Effects of mixing on methane production during thermophilic anaerobic digestion of manure: lab-scale and pilot-scale studies

The effect of mixing on anaerobic digestion of manure was evaluated in lab-scale and pilot-scale experiments at 55 degrees C. The effect of continuous (control), minimal (mixing for 10 min prior to extraction/feeding) and intermittent mixing (withholding mixing for 2h prior to extraction/feeding) on methane production was investigated in three lab-scale continuously stirred tank reactors. On comparison to continuous mixing, intermittent and minimal mixing strategies improved methane productions by 1.3% and 12.5%, respectively. Pilot-scale studies also supported the lab-scale results with an average 7% increase in biogas yields during intermittent mixing compared to continuous mixing. The effect of mixing intensities (minimal, gentle or vigorous) in batch assays at 55 degrees C showed that when the process was overloaded by high substrate to inoculum ratio (40/60), gentle (35 times per minute) or minimal mixing (10 min mixing before feeding) was advantageous compared to vigorous mixing (110 times per minute). On the other hand, under low substrate to inoculum ratio (10/90), gentle mixing was the best. The study thus indicated that mixing schemes and intensities have some effect on anaerobic digestion of manures.     

Lipase of <Emphasis Type="Italic">Aspergillus niger</Emphasis> NCIM 1207: A Potential Biocatalyst for Synthesis of Isoamyl Acetate

Commercial lipase preparations and mycelium bound lipase from Aspergillus niger NCIM 1207 were used for esterification of acetic acid with isoamyl alcohol to obtain isoamyl acetate. The esterification reaction was carried out at 30°C in n-hexane with shaking at 120 rpm. Initial reaction rates, conversion efficiency and isoamyl acetate concentration obtained using Novozyme 435 were the highest. Mycelium bound lipase of A. niger NCIM 1207 produced maximal isoamyl acetate formation at an alcohol/acid ratio of 1.6. Acetic acid at higher concentrations than required for the critical alcohol/acid ratio lower than 1.3 and higher than 1.6 resulted in decreased yields of isoamyl acetate probably owing to lowering of micro-aqueous environmental pH around the enzyme leading to inhibition of enzyme activity. Mycelium bound A. niger lipase produced 80 g/l of isoamyl acetate within 96 h even though extremely less amount of enzyme activity was used for esterification. The presence of sodium sulphate during esterification reaction at higher substrate concentration resulted in increased conversion efficiency when we used mycelium bound enzyme preparations of A. niger NCIM 1207. This could be due to removal of excess water released during esterification reaction by sodium sulphate. High ester concentration (286.5 g/l) and conversion (73.5%) were obtained within 24 h using Novozyme 435 under these conditions.     

Kinetic modelling of continuous submerged fermentation of cheese whey for single cell protein production

A mathematical model describing the kinetics of continuous production of single cell protein from cheese whey using Kluyveromyces fragilis was developed from the basic principles of mass balance. The model takes into account the substrate utilization for growth and maintenance and the effect of substrate concentration and cell death rate on the net cell growth and substrate utilization during the fermentation process. A lactose concentration below 1.91 g/L limited growth of yeast cells whereas a lactose concentration above 75 g/L inhibited the growth of the yeast. The model was tested using experimental data obtained from a continuous system operated at various retention times (12, 18 and 24 h), mixing speeds (200, 400 and 600 rpm) and air flow rates (1 and 3 vvm). The model was capable of predicting the effluent cell and substrate concentrations with R2 ranging from 0.95 to 0.99. The viable cell mass and lactose consumption ranged from 1.3 to 34.3 g/L and from 74.31% to 99.02%, respectively. A cell yield of 0.74 g cell/g lactose (close to the stoichiometric value of 0.79 g cell/g lactose) was achieved at the 12 h retention time-3 vvm air flow rate-600 rpm mixing speed combination. The total biomass output (viable and dead cells) at this combination was 37 g/L.     

Formation of xylitol in Candida guilliermondii 2581 culture

The yeast strain Candida guilliermondii 2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Comparative study of aflatoxins M1 and B1 production in solid-state and shaking liquid cultures

Production of aflatoxins M1 (AFM) and B1 (AFB) by Aspergillus flavus NRRL 3251 in solid-state and shaking liquid cultures using rice as the carbon source was compared. In general, solid-state cultures produced more aflatoxins than shaking liquid cultures on an equal rice weight basis. Solid-state cultures with continuous shaking yielded higher levels of toxins than those with intermittent shaking. However, intermittent shaking is a feasible replacement for the continuous shaking method for AFM production. A typical solid rice culture supplemented with yeast extract produced 30 and 2600 mg per kg rice of AFM and AFB, respectively, in 8 days at 29 degrees C. The optimal culture conditions for toxin production in a shaking liquid culture were also studied. Parameters under consideration included the amount of carbon (rice) and nitrogen source, initial medium pH, and aeration rate. At optimum conditions, a representative shaking liquid culture produced 18 and 1680 mg per kg rice of AFM and AFB, respectively, in 5 days at 29 degrees C. This shaking liquid culture appears feasible for scaling up and routine production of AFM and AFB for toxicological investigations.     

Xylitol Production by a Culture ofCandida guilliermondii2581

The yeast strain Candida guilliermondii2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.     

Maximizing production of Penicillium cyclopium partial acylglycerol lipase

Penicillium cyclopium partial acylglycerol lipase production was maximized in shaken batch culture. The effect of inoculum size and substrate concentration on the lipase activity released in the culture medium was visualized using a surface response methodology based on a Doehlert experimental design. The main advantage of this approach is the low number of experiments required to construct a predictive model of the experimental domain. Substrate percentage (corn steep, w/v) ranged from 0.1% to 1.9% and inoculum from 100 spores/ml to 3,200 spores/ml. We determined that an optimal set of experimental conditions for high lipase production was 1.0% substrate and 3,200 spores/ml, with initial pH 5.0, temperature 25 degrees C and shaking speed 120 rpm. Between the conditions giving the minimum and the maximum lipase production, we observed a three-fold increase in both the predicted and the measured values.     

Interaction of cultural conditions and end-product distribution in Bacillus subtilis grown in shake flasks

Summary A culture of Bacillus subtilis, in which the relative production of acetoin (Ac) and butanediol (Bu) is highly sensitive to oxygen tension as well as to mixing conditions, was used to evaluate several culture conditions in 500-ml shake flasks. The concentration ratio of these metabolites (Ac/Bu) produced in a defined period of culture time was used as a parameter for comparative purposes. The influence of working volume, shaking speed, broth viscosity and the presence of baffles were evaluated. Using unbaffled flasks it was found that working volume had the most influence on oxygenation in shake flasks, especially below 10%, where differences in Ac/Bu ratios up to ten times could be measured. Shaking speed played an important role only at values higher than 400 rpm or when small working volumes were used. The addition of xanthan gum decreased the Ac/Bu ratio nearly four times under equivalent working conditions and also diminished the influence of shaking speed. In general, Ac/Bu was higher when sulphite oxygen transfer rate (OTR) values were higher. However, the test culture was able to detect differences which were not evident using the OTR method. Comparing Ac/Bu ratios in stirred fermentors from the literature, it seems that similar oxygenation conditions can be reached in non-baffled shake flasks only at very high shaking speeds using small working volumes. With baffled flasks, our data suggest that better oxygenation and mixing can be achieved in shake flasks if compared with those obtained in stirred fermentors at conventional power inputs.     

Efficient enantioselective hydrolysis of D,L-phenylglycine methyl ester catalyzed by immobilized Candida antarctica lipase B in ionic liquid containing systems

Immobilized Candida antarctica lipase B (Novozym 435)-catalyzed enantioselective hydrolysis of D,L-phenylglycine methyl ester to enatiopure D-phenylglycine was successfully conducted in the systems with ionic liquids (ILs). Novozym 435 exhibited excellent activity and enantioselectivity in the system containing the IL BMIMxBF(4) compared to several typical organic solvents tested. It has been found that the cations and, particularly, the anions of ILs have a significant effect on the reaction, and the IL BMIMxBF(4), which shows to be the most suitable for the reaction, gave the highest initial rate and enantioselectivity among various ILs examined. The reaction became much less active and enantioselective in the systems with BMIMxHSO(4). Also, it was noticed that the enzymatic hydrolysis was strongly dependent on BMIMxBF(4) content in the co-solvent systems and the favorable content of the IL was 20% (v/v). Of the assayed four co-solvents and phosphate buffer, the lowest apparent K(m) and activation energy, and the highest V(max) of the reaction were achieved using 20% (v/v) BMIMxBF(4) co-solvent with phosphate buffer. Additionally, various influential variables were investigated. The optimum pH, substrate concentration, reaction temperature and shaking rate were 8.0, 80mM, 25-30 degrees Celsius and 150rpm, respectively, under which the initial rate, the residual substrate e.e. and the enantioselectivity were 2.46mM/min, 93.8% (at substrate conversion of 53.0%) and 38, respectively. When the hydrolysis was performed under reduced pressure, the initial rate (2.64mM/min) and the enantioselectivity (E=43) were boosted.     

Protozoan biomass relation to nutrient and chemical oxygen demand removal in activated sludge mixed liquor

The relationship between biomass concentration to nutrient and chemical oxygen demand (COD) removal in mixed liquor supplemented with sodium acetate was investigated, using three protozoan isolates and three different initial biomass concentrations (10(1), 10(2) and 10(3) cells/mL). The study was carried out in a shaking flask environment at a shaking speed of 100 rpm for 96 h at 25 degrees C. Aliquot samples were taken periodically for the determination of phosphate, nitrate, COD and dissolved oxygen, using standard methods. The results revealed remarkable phosphate removal of 82-95% at biomass concentration of 10(3)cells/mL. A high nitrate removal of over 87% was observed at all initial biomass concentration in mixed liquor. There was an observed COD increase of over 50% in mixed liquor in at the end of 96-h incubation and this was irrespective of initial biomass concentration used for inoculation. The study shows the trend in nutrient and COD removal at different biomass concentrations of the test isolates in mixed liquor.     

Evaluation of succinic acid continuous and repeat-batch biofilm fermentation by <Emphasis Type="Italic">Actinobacillus succinogenes</Emphasis> using plastic composite support bioreactors

Continuous and repeat-batch biofilm fermentations using Actinobacillus succinogenes were performed with immobilized and suspended-cell systems. For the immobilized continuous system, plastic composite supports (PCS) containing 50% (w/w) polypropylene (PP), 35% (w/w) ground soybean hulls, 5% (w/w) dried bovine albumin, 2.5% (w/w) soybean flour, 2.5% (w/w) yeast extract, 2.5% (w/w) dried red blood cells, and 2.5% (w/w) peptone, or PP tubes (8.5 cm in length) were arranged around the agitator shaft in a grid formation. Agitation was controlled at 125 rpm and 150 rpm. Samples were taken at dilution rates of 0.2, 0.4, 0.6, 0.8, 1.0, and 1.2 h^–1 and analyzed for succinic acid production and glucose consumption (g l^–1). For PCS bioreactors, the highest final succinic acid concentrations (10.1 g ^–1, 10.4 g l^–1) and percentage yields (62.6%, 71.6%) occurred at the dilution rate of 0.2 h^–1. PCS disks were evaluated in a repeat-batch biofilm reactor. Suspended-cell batch fermentations were performed in flasks and a repeat-batch bioreactor. The maximum concentration of succinic acid produced was 40 g l^–1. Peak succinic acid percentage yields in continuous and repeat-batch fermentations of A. succinogenes were observed in suspended-cell continuous fermentations at a dilution rate of 1.0 h^–1 (76.2%) and in PCS repeat-batch fermentations with an initial glucose concentration of 40 g l^–1 (86.7%).     

无载体固定化酵母细胞木薯淀粉质原料酒精连续发酵研究

以木薯粉糖化液为发酵底物,在总发酵体积(有效)为15L的悬浮床生物反应器中,对一株粟酒裂殖酵母变异株进行一级和二级连续发酵研究。结果表明,二级连续发酵系统可明显改善一级系统的不足,并取得了平均流加糖液浓度150g/L,发酵强度为97g/L.h,流出液酒精浓度727g/L,残糖浓度374g./L,总糖利用率达90%的较好结果;整个系统在连续一个月的运行中从未发现染菌现象,发酵操作稳定。     

Candida shehatae and Saccharomyces cerevisiae work synergistically to improve ethanol fermentation from sugarcane bagasse and rice straw hydrolysate in immobilized cell bioreactor

Sugarcane bagasse (SCB) and rice straw (RS), abundant lignocellulosic agro‐industrial residues in South‐East Asia, are potent feedstocks for bioethanol production as they contain significant amount of glucose and xylose monomers after fractionation and subsequent enzymatic hydrolysis. To simultaneously convert glucose and xylose to ethanol, it requires co‐cultivation of Saccharomyces cerevisiae and Candida shehatae which are hexose and pentose‐fermenting yeasts, respectively. Xylose‐fermenting strain grows slower than glucose‐fermenting one, therefore low efficiency of xylose‐to‐ethanol conversion was found. To enhance the efficiency of ethanol fermentation, the present work proposed to improve xylose assimilation by using co‐immobilization of two strains in a packed bed bioreactor and to increase oxygenation of the medium by applying a recycled batch system when the recycle stream was intervened by a mixing system in a naturally aerated vessel. Initially, conversion of glucose and xylose to ethanol using pure culture was investigated. Subsequently, influence of different immobilization techniques was investigated. Cells entrapment in Ca‐alginate beads provided considerably high ethanol yield over cells immobilized on delignified cellulose, and thus it was selected to use as inoculum in an immobilized cell bioreactor (ICB). The results showed that continuous ethanol production yielded 0.38 and 0.40 g/g corresponding to 74.5% and 78.4% theoretical yields from SCB and RS hydrolysate, respectively. However, recycled batch system produced significantly improved ethanol yield to 0.49 g/g and 0.50 g/g corresponding to 96.1% and 98.0% theoretical yields for SCB and RS hydrolysate, respectively. In this study, higher ethanol concentration and less unfermented sugar concentration was successfully achieved in the ICB with recycled batch system when using SCB and RS hydrolysate as the substrate.     

Development of a helical-ribbon impeller bioreactor for high-density plant cell suspension culture

A double helical-ribbon impeller (HRI) bioreactor with a 11-L working volume was developed to grow high-density Catharanthus roseus cell suspensions. The rheological behavior of this suspension was found to be shear-thinning for concentrations higher than 12 to 15 g DW . L(-1). A granulated agar suspension of similar rheological properties was used as a model fluid for these suspensions. Mixing studies revealed that surface baffling and bottom profiling of the bioreactor and impeller speeds of 60 to 150 rpm ensured uniform mixing of suspensions. The HRI power requirement was found to increase singnificantly for agar suspensions higher than 13 g DW . L(-1), in conjunction with the effective viscosity increase. Oxygen transfer studies showed high apparent surface oxygen transfer coefficients (k(L)a approximately 4 to 45 h(-1)) from agar suspensions of 30 g DW . L(-1) to water and for mixing speeds ranging from 120 to 150 rpm. These high surface k(I)a values were ascribed to the flow pattern of this bioreactor configuration combined with surface bubble generation and entrainment in the liquid phase caused by the presence of the surface baffles. High-density C. roseus cell suspension cultures were successfully grown in this bioreactor without gas sparging. Up to 70% oxygen enrichment of the head space was required to ensure sufficient oxygen supply to the cultures so that dissolved oxygen concentration would remain above the critical level (>/=10% air saturation). The best mixing speed was 120 rpm. These cultures grew at the same rate ( approximately 0.4 d(-1)) and attained the same high biomass concentrations ( approximately 25 to 27 g DW . L(-1), 450 to 500 g filtered wet biomass . L(-1), and 92% to 100% settled wet biomass volume) as shake flask cultures. The scale-up potential of this bioreactor configuration is discussed.     

Isomaltulose production using free cells: optimisation of a culture medium containing agricultural wastes and conversion in repeated-batch processes

The enzyme glucosyltransferase is an industrially important enzyme since it produces non-cariogenic isomaltulose (6-O-alpha-D-glucopyronosyl-1-6-D-fructofuranose) from sucrose by intramolecular transglucosylation. The experimental designs and response surface methodology (RSM) were applied for the optimisation of the nutrient concentrations in the culture medium for the production of glucosyltransferase by Erwinia sp. D12 in shaken flasks at 200 rpm and 30 degrees C. A statistical analysis of the results showed that, in the range studied, the factors had a significant effect (P < 0.05) on glucosyltransferase production and the highest enzyme activity (10.84 U/ml) was observed in culture medium containing sugar cane molasses (150 g l(-1)), corn steep liquor (20 g l(-1)), yeast extract Prodex Lac SD (15 g l(-1)) and K2HPO4 (0.5 g l(-1)) after 8 h at 30 degrees C. The production of cell biomass by the strain of Erwinia sp. D12 was carried out in a 6.6-l fermenter with a mixing rate of 200 rpm and an aeration rate of 1 vvm. Fermentation time, cellular growth, medium pH and glucosyltransferase production were observed. The greatest glucosyltransferase activity was 22.49 U/ml, obtained after 8 h of fermentation. The isomaltulose production from sucrose was performed using free Erwinia sp. D12 cells in a batch process using an orbital shaker. The influence of the parameters sucrose concentration, temperature, pH, and cell concentration on the conversion of sucrose into isomaltulose was studied. The free cells showed a high conversion rate of sucrose into isomaltulose using batch fermentation, obtaining an isomaltulose yield of 72.11% from sucrose solution 35% at 35 degrees C.     

High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.     

Selective suppression of bacterial contaminants by process conditions during lignocellulose based yeast fermentations

Background

When scaling up lignocellulose-based ethanol production, the desire to increase the final ethanol titer after fermentation can introduce problems. A high concentration of water-insoluble solids (WIS) is needed in the enzymatic hydrolysis step, resulting in increased viscosity, which can cause mass and heat transfer problems because of poor mixing of the material. In the present study, the effects of mixing on the enzymatic hydrolysis of steam-pretreated spruce were investigated using a stirred tank reactor operated with different impeller speeds and enzyme loadings. In addition, the results were related to the power input needed to operate the impeller at different speeds, taking into account the changes in rheology throughout the process.

Results

A marked difference in hydrolysis rate at different impeller speeds was found. For example, the conversion was twice as high after 48 hours at 500 rpm compared with 25 rpm. This difference remained throughout the 96 hours of hydrolysis. Substantial amounts of energy were required to achieve only minor increases in conversion during the later stages of the process.

Conclusions

Impeller speed strongly affected both the hydrolysis rate of the pretreated spruce and needed power input. Similar conversions could be obtained at different energy input by altering the mixing (that is, energy input), enzyme load and residence time, an important issue to consider when designing large-scale plants.     

Cultivation of Zymomonas mobilis 113 S at Different Mixing Regimes and their Influence on the Levan Formation

The Zymomonas mobilis 113 S strain was cultivated in a bioreactor with a working volume of 1.4 l at different stirring regimes in a 15% initial sucrose medium. The levan obtained in the fermentation process was analyzed by gel filtration. Because the sucrose/biomass ratio in the fermentation broth decreased to below 300 g/g, the insufficient concentration of sucrose might have decreased the concentration of levan. Besides the growth characteristics of the population, the mixing intensity and flow structure were also found to influence the molecular mass of levan. At 600 rpm, the microorganisms produced levan with a molecular mass lower than at 300 rpm. The stirring of a fermentation broth with levan without cells at 300 rpm and 900 rpm showed changes in the molecular mass approximately at 20 kD and 5 kD. The size of eddies in the fermenter was supposed to determine the size of a levan molecule. Because the size of the eddy may be compared with that of a levan molecule, it explains the decline in the molecular mass of levan.     

Relations between substrate feeding pattern and development of filamentous bacteria in activated sludge processes

Summary Laboratory scale activated sludge systems were operated under regimes of continuous or intermittent feeding of substrate. It was found that continuously fed systems repeatedly resulted in the development of filamentous bacteria and bulking of the sludge. Intermittently fed systems did form good settling sludges, without filamentous bacteria. The same results were found using different sludge loadings and different concentrations of mixed liquor suspended solids. High dissolved oxygen concentration did not prevent bulking in continuous systems while low dissolved oxygen concentration resulted in bulking with intermittently fed systems. It was found that the substrate removal rate of intermittently operated systems was always higher than for continuously fed systems. The hypothesis is formulated that intermittent feeding leads to higher substrate removal rates by floc forming bacteria and their predominance in intermittently fed systems, which can be compared to plug flow systems.     

Novel and highly regioselective route for synthesis of 5-fluorouridine lipophilic ester derivatives by lipozyme TL IM

For the first time, lipozyme TL IM, an inexpensive lipase from Thermomyces lanuginosa, was successfully applied to the regioselective synthesis of lipophilic 5-fluorouridine ester derivatives. The ESI-MS and (13)C NMR analysis confirmed that the end products of the acylation were 5'-O-acyl 5-fluorouridines, more powerful anti-tumor drugs than 5-fluorouridine itself. Notably, the chain length of acyl donors had an obvious effect on the initial rate and the maximum substrate conversion of the regioselective acylation. The acylation of 5-fluorouridine with vinyl laurate was used as a model to explore the influence of various factors on the reaction with respect to the initial rate, the maximum substrate conversion and the regioselectivity. The optimum water activity, the molar ratio of vinyl laurate to 5-fluorouridine, reaction temperature and shaking rate were 0.07, 15/1, 45 degrees C and 200rpm, respectively, under which the maximum substrate conversion and the regioselectivity were as high as 98.4 and >99%, respectively, after a reaction time of around 6h.