Comparison of batch,fed-batch and continuous well-mixed reactors for enzymatic hydrolysis of orange peel wastes

An alternative potential feedstock for bioethanol in the automotive sector is citrus peel waste (CPW), which can be processed through enzymatic hydrolysis and fermentation. The present work considers mathematical modeling of orange peel wastes (OPW) hydrolysis with the use of free enzymes and compares the performance of batch, fed-batch and continuous well-mixed reactors after introducing appropriate rate equations in dynamic mass balances. MATLAB^® was used for model implementation.Following the Michaelis–Menten approach, the authors used their own kinetic parameters for the pectin hydrolysis rate equation. The parameters were generated in an apposite experimental program for OPW hydrolysis to galacturonic acid with consideration of product inhibition; the corresponding values were obtained after Lineweaver–Burk linearization and are: r_max = 0.28 g/(L min), K_m = 19.80 g/L and K_IGA = 6.96 g/L, respectively. Vice-versa, the authors adopted the Kadam's group kinetic schemes and parameters for cellulose hydrolysis to cellobiose and glucose. The mathematical model of a well-mixed batch reactor was perfectly validated against the experimental results of OPW hydrolysis to galacturonic acid. In the case of a continuous well-mixed reactor, high dilution rates determine low conversion of OPW. The increased complication of fed-batch operation does not add advantages when compared to batch processing.     

Characteristics of nitrifying granules developed in an air pulsing SBR

Nitrifying bacteria are characterized by low biomass yields and slow activity rates, the enhancement of nitrifying biomass retention by promoting the formation of granules would allow improving nitrification process. The use of a pulsing sequencing batch reactor (SBR) was tested to produce nitrifying granules minimizing the air consumption. After 400 days of operation, 1.07 g VSS/L of granular biomass with a mean diameter of 0.9 mm was accumulated in the system. The maximal nitrate production rate reached a value of 0.3 kg N/(m³ d).Sporadic nitrite accumulations were registered and attributed to oxygen diffusion limitations which were solved improving the granule-liquid mass transfer with a liquid recirculation system. FISH technique was used in order to identify the main populations in the nitrifying granules; Nitrosomonas spp. and Nitrospira spp. were identified as the main ammonia and nitrite oxidizing population, respectively. With the introduction of liquid recirculation in the reactor the ammonia oxidation efficiencies improved and the proportion of Nitrospira spp. increased from 1% to 15%.     

Differential effects of mineral and organic acids on the kinetics of arabinose degradation under lignocellulose pretreatment conditions

Sugar degradation occurs during acid-catalyzed pretreatment of lignocellulosic biomass at elevated temperatures, resulting in degradation products that inhibit microbial fermentation in the ethanol production process. Arabinose, the second most abundant pentose in grasses like corn stover and wheat straw, degrades into furfural. This paper focuses on the first-order rate constants of arabinose (5 g/L) degradation to furfural at 150 and 170 °C in the presence of sulfuric, fumaric, and maleic acid and water alone. The calculated degradation rate constants (k_d) showed a correlation with the acid dissociation constant (pK_a), meaning that the stronger the acid, the higher the arabinose degradation rate. However, de-ionized water alone showed a catalytic power exceeding that of 50 mM fumaric acid and equaling that of 50 mM maleic acid. This cannot be explained by specific acid catalysis and the shift in pK_w of water at elevated temperatures. These results suggest application of maleic and fumaric acid in the pretreatment of lignocellulosic plant biomass may be preferred over sulfuric acid. Lastly, the degradation rate constants found in this study suggest that arabinose is somewhat more stable than its stereoisomer xylose under the tested conditions.     

Investigation on the formation and kinetics of glucose-fed aerobic granular sludge

Aerobic granular sludge was cultivated in a glass sequencing batch reactor (SBR) with glucose synthetic wastewater. The spherical shaped granules were observed on 4th day with the mean diameter of 0.1 mm. With the increase of chemical oxygen demand (COD) concentration of the influent, aerobic granules grew matured, the size of which ranged from 1.2 to 1.9 mm. The aerobic granular sludge could sustain high organic loading rate (about 4.0 g COD L⁻¹ d⁻¹), with good settling ability (settling velocity 36 m/h) and high biomass concentration (MLSS 6.7 ±0.2 g/L). Experimental data indicated that the substrate utilization and biomass growth kinetics followed Monod's kinetics model approximately. The corresponding kinetic coefficients of maximum specific substrate utilization rate (k), half velocity coefficient (K_s), growth yield coefficient (Y) and decay coefficient (K_d) were 13.2 d⁻¹, 275.8 mg/L, 0.183–0.250 mg MLSS/mg COD and 0.023–0.075 d⁻¹, respectively, which made aerobic granules have short setup period, high rate of substrate utilization and little surplus sludge.     

Application of polyethylene glycol immobilized Clostridium sp. LS2 for continuous hydrogen production from palm oil mill effluent in upflow anaerobic sludge blanket reactor

A novel polyethylene glycol (PEG) gel was fabricated and used as a carrier to immobilize Clostridium sp. LS2 for continuous hydrogen production in an upflow anaerobic sludge blanket (UASB) reactor. Palm oil mill effluent (POME) was used as the substrate carbon source. The optimal amount of PEG-immobilized cells for anaerobic hydrogen production was 12% (w/v) in the UASB reactor. The UASB reactor containing immobilized cells was operated at varying hydraulic retention times (HRT) that ranged from 24 to 6 h at 3.3 g chemical oxygen demand (COD)/L/h organic loading rate (OLR), or at OLRs that ranged from 1.6 to 6.6 at 12 h HRT. The best volumetric hydrogen production rate of 336 mL H₂/L/h (or 15.0 mmol/L/h) with a hydrogen yield of 0.35 L H₂/g COD_removed was obtained at a HRT of 12 h and an OLR of 5.0 g COD/L/h. The average hydrogen content of biogas and COD reduction were 52% and 62%, respectively. The major soluble metabolites during hydrogen fermentation were butyric acid followed by acetic acid. It is concluded that the PEG-immobilized cell system developed in this work has great potential for continuous hydrogen production from real wastewater (POME) using the UASB reactor.     

Biosorption of Fe3+ from aqueous solution by a bacterial dead Streptomyces rimosus biomass

The iron biosorption capacity of a Streptomyces rimosus biomass treated with NaOH was studied in batch mode. After pretreatment of biomass at the ambient temperature, optimum conditions of biosorption were found to be: a biomass particle size between 50 and 160 μm, an average saturation contact time of 4 h, a biomass concentration of 3 g/l and a stirring speed of 250 rpm. The equilibrium data could be fitted by Langmuir isotherm equation. Under these optimal conditions, 122 mg _Fe/g_biomass were fixed.     

Effect of oxygen supply on biomass and helvolic acid production in submerged fermentation of Cordyceps taii

The entomogenous fungus Cordyceps taii, a traditional Chinese medicinal mushroom, exhibits potent important pharmacological effects and it has great potential for health foods and medicine. In this work, the effects of oxygen supply on production of biomass and bioactive helvolic acid were studied in shake-flask fermentation of C. taii mycelia. The value of initial volumetric oxygen transfer coefficient (K_La) within 10.1–33.8 h⁻¹ affected the cell growth, helvolic acid production and expression levels of biosynthetic genes. The highest cell concentration of 17.2 g/L was obtained at 14.3 h⁻¹ of initial K_La. The highest helvolic acid production was 9.6 mg/L at 10.1 h⁻¹ of initial K_La. The expression levels of three genes encoding hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase and squalene synthase were down-regulated on day 2 and day 8 but up-regulated on day 14 at an initial K_La value of 10.1 h⁻¹ vs. 33.8 h⁻¹, which well corresponded to the helvolic acid biosynthesis in those conditions. The information obtained would be helpful for improving the biomass and helvolic acid production in large-scale fermentation of C. taii.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

Batch and continuous fermentative production of hydrogen with anaerobic sludge entrapped in a composite polymeric matrix

Cell immobilization techniques were adopted to biohydrogen production using immobilized anaerobic sludge as the seed culture. Sucrose-based synthetic wastewater was converted to H₂ using batch and continuous cultures. A novel composite polymeric material comprising polymethyl methacrylate (PMMA), collagen, and activated carbon was used to entrap biomass for H₂ production. Using the PMMA immobilized cells, the favorable conditions for batch H₂ fermentation were 35 °C, pH 6.0, and an 20 g COD l⁻¹ of sucrose, giving a H₂ production rate of 238 ml h⁻¹ l⁻¹ and a H₂ yield of 2.25 mol H₂ mol sucrose⁻¹. Under these optimal conditions, continuous H₂ fermentation was conducted at a hydraulic retention time (HRT) of 4–8 h, giving the best H₂-producing rate of 1.8 l h⁻¹ l⁻¹ (over seven-fold of the best batch result) at a HRT of 6 h and a H₂ yield of 2.0 mol H₂ mol sucrose⁻¹. The sucrose conversion was essentially over 90% in all runs. The biogas consisted of only H₂ and CO₂. The major soluble metabolites were butyric acid, acetic acid, and 2,3-butandiol, while a small amount of ethanol also detected. The PMMA-immobilized-cell system developed in this work seems to be a promising H₂-producing process due to the high stability in continuous operations and the capability of achieving a competitively high H₂ production rate under a relatively low organic loading rate.     

Phosphorus retention capacity of agricultural headwater ditch sediments under alkaline condition in purple soils area,China

Phosphorus (P) retention by headwater ditch sediments adsorption plays a pivotal ecological role in P buffering in freshwater ecosystems. Previous studies focused on headwater ditch sediment adsorption and its P retention capacity in acid conditions, but little information is available for headwater ditches under alkaline condition. In this study, adsorption behavior of phosphorus was investigated in headwater ditch sediments under alkaline condition using a batch equilibrium technique, thus determining phosphorus retention capacity of headwater ditch sediments collected at 11 sites at base-flow on 2 March 2006 in purple soils area of China. Results showed that headwater ditch sediments had elevated phosphorus sorption maximum (S_max) values (122.72–293.23 mg P kg^?1) and P binding energy (K) values (1.64–8.65 L mg^?1), while they had low equilibrium phosphorus concentration (EPC₀) (0.001–0.108 mg L^?1) and degree of phosphorus saturation (DSP) (1.93–10.19%). Analysis of EPC₀ and soluble P concentration indicated that sediments acted as a sink for P across all headwater ditches. Therefore, there were high intrinsic P retention capacities of headwater ditch sediments. Positive correlations of both K and S_max with oxalate-extractable Fe (r of 0.93 and 0.81, p < 0.05) and total carbon (TC) (r of 0.89 and 0.74, p < 0.05) were found, thus suggesting that organic matter and amorphous or poorly crystalline Fe would play dominant roles in P adsorption in the headwater ditch sediments under alkaline condition. Since neither S_max nor K were correlated with CCE (CaCO₃) (r of 0.15 and ?0.06, p > 0.05), the high-energy sorptive surfaces of Fe oxides were more important than CaCO₃ in P sorption of sediment under alkaline condition. At the same time, these poor correlations between CCE and K and S_max imply a non-linear relationship between P retention and the content of carbonate. The negative correlations of both K and S_max with pH (r of–0.73, and–0.58, p < 0.05) revealed that an increase in pH would not improve sediment retention capacity under alkaline conditions.     

Effect of heating rate on pDNA production by E. coli

The effects of heating rate (HR) on the performance of two-phase (batch followed by fed-batch) high cell-density cultivations (HCDC) of E. coli DH5α for the production of plasmid DNA (pDNA) were investigated. Optimal temperatures for the HCDC, as selected from shake flask experiments at constant temperatures between 30 and 45 °C, were 35 °C for biomass accumulation in the batch phase and 42 °C for inducing pDNA replication during the fed-batch. In HCDC the temperature was increased at HR of 0.025, 0.05, 0.10 and 0.25 °C/min and the performance of the cultivations were compared to a HCDC run at constant temperature (35 °C). Compared to constant 35 °C, heat-induced HCDC accumulated up to 50% less biomass within the same cultivation time and acetate and glucose accumulated to high concentrations. The overall specific productivity (Q_P) and average pDNA yield (Y_p/x) in HCDC at 35 °C were 0.22 ± 0.02 mg/g h and 5.3 ± 0.00 mg/g, respectively. Such parameters were maximum at a HR of 0.05 °C/min, reaching 0.56 ± 0.06 mg/g h and 9.3 ± 0.6 mg/g, respectively. At HR above 0.5 °C/min, Y_p/x remained relatively constant, whereas Q_P tended to decrease. The supercoiled pDNA fraction remained around 80% at all HR. Bioreactors were equipped with a capacitance/conductivity probe. In all cases biomass concentration correlated closely with the capacitance signal and acetate and glucose accumulation was accompanied by an increase in the conductivity signal. Thus, it was possible to calculate acetate and biomass concentrations, as well as μ, from online capacitance and conductivity signals using estimators. Altogether, in this study it was shown that it is possible to maximize pDNA productivity by choosing an appropriate HR and that relevant parameters can be estimated by capacitance/conductivity signals, which are useful for better process control and development.     

Production of polysaccharide–peptide complexes by submerged mycelial culture of an entomopathogenic fungus Cordyceps sphecocephala

The effects of medium components and environmental factors on the production of mycelial biomass and polysaccharide–peptide complexes (exobiopolymers) by Cordyceps sphecocephala J-201 were investigated in submerged cultures. The optimal temperature and initial pH for the production of both mycelial biomass and exobiopolymers in flask cultures were found to be 25 °C and pH 4–5, respectively. The optimal combination of the media constituents was as follows (g l⁻¹): sucrose 40, yeast extract 6, polypepton 2, KH₂PO₄ 0.46, K₂HPO₄ 1, and MgSO₄·7H₂O 0.5. The results of bioreactor culture revealed that the maximum concentration of mycelial biomass (28.2 g l⁻¹) was obtained at an agitation speed of 300 rpm and at an aeration rate of 2 vvm, whereas maximum exobiopolymer production (2.5 g l⁻¹) was achieved at a milder agitation speed (150 rpm). There was a significant variance in mycelial morphology between different aeration conditions. Looser mycelial pellets were developed, and their size and hairiness increased as the aeration rate increased from 0.5 to 2.0 vvm, resulting in enhanced exobiopolymer production. The apparent viscosities of fermentation broth increased rapidly towards the end of fermentations at the conditions of high aeration rate and agitation speed, which were mainly due to high amount of mycelial biomass rather than exobiopolymers at the later stages of fermentation. The three different exobiopolymers (FR-I, -II, and -III) were fractionated by a gel filtration chromatography on Sepharose CL-6B. The carbohydrate and protein contents in each fraction were significantly different and the molecular weights of FR-I, FR-II, and FR-III were determined to be 1831, 27, and 2.2 kDa, respectively. The compositional analysis revealed that the three fractions of crude exobiopolymers consisted of acidic and nonpolar amino acids, such as aspartic acid, glutamic acid, glycine, and valine in protein moiety, and of mainly mannose and galactose in sugar moiety.     

Solvent-free methanolysis of canola oil in a packed-bed reactor with use of Novozym 435 plus loofa

Enzymatic methanolysis of canola oil in the solvent-free system was studied in a packed-bed reactor (PBR) using small pieces of loofa plus Novozym 435. Response surface methodology (RSM) was applied to determine the effect of the transesterification conditions, namely flow rate of substrate (x₁), temperature (x₂) and methanol to canola oil molar ratio (x₃) as the regressors, on the methyl ester production. A central composite design (CCD) was employed to optimize the reaction. A second-order polynomial multiple regression model was chosen and analysis of variance (ANOVA) showed a high coefficient of determination (R²) value of 0.996, thus adjustment of the model with experimental data was ensured. The methyl ester yield increased as the flow rate of the reaction mixture in the PBR increased from its low to the middle level thereafter, increasing the flow rate corresponded to decreasing the yield. The same trends of changes were observed for the other two factors. The optimum process conditions for biodiesel production in the PBR were found to be: x₁ = 6.3 mL/min, x₂ = 38 °C and x₃ = 4.3. The same batch was successfully used repeatedly in the PBR for six enzymatic cycles (432 h), where the methyl ester yield was maintained above 97%.     

Efficient production of (R)-(−)-mandelic acid with highly substrate/product tolerant and enantioselective nitrilase of recombinant Alcaligenes sp.

For efficient production of (R)-(−)-mandelic acid, a nitrilase gene from Alcaligenes sp. ECU0401 was cloned and overexpressed in Escherichia coli. After simple optimization of the culture conditions, the biocatalyst production was greatly increased from 500 to 7000 U/l. The recombinant E. coli whole cells showed strong tolerance against a high substrate concentration of up to 200 mM, and the concentration of (R)-(−)-mandelic acid after only 4 h of transformation reached 197 mM with an enantiomeric excess (ee_p) of 99%. In a fed-batch reaction with 600 mM mandelonitrile as the substrate, the cumulative production of (R)-(−)-mandelic acid after 17.5 h of conversion reached 520 mM. The recombinant E. coli cells could also be repeatedly used in the biotransformation, retaining 40% of the initial activity after 10 batches of reaction. The highly substrate/product tolerable and enantioselective nature of this recombinant nitrilase suggests that it is of great potential for the practical production of optically pure (R)-(−)-mandelic acid.     

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

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

Factors affecting the biotransformation of trimethylammonium compounds into l-carnitine by Escherichia coli

The biotransformation of d-carnitine and crotonobetaine into l-carnitine with wild and transformed E. coli strains under batch and continuous operation was optimised. In batch, the best conditions for the transformed strain were 30% oxygen saturation, a temperature of 41 °C and a minimal medium, whereas anaerobic cultures in either complex or minimal media at 37 °C and pH 7.5 were optimal for the wild strain. Studies on the expression of the enzymes involved in trimethylammonium metabolism showed that l-carnitine dehydratase activity was always higher than that of d-carnitine racemase. Experiments with the transformed strain in continuous cell-recycle reactors showed that, despite the higher productivity that could be achieved (0.65–1.2 g/L h), plasmid-bearing cells were segregated even when a selective medium was used. This fact was also confirmed by studying the evolution of the d-carnitine racemase level. Immobilization of the transformed strain in κ-carrageenan gels allowed continuous operation for l-carnitine production with no plasmid loss. In continuous processes with cell-retention systems, the wild strain showed higher productivity and stability than the transformed strain. Moreover, crotonobetaine was a better substrate for both strains used. Recycling with hollow-fiber cartridges provided the highest biomass level even though the l-carnitine dehydratase/biomass ratio was lower. However, membrane composition and cut-off had less influence on reactor performance as similar levels of productivity were attained. In spite of this, continuous processes attained a l-carnitine production as high as 11.5 g/L h as a result of the high enzyme induction and biomass levels.     

Biodiesel-fuel production in a packed-bed reactor using lipase-producing Rhizopus oryzae cells immobilized within biomass support particles

A packed-bed reactor (PBR) system using fungus whole-cell biocatalyst was developed for biodiesel fuel production by plant oil methanolysis. Lipase-producing Rhizopus oryzae cells were immobilized within 6 mm × 6 mm × 3 mm cuboidal polyurethane foam biomass support particles (BSPs) during batch cultivation in a 20-l air-lift bioreactor. Emulsification of the reaction mixture containing soybean oils and water improved the methanolysis reaction rate. Using a high flow rate for the reaction mixture in the PBR caused exfoliation of the immobilized cells from the BSPs, while the inefficient mixing of the reaction mixture at low flow rates allowed the BSPs to be covered with a hydrophilic layer of high methanol concentration, leading to a significant decrease in lipase activity. A high methyl ester content of over 90% was achieved at a flow rate of 25 l/h in the first cycle of repeated batch methanolysis and a high value of around 80% was maintained even after the tenth cycle. Comparison with methanolysis reaction in a shaken bottle suggested that the PBR enhances repeated batch methanolysis by protecting immobilized cells from physical damage and excess amounts of methanol. The process presented here is therefore considered to be promising for industrial biodiesel-fuel production.     

Evaluation of biocomposite-based supports for immobilized-cell xylitol production compared with a free-cell system

This study was conducted to evaluate the importance of aeration in free and immobilized cell systems in an aerated bioreactor for xylitol production from an oat hull hemicellulosic hydrolysate using an integrated process. The aeration rate (AR) or oxygen mass transfer coefficient (k_La) demonstrated a significant role in controlling cell (Candida guilliermondii FTI 20037) regeneration and bioconversion performance in free and immobilized cell systems. In the free cell system, an aeration rate of 1.25 vvm corresponding to k_La of 15.8 1/h resulted in maximum values of product yield (Y_p/s: 0.87 g/g), productivity (Q_p: 0.57 g/l/h), and final xylitol concentration (P_f: 55 g/l) from the hydrolysate with a 74.5 g/l xylose concentration. However, in the aerated immobilized cell system, maximum and almost similar results (almost P_f: 54 g/l, Q_p: 0.57 g/l/h and Y_p/s: 0.84 g/g) were obtained with aeration rates from 1.25 to 1.5 vvm using composites based on polypropylene (PP) and partially delignified fiber (PDF). Composites based on acid treated fiber (ATF) containing a high amount of lignin showed some inhibitory impact on xylose uptake and xylitol formation (P_f: 47 g/l and Q_p < 0.49 g/l/h) with the optimal aeration rate of 1.5 vvm in the initial cycle of the bioconversion; this inhibition impact could be resolved in the next consecutive cycles. The surface modifier polyethyleneimine (PEI) slightly enhanced cell retention in the immobilized form on the ATF-based cell support. This investigation helps fill in the knowledge gaps existing on the integrated processing of the lignocellulosic biomass for xylitol bioproduction and biorefinery industry; however, more scale-up studies are recommended for commercialization.     

The use of a mirror reduces isolation stress in horses being transported by trailer

Horse trailers are a common form of transportation for horses and ponies and often require the animal to travel alone or with a single companion. The current study investigated the effect of transporting horses alone, in company or with an acrylic safety mirror (measuring 81 cm × 61.5 cm) that provided surrogate companionship. The behavioural and physiological responses of 12 mature horses during a 30-min journey by trailer under the three treatments were compared. Behaviours (vocalisation, eating, head-tossing, pawing, and head-turning) were recorded. In order to assess circulatory changes that occur as part of the response to transport, heart rate (HR), rectal (T_r) and ear-pinna (T_p) temperatures were recorded. When travelling with a live companion significantly less time was spent vocalising (p < 0.001), head-turning (p < 0.001), head-tossing (p < 0.01) and pawing (p < 0.01); eating behaviour increased (p < 0.05). Physiological responses (increases in HR and T_r and decreases in T_p) were also significantly reduced when travelling with a live companion (p < 0.01). Travelling with the mirror did not significantly affect physiological responses compared with travelling alone, but the rise in T_r and fall in T_p was reduced (p = 0.052 and p = 0.051, respectively) and can be considered a trend. When travelling with a mirror significantly less time was spent turning the head (p < 0.01), vocalising (p < 0.05) and head-tossing (p < 0.05); eating behaviour increased (p < 0.05). The only significant difference between travelling with a live companion and a mirror was that the time spent turning the head round was less with a live companion (p < 0.05). The provision of surrogate companionship in the form of a mirror was found to be preferable to travel alone, but where possible a live companion is recommended. Isolation during transportation was found to suppress feeding behaviour. Although peripheral blood flow (T_p) has been used to assess transport stress in other species it has not previously been used in the horse. Further evaluation of this non-invasive measure is now required.     

Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass

Sequential-co-culture technique was investigated in this study for the production of bioethanol from relatively cheaper lignocellulosic biomass of Kans grass (Saccharum spontaneum). The consortium of Pichia stipitis and Zymomonas mobilis was used to develop a suitable sequential-co-culture system. The Kans grass biomass was hydrolyzed in such a manner that the two sugar fractions, xylose rich and glucose rich were generated (a separate study). The P. stipitis cells and respective fermentation media (xylose rich) were fed to the fermentation vessel, after the set fermentation time Z. mobilis cells and respective media were fed to the same vessel. Different strategies have been followed and experiments were conducted initially at flask level. The selected strategy was then applied at bioreactor level using both synthetic fermentation media and Kans grass hydrolysate media to compare the kinetic parameters. The sequential addition of cultures with their respective media and imposed process conditions, showed better utilization of total sugars added (>95%). Microaerobic condition for P. stipitis and strictly anaerobic condition for Z. mobilis fermentation were found significant. The average ethanol yield (Y_p/s) and overall volumetric productivity (r_po) were found as 0.453 g_p/g_s and 1.580 g/l/h respectively for Kans grass hydrolysate media and 0.474 g_p/g_s and 2.901 g/l/h respectively for synthetic fermentation media.