Simulation and optimization of fed-batch culture for ethanol production from molasses

Two simulation methods for ethanol production from molasses by a flocculating yeast, Saccharomyces cerevisiae AM12, were investigated and molasses feeding was optimized. The first method was based on a deterministic model with fixed kinetic parameters and the second was based on regression analysis. The amount of ethanol produced in a fed-batch culture with multiple additions of molasses was simulated by both of these two methods. Simulated results of a fed-batch culture were compared with those of a simple batch culture by a model of regression analysis. The intermittent addition of molasses gave better production than a single addition at the beginning; more frequent addition may further improve production. The experimental results suggested the same. The effect of the amount of the added molasses on ethanol production was investigated by simulation. Repeated batch culture with and without intermittent addition of molasses in each batch was also done.List of Symbols C _e deviation of calculated results from experimental results - F m³ volume of feed medium added to the fermentor - P kg/m³ concentration of ethanol - P _M kg total amount of ethanol - S kg/m³ concentration of sugar - S ₀ kg/m³ concentration of sugar in the molasses feed medium - S _M kg total amount of sugar - V m³ culture volume - X kg/m³ concentration of cells - X _M kg total amount of cells - x _c calculated data - x _e experimental data - h^–1 specific rate of growth - kg-sugar/(kg-cell h) specific rate of sugar consumption - kg-ethanol/(kg-cell h) specific rate of ethanol production     

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.     

The effects of rearing density on growth,size heterogeneity and inter-individual variation of feed intake in monosex male Nile tilapia Oreochromis niloticus L.

The growth dispersion of farmed fish is a subject of increasing interest and one of the most important factors in stocking density. On a duration of 60 days, the effect of stocking density on the growth, coefficient of variation and inter-individual variation of feed intake (CV_FI) of juvenile Nile tilapia Oreochromis niloticus L. (14.9 ± 1.2 g) were studied in an experimental tank-based flow-through system. Groups of fish were stocked at four stocking densities: 200, 400, 600 and 800 fish/m³, corresponding to a density of ∼3, 6, 9 and 12 kg/m³ and referred to as D₁, D₂, D₃ and D₄, respectively. Each treatment was applied to triplicate groups in a completely randomized design. No treatment-related mortality was observed. The fish densities increased throughout the experiment from 3 to 23.5, 6 to 43.6, 9 to 56.6 and 12 to 69 kg/m³. Results show that mass gain and specific growth rate (SGR, %M/day) were negatively correlated with increased stocking density. Groups of the D₁ treatment reached a mean final body mass (FBM) of 119.3 g v. 88.9 g for the D₄ groups. Feed conversion ratios (FCRs) were 1.38, 1.54, 1.62 and 1.91 at D₁, D₂, D₃ and D₄ treatments, respectively. Growth heterogeneity, expressed by the inter-individual variations of fish mass (CV_M), was significantly affected by time (P < 0.001), stocking density (P < 0.001) and their interaction (P < 0.05). The difference in CV_M was particularly conspicuous towards the end of the experiment and was positively correlated with stocking density. Similarly, radiographic study shows that CV_FI was also found to be significantly greater for groups reared at high stocking densities (D₃ and D₄) than the other treatments (D₁ and D₂). These differences in both CV_M and CV_FI related to the stocking density need to be taken into account by husbandry practices to assure the production of more homogeneous fish size. A simple economic analysis indicates a parabolic relationship between profit and density with optimal final density at the peak of the curve. Given reasonable assumptions about production costs, the optimal final density (D_opt) is 73.7 kg/m³. A sensitivity analysis shows that changes in the fixed cost have no effects on the optimal final density. However, small change in variable costs, such as feed and juvenile costs, may have substantial effect on the optimal density.     

Experience in using an ethanol sensor to control molasses feed-rates in baker's yeast production

An ethanol sensor has been tested for feed-rate control of baker's yeast prouction. The yeast was grown on molasses in an 8 dm³ fed-batch reactor up to a cell concentration of 60–70 kg/m³. Studies were made on three levels: reliability of the sensor system, characterisation of the control problem, and evaluation of ethanol-controlled cultivations in terms of yield and production rate. Arguments are given for the conceptual advantages of ethanol control compared to other methods of substrate control. It is also shown that ethanol control allows for a simple regulator structure. In fact, a PID regulator, with constant parameters, was used around an exponential dosage scheme. Tuning of the regulator parameters was performed by using simulation on a simplified model of the process. Several cultivations have been carried out. Results from four comparable cultivations are given in detail, and the experience from many others is summarized.     

Startup of anaerobic biofilm fluidized bed reactors on molasses and phenol under various conditions

Fluidized sand bed anaerobic biofilm reactors were operated in parallel to study the effects of inoculum, loading, residence time and carrier type on the startup dynamics for the degradation of molasses and phenol. Degradation rates generally depended most directly on concentrations rather than on other operating variables. Residence times did not appear to directly influence startup. Short residence times and high loadings gave the highest specific activities for both substrates. The type of inoculum was found to be most important for the molasses system, and inoculation on fresh carrier was found to be better than reinoculation. The two times higher specific biomass retention on Siran porous glass gave essentially the same degradation rates on a volume basis.List of Symbols L kg/h loading of reactor - M kg/kg biomass per carrier mass - Red. % reduction of feed concentration due to degradation - R kg/(m³ · h) reaction rate - S kg/m³ substrate concentration in reactor and effluent - S ₀ kg/m³ substrate concentration in feed - t h time     

An adaptive on-line control feed rate system in a laboratory scale bakers yeast process

Summary A new control policy for the on-line optimization of the nutrient supply in bakers yeast process is proposed. A feed rate corresponding to minimal substrate uptake time was shown to be optimal for cell yield and specific growth rate. Cultivation results of baker's yeast are presented.Nomenclature c glucose concentration in wort (mol.l^–1) - C total glucose used (mol) - c_e ethanol concentration in wort (mg.l^–1) - c_p glucose concentration in fresh medium (mol.l^–1) - dt/dc glucose consumption time (sec.mol^–1) - F substrate feed rate (litre.hr^–1) - q_c glucose uptake rate (mol.hr^–1) - Q_c specific glucose uptake rate (moll.g^–1.hr^–1) - qO₂ oxygen uptake rate (mol.hr^–1) - QO₂ specific oxygen uptake rate (mol.g^–1.hr^–1) - r_x productivity (g.l^–1.hr^–1) - t time (hr) - x biomass concentration (g.l^–1) - X total biomass (g) - Y_x/c cell yield (g.g^–1): (g.mol^–1) - Y_o/c consumed oxygen to glucose ratio (mol.mol^–1)     

Construction of a Stable α-Galactosidase-Producing Baker's Yeast Strain

Molasses is widely used as a substrate for commercial yeast production. The complete hydrolysis of raffinose, which is present in beet molasses, by Saccharomyces strains requires the secretion of α-galactosidase, in addition to the secretion of invertase. Raffinose is not completely utilized by commercially available yeast strains used for baking, which are Mel⁻. In this study we integrated the yeast MEL1 gene, which codes for α-galactosidase, into a commercial mel⁰ baker's yeast strain. The Mel⁺ phenotype of the new strain was stable. The MEL1 gene was expressed when the new Mel⁺ baker's yeast was grown in molasses medium under conditions similar to those used for baker's yeast production at commercial factories. The α-galactosidase produced by this novel baker's yeast strain hydrolyzed all the melibiose that normally accumulates in the growth medium. As a consequence, additional carbohydrate was available to the yeasts for growth. The new strain also produced considerably more α-galactosidase than did a wild-type Mel⁺ strain and may prove useful for commercial production of α-galactosidase.     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

Anaerobic treatment of baker's yeast effluent using a hybrid digester with polyurethane as support material

Summary A high-strength baker's yeast effluent was anaerobically treated using a hybrid digester under mesophilic conditions. The digester was subjected to a substrate COD concentration of 21 767 mg/I at three different HRTs. At HRTs of 3.0, 2.0 and 1.0 d, the digester reduced the substrate COD by 76, 61 and 33%, respectively. Although the best COD removal was obtained at an OLR of 7.30 kg COD/m³.d, the highest COD removal rate (6.51 kg COD/M³-d) was found at 10.65 kg COD/m³.d at an HRT of 2.0 d. The low methane yield and VFA accumulation found in the digester effluent, indicated inhibition on methanogenic level and this was considered to be the rate-limiting step during the anaerobic treatment process. The overall efficiency of the digester indicated that this digester design and support medium was suitable for the treatment of a high-strength, sulfate-rich baker's yeast effluent.     

Influence of exponentially decreasing feeding rates on fed-batch ethanol fermentation of sugar cane blackstrap molasses

Summary The ethanol yield was not affected and the ethanol productivity was increased when exponentially decreasing feeding rates were used instead of constant feeding rates in fed batch ethanol fermentations. The influences of the initial sugar feeding rate on the ethanol productivity, on the constant ethanol production rate during the feeding phase and on the initial ethanol production specific rate are represented by Monod-like equations.Nomenclature F reactor feeding rate (L.h^–1) - F_o initial reactor feeding rate (L.h^–1) - K time constant; see equation (l) (h^–1) - M_E mass of ethanol in the fermentor (g) - M_s mass of TRS in the fermentor (g) - M_x mass of yeast cells (dry matter) in the fermentor (g) - P ethanol productivity (g.L^–1.h^–1) - R ethanol constant production rate during the feeding phase (g.h^–1) - s standard deviation - S_o TRS concentration in the feeding mash (g.L^–1) - t time (h) - T fermentor filling-up-time (h) - T time necessary to complete the fermentation (h) - TRS total reducing sugars calculated as glucose (g.L^–1) - V_o volume of the inoculum (L) - V_f final volume of medium in the fermentor (L) - X_o yeast concentration of the inoculum (dry matter) (g.L^–1) - ethanol yield (% of the theoretical value) - initial specific rate of ethanol production (h^–1)     

Yeast strains for concentrated substrates

Summary The screening of twenty yeast strains for ethanol productivity at high osmotic pressure at temperatures ranging from 32°C to 45°C is described. Shake flask fermentations of 30°, 40°, and 50° Bx cane molasses were performed. The effect of temperature on productivity at a non-inhibitory ethanol level is weakly pronounced. Most strains fermented poorly at 50° Bx molasses but two Schizosaccharomyces pombe and one commercial baker's yeast, Saccharomyces cerevisiae performed well at all concentrations of molasses. In an extended study with Schizosaccharomyces pombe (CBS 352) and Saccharomyces cerevisiae (SJAB, fresh yeast), simulating a continuous run it was shown that Schizosaccharomyces pombe was less sensitive to high DS than Saccharomyces cerevisiae. At 25% DS the productivity of Schizosaccharomyces pombe is almost twice that of Saccharomyces cerevisiae.     

Distribution and diffusion of alamethicin in a lecithin/water model membrane system

Summary Attenuated total reflection infrared spectroscopy has been used to determine the equilibrium distribution of the peptide antibiotic alamethicinR _F30 between dipalmitoyl phosphatidylcholine bilayers and the aqueous environment. The distribution coefficientK=c _eq ^W /c _eq ^M turned out to be concentration dependent, pointing to alamethicin association in the membrane with increasing concentration in the aqueous phase (c _eq ^W ). This concentration was varied within 28 and 310nm, i.e., in a range typical for black film experiments. Furthermore, diffusion coefficients of alamethicin in the hydrophobic phase of the membrane (D _M) and across the membrane/water interface (D _I) have been estimated from the time course of the equilibration process. It was found that the diffusion rate of the uncharged analogueR _F50 is about 10 times higher than that of theR _F30 component, exhibiting one negative charge at theC-terminus. The time constants for transmembrane diffusion of alamethicinR _F30 varied between 2.2 hr at low concentration and 3.2 hr at higher concentration. The corresponding low concentration value of theR _F50 component was found to be 0.25 hr.     

Continuous production of citric acid from sugarcane molasses using a combination of submerged immobilized and surface stabilized cultures of Aspergillus niger KCU 520

Summary A high performance fermentation process for the continuous production of citric acid from sugarcane molasses by using the combination of submerged calcium alginate-immobilized and surface-stabilized cultures of Aspergillus niger KCU 520 in a continuous flow horizontal bioreactor is described. The citric acid productivity was dependent on the dilution rate with an optimum value of 0.015/h. Presaturation of fermentation medium with sterile air, in addition to surface aeration, before feeding into the bioreactor enhanced the citric acid productivity. The highest productivity, citric acid product concentration and yield obtained were 1.7 kg M^–3h^–1, 110kg M^–3 and 91% respectively. The cultures were continuously used for 30 days without any apparent loss in citric acid productivity.     

Optimal control of batch processes involving simultaneous enzymatic and microbial reactions

Optimal enzyme feed rate profiles have been calculated, based on a model for a fed-batch simultaneous enzymatic and microbial reaction (SEMR) process. The model parameters corresponded to a relatively slow citric acid fermentation. The profiles were calculated using an iterative algorithm based on the minimum principle. Penalty functions were used to enforce inequality constraints on the enzyme feed rate. Significant improvements in the objective function relative to that for the best constant enzyme feed rate were found. The effect on the optimal profiles of changes in the parameters of the model and the objective function were investigated, as was the effect of introducing the stationary state assumption to eliminate glucose concentration as a state variable. Major differences between bang-bang control variable profiles and singular arcs were found, with the singular arc solution slightly better than the optimal bang-bang control.List of Symbols a N-vector of initial conditions - b ₁–b₁₀ parameters defined in Table 2 - c vector of cost parameters - c ₁–c₆ penalty function parameters - E enzyme concentration (U/l) - f N-vector of functions - F enzyme feed rate (U/l-h) - g N-vector of functions - G glucose concentration (g/l) - H Hamiltonian - J objective function - J ^* modified objective function - L number of integration steps per time interval - L number of control variables - M number of time intervals - n iteration index - N number of state variables - P product concentration (g/l) - r ₁ glucose formation rate (g/l-h) - r ₂ product formation rate (g/l-h) - t time (h) - T final time (h) - u L-vector of control variables - x N-vector of state variables - z N-vector of adjoint variables - Z total enzyme fed (U/l) Greek convergence parameter The support of one of the authors by the National Science Foundation (Grant CBT-84-20552) is gratefully acknowledged.     

Theoretical analysis of the baker's yeast production: An experimental verification at a laboratory scale

The scale-down procedure can be used to optimize and scale up fermentation processes. The first step in this procedure, a theoretical analysis of the process at a large scale, must give information about the regime, or bottle necks, ruling the process. In order to verify the theoretical results the process analysis has been applied to the fed-batch baker's yeast production at a laboratory scale. The results of this analysis are compared with results from fed-batch experiments. It was concluded that if only one mechanism is ruling the process, for instance mass transfer, the results of the analysis are quite clear. If more than one mechanism is important, for example mass transfer and liquid mixing, additional knowledge is needed to predict the behaviour of the process.Concerning the baker's yeast production, it was concluded that if oxygen limitation occurs, liquid mixing is of little importance.List of Symbols C kg/m³ concentration - C ^* kg/m³ saturation concentration - D m diameter - D _E m²/s effective dispersion coefficient - d m holes of the sparger - F _sm³/s substrate flow to the fermentor - g m/s² gravitational acceleration - H m height - k _La s^–1 volumetric mass transfer coefficient based on the liquid volume - L m length - m _skg/(kg·s) maintenance coefficient - OTR kg/(m³·s) oxygen transfer rate - OUR kg/(m³·s) oxygen uptake rate - r kg/(m³·s) reaction rate - t s time - V m³ volume - v m/s velocity - v _sm/s superficial gas flow rate - y _ijkg/kg yield of componentj oni - s^–1 specific growth rate - s time constant - _gm³/s gas flow rate Indices 0 value att=0 - cir liquid circulation - e ethanol - f feed concentration - g gas phase - in flow going to the fermentor - l liquid phase - m mixing - mt mass transfer - o, O₂ oxygen - oc oxygen consumption - out flow coming out the fermentor - s substrate - sa substrate addition - sc substrate consumption - x biomass     

Candida krusei in Baker's yeast production

Summary Candida krusei is a harmful contaminant in baker's yeast manufacture, because it grows much faster than Saccharomyces cerevisiae under production conditions. This investigation showed that C. krusei utilizes the ethanol produced by baker's yeast as sole carbon source when molasses is used as a substrate. When the alcohol concentration in the effluent air is used as a parameter for controlling the aeration of the culture, conditions become favourable for the dominance of wild yeast because some of the ethanol produced by the baker's yeast is consumed immediately by C. krusei and aeration is then automatically reduced, leading to increased growth of the wild yeast.     

Effects of pulse feeding of beet molasses on recombinant benzaldehyde lyase production by Escherichia coli BL21(DE3)

The effect of fed-batch operation (FBO) strategy was investigated using pretreated-beet molasses, containing galactose that induces the lac promoter, on benzaldehyde lyase (BAL) production by recombinant Escherichia coli BL21(DE3)pLySs. After batch cultivation with 30 g l^?1 pretreated-beet molasses consisting of 7.5 g l^?1 glucose and 7.5 g l^?1 fructose, three FBO strategies were applied at dissolved oxygen (=40%) cascade to air-flow rate. In FBO1 when air-flow rate decreased considerably, feed was given to the system in pulses in such a way that pretreated-beet molasses concentration increased by 10 kg m^?3 (containing 2.5 g l^?1 glucose+2.5 g l^?1 fructose); however, decrease in air-flow rate demonstrated only the absence of glucose but not fructose. Thus, in FBO2 when fructose and glucose were completely utilized, pretreated-beet molasses was pulse-fed and its concentration increased by 10 g l^?1. In FBO3 with the decreased amount of pretreated-beet molasses (6 g l^?1), shift response time from glucose to fructose consumption was avoided, and glucose and fructose consumptions were well correlated with air-flow rate, and the highest C _X (8.04 g l^?1) and BAL (2,315 U ml^?1) production were obtained (t?=?24 h) with the highest substrate yield on cell and product formation.     

Growth and physiology of a yeast cultivated in batch and continuous culture systems

Inoculum size has been found to affect significantly the maximum attainable specific growth rate during batch cultivation ofCandida utilis. Lower inoculum size resulted in an increased growth rate and relatively longer lag. The culture is found to be most active in the beginning of the exponential phase as regards its RNA synthesis rate. Batch data were used for predicting the conditions of the yeast population in single-stage continuous culture system. Predicted and the experimental values showed a reasonable agreement. In single-stage chemostat the physiology of the yeast was studied on the basis RNA, DNA and protein synthesis rates at various growth rates. The results indicate that the productivity of cells and the rate of synthesis of macromolecules is highest at the dilution rate values of 0.33 to 0.35 hr⁻¹. In order to attain so-called unrestricted conditions of growth a pluristage pluristream continuous system was employed. It is assumed that under such conditions the specific growth rate and the synthetic activity of yeasts may reach its maximum on a given medium. The results presented do not show such conditions of growth under the experimental conditions employed (D ₁=0.35 hr⁻¹ andD ₂=0.2 to 1.7 hr⁻¹) withCandida utilis cultivated on beet molasses medium. Second stage of a two-stage two-stream continuous system is constantly fed with the cells from the foregoing stage; this category of cells on entering the new conditions of the second stage is expected to show some adaptation period. Experiments are reported to this effect.     

Condition cycles in juvenile Pagrus auratus

Nutritional condition was measured in juvenile snapper Pagrus auratus (<200 mm fork length) using three indices: relative condition index (I_c), hepatosomatic index (I_H) and digesto-somatic index (I_D). In a laboratory starvation experiment, all three indices declined substantially over a 24-day period, but I_H was most sensitive. In wild snapper I_c and I_H showed no diel cycles. I_D for 0+ snapper showed a strong diel cycle consistent with continuous feeding during daylight hours and lack of feeding during the night. I_D for 1+ snapper showed no diel cycle. Subsequent analyses were restricted to daytime samples for I_H and morning samples for I_D to minimize the confounding effect of time of day. I_c, I_H and I_D were monitored at one site at approximately bi-monthly intervals over a period of 3.25 years. All three indices varied significantly, but only I_D and I_H displayed seasonal cycles. I_D peaked in late summer-autumn and dropped to a minimum in winter, due to seasonal fluctuations in the feeding rate that probably reflected variations in metabolic and growth rates. I_H peaked in autumn-winter and declined to a minimum in summer, thus lagging 4–6 months behind I_D. I_H varied significantly among four sampling sites for all five combinations of sampling periods and year classes, whereas I_c varied significantly among sites for only one of the five combinations. The Kawau Bay site, which supported the highest density of snapper, had the highest I_H for all except one of the combinations. This suggests that juvenile snapper aggregate selectively at sites that provide optimal feeding conditions. However, no relationship was found between I_H and growth rate, indicating that better nutritional condition may not translate into faster growth.     

Cultivation of baker's yeast by a fermenter with a basket-shaped unit for agitation

A simple and new basket-shaped unit for agitation made of stainless wire was developed. A fermenter equipped with this unit attained higher k_La, than 3500 h^–1 under the condition with an aeration rate 2vvm and a rotation speed of the unit 1100rpm. In the cultivation of baker's yeast, the cell concentration reached about 110g per liter on dry weight basis in 17.5h.