phylogenetic investigation of field-scale bioreactors treating AMD and the first reported application of multivariate statistical analysis of remediation system microbial communities applying UniFrac software.     

Baffles increase performance of solid-state fermentation in rotating drum bioreactors

Summary Rhizopus oligosporus grew better on wheat bran in a rotating drum when baffles were fitted. The maximum O₂ uptake rates for the baffled and unbaffled runs were 9.0 and 5.7 mmol/min.kg initial dry substrate respectively. The RQ remained at 1.0 throughout the baffled run but varied between 1.0 and 1.2 for the unbaffled run.     

Comparative technoeconomic analysis of a softwood ethanol process featuring posthydrolysis sugars concentration operations and continuous fermentation with cell recycle

Economical production of second generation ethanol from Ponderosa pine is of interest due to widespread mountain pine beetle infestation in the western United States and Canada. The conversion process is limited by low glucose and high inhibitor concentrations resulting from conventional low‐solids dilute acid pretreatment and enzymatic hydrolysis. Inhibited fermentations require larger fermentors (due to reduced volumetric productivity) and low sugars lead to low ethanol titers, increasing distillation costs. In this work, multiple effect evaporation (MEE) and nanofiltration (NF) were evaluated to concentrate the hydrolysate from 30 g/l to 100, 150, or 200 g/l glucose. To ferment this high gravity, inhibitor containing stream, traditional batch fermentation was compared with continuous stirred tank fermentation (CSTF) and continuous fermentation with cell recycle (CSTF‐CR). Equivalent annual operating cost (EAOC = amortized capital + yearly operating expenses) was used to compare these potential improvements for a local‐scale 5 MGY ethanol production facility. Hydrolysate concentration via evaporation increased EAOC over the base process due to the capital and energy intensive nature of evaporating a very dilute sugar stream; however, concentration via NF decreased EAOC for several of the cases (by 2 to 15%). NF concentration to 100 g/l glucose with a CSTF‐CR was the most economical option, reducing EAOC by $0.15 per gallon ethanol produced. Sensitivity analyses on NF options showed that EAOC improvement over the base case could still be realized for even higher solids removal requirements (up to two times higher centrifuge requirement for the best case) or decreased NF performance. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:946–956, 2015     

Nonlinear adaptive optimization of biomass productivity in continuous bioreactors

A novel on-line adaptive optimization algorithm is developed and applied to continuous biological reactors. The algorithm makes use of a simple nonlinear estimation model that relates either the cell-mass productivity or the cell-mass concentration to the dilution rate. On-line estimation is used to recursively identify the parameters in the nonlinear process model and to periodically calculate and steer the bioreactor to the dilution rate that yields optimum cell-mass productivity. Thus, the algorithm does not require an accurate process model, locates the optimum dilution rate online, and maintains the bioreactors at this optimum condition at all times. The features of the proposed new algorithm are compared with those of other adaptive optimization techniques presented in the literature [1–5]. A detailed simulation study using three different microbial system models [3, 6–7] was conducted to illustrate the performance of the optimization algorithm.List of Symbols A(q ^–1) polynomial in q ^–1 - b bias term - c _F nutrient cost term - B(q ^–1) polynomial in q ^–1 - C(q ^–1) polynomial in q ^–1 - CMPR kg/(m³ · h) cell mass productivity - D 1/h dilution rate - D _opt 1/h optimum dilution rate - E(q ^–1) polynomial in q ^–1 - h exponential filter constant - J objective function - k time index - K _m Monod's constant - n optimization interval - P covariance matrix - q ^–1 backward shift operator - r defined by equation (28) - S kg/m³ substrate concentration - S _F kg/m³ feed substrate concentration - T _s h sampling period - u vector containing previous input values - V dm³ fermenter volume - X kg/dm³ cell mass concentration - Y output variable - Y vector containing previous output values - Y _x/s g/g yield coefficient - optimization tuning constant - vector linear or nonlinear combination of u and Y - denominator covariance matrix update equation - forgetting factor - parameter vector - 1/h specific growth rate - _m 1/h maximum specific grow rate     

The ethanolic fermentation pathway supports respiration and lipid biosynthesis in tobacco pollen

Rapid pollen tube growth requires a high rate of sugar metabolism to meet energetic and biosynthetic demands. Previous work on pollen sugar metabolism showed that tobacco pollen carry out efficient ethanolic fermentation concomitantly with a high rate of respiration (Bucher et al., 1995). Here we show that the products of fermentation, acetaldehyde and ethanol, are further metabolised in a pathway that bypasses mitochondrial PDH. The enzymes involved in this pathway are pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthetase. Radiolabelling experiments show that during tobacco pollen tube growth label of 14C-ethanol is incorporated into CO2 as well as into lipids and other higher molecular weight compounds. A role for the glyoxylate cycle appears unlikely since activity of malate synthase, a key enzyme of the glyoxylate cycle, could not be detected.     

The effect of pH on kinetic and yield parameters during the ethanolic fermentation of D-xylose with Pachysolen tannophilus

We have studied the ethanolic fermentation of D-xylose with Pachysolen tannophilus in batch cultures. We propose a model to predict variations in D-xylose consumed, and biomass and ethanol produced, in which we include parameters for the specific growth rate, for the consumption of D-xylose and production of ethanol either related or not to growth.The ideal initial pH for ethanol production turned out to be 4.5. At this pH value the net specific growth rate was 0.26 h^–1, biomass yield was 0.16 g.g^–1, the cell-maintenance coefficient was 0.073 g.g^–1.h^–1, the parameter for ethanol production non-related to growth was 0.064 g.g^–1,h^–1 and the maximum ethanol yield was 0.32 g.g^–1.List of Symbols A _c Carbon atomic weight - a _d1/h Specific cell-maintenance rate defined in Eq. (8) - c Mass fraction of carbon in the biomass - E g/l Ethanol concentration - f _x Correction factor defined in Eq. (13) - f _x Correction factor defined in Eq. (13) - f _xi Correction factor defined in Eq. (14) - k _d1/h Death constant - M _E Ethanol molecular weight - M _s Xylose molecular weight - M _xi Xylitol molecular weight - m g xylose/g biomass Maintenance coefficient for substrate - m _dg xylose/g biomass Maintenance coefficient when k _d - q _Eg ethanol/g biomass. Specific ethanol production rate - s g/l Residual xylose concentration - s ₀ g/l Initial xylose concentration - t h Time - x g/l Biomass concentration - x ₀ g/l Initial biomass concentration - Y _E/sg ethanol/g xylose Instantaneous ethanol yield - ¯Y _E/sg ethanol/g xylose Mean ethanol yield - Y _E ^s/T g ethanol/g xylose Theoretical ethanol yield - Y _E ^s/* g ethanol/g xylose Corrected instantaneous ethanol yield - ¯Y _E ^s/* g ethanol/g xylose Corrected mean ethanol yield - Y _x/sg biomass/g xylose Biomass yield - ¯Y _xi/sg xylitol/g xylose Mean xylitol yield Greek Letters g ethanol/g biomass Growth-associated product formation parameter - g ethanol/g biomass.h Non-growth-associated product formation parameter - _dg ethanol/g biomass.h Non-growth-associated product formation parameter when k _d0 - h Variable defined in Eq. (6) or Eq. (7) - 1/h Specific growth rate - _m1/h Maximum specific growth rate