Optimal Bioreactor Operational Policies for the Enzymatic Hydrolysis of Sugarcane Bagasse

The consolidation of the industrial production of second-generation (2G) bioethanol relies on the improvement of the economics of the process. Within this general scope, this paper addresses one aspect that impacts the costs of the biochemical route for producing 2G bioethanol: defining optimal operational policies for the reactor running the enzymatic hydrolysis of the C6 biomass fraction. The use of fed-batch reactors is one common choice for this process, aiming at maximum yields and productivities. The optimization problem for fed-batch reactors usually consists in determining substrate feeding profiles, in order to maximize some performance index. In the present control problem, the performance index and the system dynamics are both linear with respect to the control variable (the trajectory of substrate feed flow). Simple Michaelis–Menten pseudo-homogeneous kinetic models with product inhibition were used in the dynamic modeling of a fed-bath reactor, and two feeding policies were implemented and validated in bench-scale reactors processing pre-treated sugarcane bagasse. The first approach applied classical optimal control theory. The second policy was defined with the purpose of sustaining high rates of glucose production, adding enzyme (Accellerase® 1500) and substrate simultaneously during the reaction course. A methodology is described, which used economical criteria for comparing the performance of the reactor operating in successive batches and in fed-batch modes. Fed-batch mode was less sensitive to enzyme prices than successive batches. Process intensification in the fed-batch reactor led to glucose final concentrations around 200 g/L.     

Power consumption evaluation of different fed-batch strategies for enzymatic hydrolysis of sugarcane bagasse

The minimization of costs in the distillation step of lignocellulosic ethanol production requires the use of a high solids loading during the enzymatic hydrolysis to obtain a more concentrated glucose liquor. However, this increase in biomass can lead to problems including increased mass and heat transfer resistance, decreased cellulose conversion, and increased apparent viscosity with the associated increase in power consumption. The use of fed-batch operation offers a promising way to circumvent these problems. In this study, one batch and four fed-batch strategies for solids and/or enzyme feeding during the enzymatic hydrolysis of sugarcane bagasse were evaluated. Determinations of glucose concentration, power consumption, and apparent viscosity were made throughout the experiments, and the different strategies were compared in terms of energy efficiency (mass of glucose produced according to the energy consumed). The best energy efficiency was obtained for the strategy in which substrate and enzyme were added simultaneously (0.35 kg_glucose kWh^?1). This value was 52 % higher than obtained in batch operation.     

Fuzzy logic control of a fed-batch fermentor

A rule-based fuzzy logic control is developed for control of penicillin concentration in a fed-batch bioreactor. The membership functions, fuzzy ranges for the error and for the controller output are defined. A fuzzy rule base is constructed relating error to the control output based on operators' knowledge. The performance of the fuzzy-logic controller is evaluated by simulating a mathematical model of the fed-batch bioreactor.     

Fuzzy logic control of a fed-batch fermentor with significant measurement delay

A rule based fuzzy logic controller is developed for control of product concentration in a fed-batch fermentor with a significant measurement delay. The performance of the delay compensated fuzzy logic controller is compared by simulation with that of a delay uncompensated fuzzy controller and with that of a conventional proportional and derivative (PD) controller.     

Carotenoid production from hydrolyzed molasses by Dietzia natronolimnaea HS-1 using batch,fed-batch and continuous culture

The different cultivation strategies of batch, fed-batch and continuous culture for the synthesis of biomass and carotenoids by Dietzia natronolimnaea HS-1 from waste molasses and its hydrolysate were compared. The efficiency of three various pretreatments (enzymatic, acidic and acidic at high temperature) for the determination of the best hydrolysate was also studied by evaluating the conversion rate of sucrose. The analytical procedures initially showed that canthaxanthin (CTX) and enzymatic hydrolysis were the most abundant pigment biosynthesized and the most suitable process for the substrate production, respectively. An increase in reducing sugar concentration of the enzymatic hydrolysate molasses (EHM) from 25 to 50 g/l led to a drastic decrease in biomass formation and substrate utilization. EHM (25 g/l) was a better substrate for the cell growth and product formation than the waste molasses (25 g/l). The application of EHM instead of molasses enhanced the biomass production in fed-batch culture more than batch and continuous cultures. However, the continuous cultivation had the highest biomass (12.98 g/l), carotenoid (27.33 mg/l) and CTX (25.04 mg/l) yields with 25 g/l of EHM. The CTX isolated from D. natronolimnaea HS-1 may be used as a natural antioxidant for possible production of healthy-functional foods in the future.     

Analysis of conversion and operation strategies for enzymatic hydrolysis of lignocellulosic biomass in a series of CSTRs with distributed feeding

Design considerations for enzymatic hydrolysis of lignocellulosic biomass in two and three continuous stirred tank reactors (CSTRs) in series with distributed feeding of substrate and enzyme, followed by a series of CSTRs, are discussed. A previously developed, fitted, and validated kinetic model is extended to accommodate distributed feeding and used along with the micromixing limiting situations of macrofluid and microfluid to describe the reaction system. The capabilities of the reaction system proposed are explored for a range of cumulative substrate concentration from 5 to 20% w/w (dry basis). Continuous distributed feeding does not show advantages in terms of cellulose conversion when compared with the operation where an equivalent mass of substrate is added at the first reactor of the series, but the potential to increase substrate concentration beyond the concentrations that can be handled in conventional CSTRs, and therefore, the volumetric productivity of reactors, is evident.     

Fuzzy neural network for the control of high cell density cultivation of recombinant Escherichia coli

A five-layer fuzzy neural network (FNN) was developed for the control of fed-batch cultivation of recombinant Escherichia coli JM103 harboring plasmid pUR 2921. The FNN was believed to represent the membership functions of the fuzzy subsets and to implement fuzzy inference using previous experimental data. This FNN was then used for compensating the exponential feeding rate determined by the feedforward control element. The control system is therefore a feedforward-feedback type. The change in pH of the culture broth and the specific growth rate were used as the inputs to FNN to calculate the glucose feeding rate. A cell density of 84 g DWC/l in the fed-batch cultivation of the recombinant E. coli was obtained with this control strategy. Two different FNNs were then employed before and after induction to enhance plasmid-encoded β-galactosidase production. Before induction the specific growth rate was set as 0.31 h⁻¹, while it was changed to 0.1 h⁻¹ after induction. Compared to when only one FNN was used, the residual glucose concentration could be tightly controlled at an appropriate level by employing two FNNs, resulting in an increase in relative activity of β-galactosidase which was about four times greater. The present investigation demonstrates that a feedforward-feedback control strategy with FNN is a promising control strategy for the control of high cell density cultivation and high expression of a target gene in fed-batch cultivation of a recombinant strain.     

Production of recombinant β-galactosidase in bioreactors by fed-batch culture using DO-stat and linear control

β-Galactosidase enzymes continue to play an important role in food and pharmaceutical industries. These enzymes hydrolyze lactose in its constituent monosaccharides, glucose and galactose. The industrial use of enzymes presents an increase in process costs reflecting in higher final product value. An alternative to enhance processes’ productivity and yield would be the use of recombinant enzymes and their large-scale fed-batch production. The overexpression of recombinant β-galactosidase from Kluyveromyces sp. was carried out in 2-L bioreactors using Escherichia coli strain BL21 (DE3) as host. Effect of induction time on recombinant enzyme expression was studied by adding 1?mM isopropyl thiogalactoside (IPTG) at 12?h, 18?h and 24?h of cultivation. Glucose feeding strategies were compared employing feedback-controlled DO-stat and ascendant linear pump feeding in bioreactor fed-batch cultivations. Linear feeding strategy with IPTG addition at 18?h of cultivation resulted in approximately 20?g/L and 17,745?U/L of biomass and β-galactosidase activity, respectively. On the other hand, although the feedback-controlled DO-stat feeding strategy induced at 12?h of cultivation led to lower final biomass of 18?g/L, it presented an approximately 2.5 increase in enzymatic activity, resulting in 42,367?U/L, and most importantly it led to the most prominent specific enzymatic activity of approximately 40?U/mg_protein. Comparing to previous results, these results suggest that the DO-stat feeding is a promising strategy for recombinant β-galactosidase enzyme production.     

Optimization of Dilute Acid Pretreatment and Enzymatic Hydrolysis of <Emphasis Type="Italic">Phalaris aquatica</Emphasis> L. Lignocellulosic Biomass in Batch and Fed-Batch Processes

Phalaris aquatica L., a rich in holocellulose (69.80 %) and deficient in lignin (6.70 %) herbaceous, perennial grass species, was utilized in a two-step (biomass pretreatment-enzymatic hydrolysis) saccharification process for sugars recovery. The Taguchi methodology was employed to determine the dilute acid pretreatment and enzymatic hydrolysis conditions that optimized hemicellulose conversion (75.04 %), minimized the production of inhibitory compounds (1.41 g/L), and maximized the cellulose to glucose yield (69.69 %) of mixed particulate biomass (particles <1000 μm) under batch conditions. The effect of biomass particle size on saccharification process efficiency was also investigated. It was found that small-size biomass particles (53–106 μm) resulted in maximum hemicellulose conversion (81.12 %) and cellulose to glucose yield (93.24 %). The determined optimal conditions were then applied to a combined batch pretreatment process followed by a fed-batch enzymatic hydrolysis process that maximized glucose concentration (62.24 g/L) and yield (92.48 %). The overall efficiency of the saccharification process was 88.13 %.     

补料方式对酵母菌生产谷胱甘肽的影响

比较了酵母菌发酵生产谷胱甘肽（GSH）的几种补料分批培养方式。实验发现补料可以明显地促进酵母菌的生长和谷胱甘肽的合成,同时还发现不同的补料方式对发酵液中的菌体浓度和GSH浓度有不同的影响。采用指数流加方式可获得极高的菌体浓度,但菌体中的GSH浓度较低;而采用恒－pH补料分批培养既可以达到较高菌体浓度,菌体中又含有较高的GSH含量,因此,其总的GSH产量最高,可达到977.8mg/L。     

A robust fed-batch feeding strategy independent of the carbon source for optimal polyhydroxybutyrate production

A three-stage control strategy independent of the organic substrate was developed for automated substrate feeding in a two-phase fed-batch culture of Cupriavidus necator DSM 545 for the production of the biopolymer polyhydroxybutyrate (PHB). The optimal feeding strategy was determined using glucose as the substrate. A combined substrate feeding strategy consisting of exponential feeding and a novel method based on alkali-addition monitoring resulted in a maximal cell concentration in the biomass growth phase. In the PHB accumulation phase, a constant substrate feeding strategy based on the estimated amount of biomass produced in the first phase and a specific PHB accumulation rate was implemented to induce PHB under limiting nitrogen at different biomass concentrations. Maximal cell and PHB concentrations of 164 and 125 g/L were obtained when nitrogen feeding was stopped at 56 g/L of residual biomass; the glucose concentration was maintained within its optimal range. The developed feeding strategy was validated using waste glycerol as the sole carbon source for PHB production, and the three-stage control strategy resulted in a PHB concentration of 65.6 g/L and PHB content of 62.7% while keeping the glycerol concentration constant. It can thus be concluded that the developed feeding strategy is sensitive, robust, inexpensive, and applicable to fed-batch culture for PHB production independent of the carbon source.     

A fuzzy logic controller to control nutrient dosage in a fed-batch baker's yeast process

A fuzzy logic controller designed to control glucose feeding in a fed-batch baker's yeast process is presented. Feeding is carried out in portions and the controller determines the time at which glucose should be added and computes the size of the portion to provide the maximum glucose uptake rate. Moreover, the controller detects and prevents the occurrence of overdosage. The experimental results indicate that yield and specific growth rate obtained with the controller approached 55% and 0.13 h^–1, respectively.     

Effect of nitrate feeding strategies on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana

Controlled nitrate feeding strategies for fed-batch cultures of microalgae were applied for the enhancement of lipid production and microalgal growth rates. In particular, in this study, the effect of nitrate feeding rates on lipid and biomass productivities in fed-batch cultures of Nannochloropsis gaditana were investigated using three feeding modes (i.e., pulse, continuous, and staged) and under two light variations on both lipid productivity and fatty acid compositions. Higher nitrate levels negatively affected lipid production in the study. Increasing the light intensity increased the lipid contents of the microalgae in all three fed-batch feeding modes. A maximum of 58.3% lipid- to dry weight ratio was achieved when using pulse-fed cultures at an illumination of 200 μmol photons m⁻² s⁻¹ and 10 mg/day of nitrate feeding. This condition also resulted in the maximum lipid productivity of 44.6 mg L⁻¹ day⁻¹. The fatty acid compositions of the lipids consisted predominantly of long-chain fatty acids (C:16 and C:18) and accounted for 70% of the overall fatty acid methyl esters. Pulse feeding mode was found to significantly enhance the biomass and lipid production. The other two feeding modes (continuous and staged) were not ideal for lipid and biomass production. This study demonstrates the applicability of pulse feeding strategies in fed-batch cultures as an appropriate cultivation strategy that can increase both lipid accumulation and biomass production.     

High-cell-density fermentation of recombinant Saccharomyces cerevisiae using glycerol

To obtain a high cell density of recombinant Saccharomyces cerevisiae (INVSc 1 strain bearing a 2 microm plasmid, pYES2 containing a GAL1 promoter for expression of the beta-galactosidase gene), the yeast was grown with glycerol as the substrate by fed-batch fermentation. The feeding strategy was based on an on-line response of the medium pH to the consumption of glycerol. The approach was to feed excess carbon into the medium to create a benign environment for rapid biomass buildup. During cell growth in the presence of glycerol, the release of protons in the medium caused a decrease in pH and the consumption rate of ammonium phosphate served as an on-line indicator for the metabolic rate of the organism. The extent of glycerol feeding in a fed-batch mode with pH control at 5.0 +/- 0.1 was ascertained from the automatic addition of ammonium phosphate to the medium. The glycerol feeding to ammonium phosphate addition ratio was found to be 2.5-3.0. On the basis of the experiments, a maximum dry cell biomass of 140 g per liter and a productivity of 5.5 g DCW/L/h were achieved. The high cell density of S. cerevisiae obtained with good plasmid stability suggested a simple and efficient fermentation protocol for recombinant protein production.     

Indigestible dextrin stimulates glucoamylase production in submerged culture of <Emphasis Type="Italic">Aspergillus kawachii</Emphasis>

Submerged batch cultures of Aspergillus kawachii grown on indigestible dextrin were investigated for potential improvements in glucoamylase (GA) production. In flask culture, specific GA productivities per dry weight biomass using dextrin and indigestible dextrin were 11.0 and 56.1 mU/mg-DW, respectively. Indigestible dextrin was a poor substrate for enzymatic hydrolysis. Rates of glucose formation from dextrin and indigestible dextrin by enzymatic hydrolysis were 0.477 and 0.100 mg-glucose/ml/h, respectively. For this reason, residual glucose concentrations in batch cultures grown on indigestible dextrin remained below 1.32 mg/ml where glucose-limiting conditions were easily maintained. Batch culture using indigestible dextrin had the same residual glucose profile as dextrin fed-batch culture, and nearly the same GA activity was obtained after 42.5 h of growth. However, between 42.5 and 66 h, the GA production rate of the indigestible dextrin batch culture (11.5 mU/ml/h) was higher than that of the dextrin fed-batch culture (6.5 mU/ml/h). During this period, a high amount of residual maltooligosaccharide was detected in the culture supernatant grown on indigestible dextrin. The high GA productivity observed in the indigestible dextrin batch culture may have resulted from the absence of glucose and the simultaneous presence of maltooligosaccharides throughout growth.     

Lipid production in batch and fed-batch cultures of Rhodosporidium toruloides from 5 and 6 carbon carbohydrates

ABSTRACT: BACKGROUND: Microbial lipids are a potential source of bio- or renewable diesel and the red yeast Rhodosporidium toruloides is interesting not only because it can accumulate over 50% of its dry biomass as lipid, but also because it utilises both five and six carbon carbohydrates, which are present in plant biomass hydrolysates. METHODS: R. toruloides was grown in batch and fed-batch cultures in 0.5 l bioreactors at pH 4 in chemically defined, nitrogen restricted (C/N 40 to 100) media containing glucose, xylose, arabinose, or all three carbohydrates as carbon source. Lipid was extracted from the biomass using chloroform-methanol, measured gravimetrically and analysed by GC. RESULTS: R. toruloides was grown on glucose, xylose, arabinose or mixtures of these carbohydrates in batch and fed-batch, nitrogen restricted conditions. Lipid production was most efficient with glucose (up to 25 g lipid L1, 48 to 75% lipid in the biomass, at up to 0.21 g lipid L1h1) as the sole carbon source, but high lipid concentrations were also produced from xylose (36 to 45% lipid in biomass). Lipid production was low (15-19% lipid in biomass) with arabinose as sole carbon source and was lower than expected (30% lipid in biomass) when glucose, xylose and arabinose were provided simultaneously. The presence of arabinose and/or xylose in the medium increased the proportion of palmitic and linoleic acid and reduced the proportion of oleic acid in the fatty acids, compared to glucose-grown cells. High cell densities were obtained in both batch (37 g L1, with 49% lipid in the biomass) and fed-batch (35 to 47 g L1, with 50 to 75% lipid in the biomass) cultures. The highest proportion of lipid in the biomass was observed in cultures given nitrogen during the batch phase but none with the feed. However, carbohydrate consumption was incomplete when the feed did not contain nitrogen and the highest total lipid and best substrate consumption were observed in cultures which received a constant low nitrogen supply. CONCLUSIONS: Lipid production in R. toruloides was lower from arabinose and mixed carbohydrates than from glucose or xylose. Although high biomass and lipid production were achieved in both batch and fed-batch cultures with glucose as carbon source, for lipid production from mixtures of carbohydrates fed-batch cultivation was preferable. Constant feeding was better than intermittent feeding. The feeding strategy did not affect the relative proportion of different fatty acids in the lipid, but the presence of C5 sugars did.     

Design and simulation of a fuzzy controller for fed-batch yeast fermentation

In baker's yeast fermentation, the process is non-linear and the response of the system to changes in glucose feeding has a very long delay time. Therefore, a conventional system can not give satisfactory results. In this paper, a fuzzy controller designed to control a fed-batch fermenter is presented. The fuzzy controller uses Respiratory Quotient (RQ) as a controller input and produces glucose feeding rate as control variable. The controller has been tested on a simulated fed-batch fermenter. The results show that the maximum yeast production is possible by keeping the specific growth rate (μ) and the glucose concentration (C _s) at preset values (μ _Cand C _s,c) and minimizing the ethanol production.     

Growth and carotenoid production of Phaffia Rhodozyma in fed-batch cultures with different feeding methods

The growth and carotenoid production of Phaffia rhodozyma in fed-batch cultures with different feeding methods and grown at specific growth rates similar to the batch culture was compared. With constant feeding, exponential feeding, DO-stat and pH-stat fed-batch cultures of Phaffia rhodozyma, the highest biomass (17.4 g/l) and lowest carotenoid content (307 g/g cell) of Phaffia rhodozyma was from the DO-stat fed-batch culture. The lowest biomass (14.7 g/l) and highest carotenoid content (412 g/g cell) was from the exponential, fed-batch culture.     

Effect of feeding strategy on Zymomonas mobilis CP4 fed-batch fermentations and mathematical modeling of the system

In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process. Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000     

Development and validation of a kinetic model for enzymatic saccharification of lignocellulosic biomass

A multireaction kinetic model was developed for closed-system enzymatic hydrolysis of lignocellulosic biomass such as corn stover. Three hydrolysis reactions were modeled, two heterogeneous reactions for cellulose breakdown to cellobiose and glucose and one homogeneous reaction for hydrolyzing cellobiose to glucose. Cellulase adsorption onto pretreated lignocellulose was modeled via a Langmuir-type isotherm. The sugar products of cellulose hydrolysis, cellobiose and glucose, as well as xylose, the dominant sugar prevalent in most hemicellulose hydrolyzates, were assumed to competitively inhibit the enzymatic hydrolysis reactions. Model parameters were estimated from experimental data generated using dilute acid pretreated corn stover as the substrate. The model performed well in predicting cellulose hydrolysis trends at experimental conditions both inside and outside the design space used for parameter estimation and can be used for in silico process optimization.