Agricultural and Biological Engineering

Investigations on the leachate bioavailability, leaching rate, and lactic acid accumulation properties of plastic composite supports (PCS) were essential for large-scale or long-term lactic acid fermentation. Leachates from PCS and polypropylene discs (controls) were analyzed by the micro-Kjeldahl method; by absorbances at 260, 275, and 280 nm; and by bioassays with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The amount of leached nitrogen in a 20-ml initial soaking solution had a high correlation with the soaking solution's cell density (r ‫؍‬ 0.87) and absorbance at 260 nm (r ‫؍‬ 0.95). Leaching rates of various PCS were evaluated by 20 20-ml simulated repeated-batch fermentations (RBF). PCS with only yeast extract as the minor agricultural ingredient had a high leaching rate and leached out 51 to 60% of the total nitrogen during the first RBF. PCS blended with dried bovine albumin, dried bovine erythrocytes, and/or soybean flour had slowed nutrient leaching (20 to 30% of the initial leached nitrogen). Hence, they could still maintain 1 g of lactic acid per liter and measurable cell density (absorbance at 620 nm, 0.4 to 0.6) at the 20th 20-ml RBF. Lactic acid accumulation properties of PCS were evaluated by soaking the supports in a 30% lactic acid solution for 72 h at 45°C. The lactic acid-soaked supports were rinsed three times and then heat treated (121°C, 15 min) in 15 ml of deionized water. The results showed that lactic acid accumulation in PCS was mainly due to absorption and had no correlation with lactic acid production or biofilm formation.

This paper describes continuous and fed-batch fermentation protocols for enhanced production of organically bound chromium yeast. During continuous fermentation, several inorganic chromium compounds were evaluated. Sodium chromate demonstrated the best chromium incorporation into yeast biomass without any precipitation in the fermentation medium. During fed-batch fermentation, several sodium chromate concentrations were evaluated at 1.1, 3.5, 5.9, 7.1, 8.3, and 10.8 g/L with continuous or single-dose addition. For single-dose addition of sodium chromate, some precipitation was observed for all concentrations, which reduced the available chromium in the fermentation medium. Adapted strain C11-1, obtained during continuous fermentation, produced higher biomass than the wild type but significantly lowered chromium incorporation. Pilot-scale fermentation demonstrated similar total chromium incorporation (2966 ppm) with lower biomass production compared to benchtop fermentation performed at the same sodium chromate addition.

A combination of lactobacilli and biofilm-forming bacteria were evaluated in continuous fermentations for lactic acid production using various supports. Twelve different bacteria, including species of Bacillus, Pseudomonas, Streptomyces, Thermoactinomyces, and Thermomonospora were tested for biofilm-forming capabilities. Solid supports that were evaluated in either batch or continuous fermentations were pea gravels, 3M-macrolite ceramic spheres, and polypropylene mixed with 25% of various agricultural materials (e.g. corn starch, oat hulls) and extruded to form chips (pp-composite). Biofilm formation was evaluated by the extent of clumping of solid supports, weight gain and (in some instances) Gram stains of the supports after drying overnight at 70° C. The supports consistently producing the best biofilm were pp-composite chips followed by 3M-Macrolite spheres then by pea gravels. The best biofilm formation was observed with P. fragi (ATCC 4973), S. viridosporus T7A (ATCC 39115), and Thermoactinomyces vulgaris (NRRL B-5790), grown optimally at 25, 37, and 45° C, respectively, on various pp-composite chips. Lactic acid bacteria used in the fermentations were Lactobacillus amylophilus (NRRL B-4437), L. casei (ATCC 11443), and L. delbrueckii mutant DP3; these grow optimally at 25, 37 and 45° C, respectively. Lactic acid and biofilm bacteria with compatible temperature optima were inoculated into 50-ml reactors (void volume 25 ml) containing sterile pp-composite supports. Lactic acid production and glucose consumption were determined by HPLC at various flow rates from 0.06 to 1.92 ml/min. Generally, mixed-culture biofilm reactors produced higher levels of lactic acid than lactic acid bacteria alone. S. viridosporus T7A and L. casei on pp-composite chips were the best combination of those tested, and produced 13.0 g/l lactic acid in the reactors without pH control. L. casei produced 10.3 g/l lactic acid under similar conditions.

This study compares the performances of animal-based byproducts with other complex nutrients. Productions of R-amylase by Bacillus subtilis, glucoamylase by Aspergillus niger, endocellulase, exocellulase, and xylanase by Trichoderma viride, protease by B. subtilis, and B. licheniformis were evaluated in shake-flask fermentation. Culture broths were analyzed daily for enzyme activity for up to 9 days. Significant increases in R-amylase, cellulase, and xylanase activities were observed. Data strongly suggest that many of these byproducts can be used to replace the more expensive complex nutrients in industrial enzyme production with a corresponding increase in enzyme production.

In this study, different carob extract-based media containing Medium A (included all ingredients), Medium B (included yeast extract and salts), Medium C (included (NH4)2SO4 and salts), Medium D (included only salts) and Medium E (included no ingredients) were evaluated for ethanol fermentation by Saccharomyces cerevisiae in a biofilm reactor and their results were used for kinetic modeling. The logistic model for cell growth, Luedeking-Piret model for ethanol production and Modified Luedeking-Piret model for substrate consumption were studied. Kinetic parameters were determined by fitting the observed values of the models. The findings indicated that the predicted data with the suggested kinetic model for each medium fitted very well the experimental data. Estimated kinetics were also in good agreement with experimental kinetics. The techno-economic analysis was performed with the unit costs of the components used in the medium and ethanol. Medium-based process economic feasibility ...

Bio-ethanol is one of the energy sources that can be produced by renewable sources. Waste potato mash was chosen as a renewable carbon source for ethanol fermentation because it is relatively inexpensive compared with other feedstock considered as food sources. However, a pretreatment process is needed: specifically, liquefaction and saccharification processes are needed to convert starch of potato into fermentable sugars before ethanol fermentation. In this study, hydrolysis of waste potato mash and growth parameters of the ethanol fermentation were optimized to obtain maximum ethanol production. In order to obtain maximum glucose conversions, the relationship among parameters of the liquefaction and saccharification process was investigated by a response surface method. The optimum combination of temperature, dose of enzyme (α-amylase) and amount of waste potato mash was 95 °C, 1 mL of enzyme (18.8 mg protein/mL) and 4.04 g dry-weight/100 mL DI water, with a 68.86% loss in dry weight for liquefaction. For saccharification, temperature, dose of enzyme and saccharification time were optimized and optimum condition was determined as 60 °C-72 h-0.8 mL (300 Unit/mL) of amyloglucosidase combination, yielded 34.9 g/L glucose. After optimization of hydrolysis of the waste potato mash, ethanol fermentation was studied. Effects of pH and inoculum size were evaluated to obtain maximum ethanol. Results showed that pH of 5.5 and 3%

Pullulan fermentations by Aureobasidium pullulans with various initial ammonium ion concentrations were evaluated in a 2-L bioreactor. The results demonstrated that A. pullulans produced highest pullulan (23.1 g/L) when the initial ammonium sulfate was 7 at 5 g/L. The purity of produced pullulan was 94.6%. Seven gram per liter of ammonium sulfate produced more biomass due to the higher level of nitrogen source, but the pullulan-degrading enzyme activity was detected after the depletion of sucrose, which reduced pullulan concentration. From the results of fed-batch fermentation, addition of 10 g/L of sucrose suppressed A. pullulans from producing pullulan-degrading enzyme. Additionally, the modified-Gompertz equation demonstrated its generality to fit all pullulan production, biomass production, and sucrose consumption curves at three ammonium sulfate levels. After incorporating the degrading factor, a re-modified-Gompertz equation was obtained that can adequately describe the decrease of pullulan at the late fermentation stage.

The agar diffusion bioassay is the most widely used method for the quantification of nisin, due to its high sensitivity, simplicity, and cost-effectiveness. This method is based on the measurement of the inhibition zone produced in nisin-sensitive microorganisms. The size of the zone is affected by many factors, such as nisinsensitive strain, amount of added agar and surfactant, and pre-diffusion step. This research aims to evaluate the effects of nisin-sensitive strains and pre-diffusion on the accuracy and precision of nisin quantification. Three strains of nisin-sensitive microorganisms (Micrococcus luteus, Lactobacillus sakei, Brochothrix thermosphacta) were tested along with three different incubation processes. The best combination was the method using L. sakei as an indicator strain with pre-diffusion at 4°C for 24 h. Compared with M. luteus and B. thermosphacta, L. sakei gave more accurate and reproducible results. Moreover, the pre-diffusion step resulted in larger inhibition zones and more precise results. Finally, the best combination was validated and compared with the method that is usually used and the result showed that the method using L. sakei with pre-diffusion gave more accurate and precise results.

The presence of pathogens in various organic wastes requires careful attention to the composting process to minimise the chance of their survival. An optimised composting mixture from previous in-vessel composting studies (50% food waste, 40% manure, and 10% bulking agent) was evaluated using windrow composting. Conventional layering and mixing methods were used for constructing the windrows. Temperature was monitored as the indication of the efficiency of composting. Initial and final physico-chemical characteristics (moisture content, C/N ratio, pH, and volatile solids) and microbial survival were also determined. There was no significant difference between the observed peak temperatures in spreader and mixer windrows (probability P40Á05). Leaching problems and structural instability were observed within the first 15-20 days. In spreader windrows, populations of faecal coliform, faecal streptococcus, Salmonella, and Escherichia coli O157:H7, in most probable number (MPN), were reduced from 418-432 MPN/g[dry compost] to 5Á2-8Á4, 78Á8-396Á0, 18Á0-59Á4, 5Á2-5Á7 MPN/g[dry compost], whereas for mixer windrows were decreased from initial values of 431-447 MPN/g[dry compost] to final values of 7Á8-8Á0, 413Á4-425Á5, 0Á5-33Á6, 6Á1-29Á2 MPN/g[dry compost], respectively. The results from this study suggested the use of faecal coliforms as indicators for Salmonella (coefficient of determination r 2 ¼ 0Á94-0Á99) and E. coli O157:H7 (r 2 ¼ 0Á89-0Á98), but did not recommend faecal streptococcus as an indicator microorganism for Salmonella (r 2 ¼ 0Á37-0Á39) and E. coli O157:H7 (r 2 ¼ 0Á11-0Á48). In conclusion, the optimum compost mixture determined from in-vessel system was composted more successfully in windrows than the in-vessel system due to higher temperatures maintained for a longer period of time and extended processing time in windrows. Further study is needed to eliminate the leaching and structural instability problems.

Lactic acid fermentations were performed with plastic-composite-support (PCS) disks in solvent-saturated media with Lactobacillus casei subsp. rhamnosus (ATCC 11443). The PCS disks contained 50% (w/w) polypropylene, 35% (w/w) ground soybean hulls, 5% (w/w) yeast extract, 5% (w/w) soybean flour, and 5% (w/w) bovine albumin. Bioassays were performed by growing L. casei in solvent-saturated media after soaking the PCS disks. Eighteen different solvent and carrier combinations were evaluated. Overall, L. casei biofilm fermentation demonstrated the same lactic acid production in solventsaturated medium as suspended cells in medium without solvents (control). To evaluate PCS solvent-detoxifying properties, two bioassays were developed. When solvent-saturated medium in consecutive equal volumes (10 mL then 10 mL) was exposed to PCS, both media demonstrated lactic acid fermentation equal to the control. However, when solvent-saturated medium with two consecutive unequal volumes (10 mL then 90 mL) was exposed to PCS, some degree of toxicity was observed. Furthermore, iso-octane, tributylphosphate (TBP), and Span 80 were optimized for recovery as 91%, 5%, and 4% (v/v), respectively, with a 1:1 ratio of 1.2 M Na 2 CO 3 stripping solution. Also, recovery by emulsion liquid extraction in the hollow-fiber contactor was minimal due to low recovery at pH 5.0 and incompatibility of the solvent and hollow-fiber material. These results suggest that PCS biofilm reactors can benefit lactic acid fermentation by eliminating the toxic effect from solvent leakage into the fermentation medium from liquid-liquid extractive integrated fermentations.

Fermentation process control is currently limited by its inability to measure parameters such as substrate, product, and biomass concentrations rapidly for consistent on-line feedback. Physical and chemical parameters, such as temperature and pH, currently can be obtained on-line using appropriate sensors. However, to obtain information on the concentration of the substrate, product, and biomass, samples must be taken off-line for measurement. With the use of spectroscopic techniques, real-time monitoring of process constituents such as product and substrate is possible. Spectroscopic techniques are rapid and nondestructive, require minimal or no sample preparation, and can be used to simultaneously assess several constituents in complex matrices. The production of ethanol is the largest fermentation process in terms of production volume and economic value as a result of its prominence in the food, agricultural, and fuel industries. This study attempts to develop an on-line ethanol fermentation monitoring technique using Fourier transform infrared (FTIR) spectroscopy with a flow-through ATR capability. Models developed using multivariate statistics, employed to obtain on-line FTIR measurements, were successfully validated by offline HPLC analysis and spectrophotometry data. Standard errors of prediction (SEP) values of 0.985 g/L (R 2) 0.996), 1.386 g/L (R 2) 0.998), and 0.546 (R 2) 0.972) were obtained for ethanol, glucose, and OD, respectively. This work demonstrates that FTIR spectroscopy could be used for rapid on-line monitoring of fermentation.

An attempt to enhance recombinant human lysozyme production by Kluyveromyces lactis K7 was performed in this study. In this study, the production of recombinant human lysozyme was investigated using shake flasks and bioreactor under different cultivation conditions. It was demonstrated that 25°C could enhance human lysozyme production when compared with other temperatures tested. This study also demonstrated that higher biomass did not necessarily produce higher human lysozyme, and it was clear that human lysozyme production was enhanced under unfavorable conditions such as low acidity and oxygen limitation. Cultivation condition with no pH control demonstrated better production than with pH controlled near neutrality. Oxygen limitation also resulted in higher recombinant human lysozyme production. Overall human lysozyme was increased from 64.1 U/ml in flask to 123.6 U/ml in fed-batch fermentation using 4.5% (w/v) glucose initially and fed with concentrated lactose to achieve 9% lactose concentration.

In this study, nisin production has been enhanced by using batch and fed-batch fermentation with calcium-alginate immobilized cell culture. Due to the inhibitory effects of original phosphate rich growth medium on the immobilizing beads, an altered complex growth medium for nisin production was used. Various pH profiles were evaluated for both batch and fed-batch fermentations. For batch fermentations, a 2.1 fold higher nisin activity was obtained by allowing the pH to drop freely after 4 hrs of fermentation at constant pH. A periodic pH profile exhibited a detrimental effect on nisin production during batch fermentations. For fed-batch fermentations, a 2.9 fold higher nisin activity was obtained by allowing the pH to remain at pH of 6.8. Approximately the same maximum concentration, 3300 IU/ml, of nisin was observed when the best pH profile for batch and fed batch experiments were compared. The results also showed that immobilized cell culture can be used in order to improve nisin fermentation for both batch and fed-batch fermentation.

Pullulan is a linear homopolysaccharide which is composed of glucose units and often described as α-1, 6linked maltotriose. The applications of pullulan range from usage as blood plasma substitutes to environmental pollution control agents. In this study, a biofilm reactor with plastic composite support (PCS) was evaluated for pullulan production using Aureobasidium pullulans. In test tube fermentations, PCS with soybean hulls, defatted soy bean flour, yeast extract, dried bovine red blood cells, and mineral salts was selected for biofilm reactor fermentation (due to its high nitrogen content, moderate nitrogen leaching rate, and high biomass attachment). Three pH profiles were later applied to evaluate their effects on pullulan production in a PCS biofilm reactor. The results demonstrated that when a constant pH at 5.0 was applied, the time course of pullulan production was advanced and the concentration of pullulan reached 32.9 g/L after 7-day cultivation, which is 1.8-fold higher than its respective suspension culture. The quality analysis demonstrated that the purity of produced pullulan was 95.8% and its viscosity was 2.4 centipoise. Fourier transform infrared spectroscopy spectra also supported the supposition that the produced exopolysaccharide was mostly pullulan. Overall, this study demonstrated that a biofilm reactor can be successfully implemented to enhance pullulan production and maintain its high purity.

Axial Flow Chromatography (AFC) is widely used for the purification of therapeutic Monoclonal Antibodies (MAbs). However, AFC columns can generate high pressure drops across the resin bed, preventing operation of the column at high flow rates especially at pilot or manufacturing-scales. Radial Flow Chromatography (RFC) was developed to provide lower pressure drops during chromatographic separations. In these studies, small and pilot-scale columns were used to evaluate purification of a MAb using both AFC and RFC technologies. A bench-scale, wedge RFC column (250 mL) was compared to a bench-scale AFC column at various linear velocities with resulting Residence Times (RT) using Protein A resin for the recovery of a monoclonal antibody. The bench RFC column was successfully operated at 4.5 min RT for equilibration and loading steps and 2 min RT for washing, elution and cleaning steps without compromising yield. The RFC column had approximately 50% lower pressure drop than the AFC column at similar flow rates. The process was then scaled-up to 5 L using a pilot-scale RFC column. The 5-L RFC column was operated at 4.5 min RT for equilibration and loading and 2 min. RT for washing, elution and cleaning with no loss of yield. However, pressure drop across the 5 L RFC column was higher than expected, but antibody recovery yields were similar for both column types. Subsequent investigations revealed a potential design issue with the RFC column. Overall, RFC has great potential to be used for pilot or manufacturing scale without high pressure drop concerns, which will certainly improve processing efficiency.

This paper describes a rapid method to identify the best solvent and carrier compound combinations with the highest extraction capability and the lowest microbial toxicity characteristics for product recovery from microbial fermentation. The extraction system has an aqueous phase, and an emulsion phase, which was a blend of sodium carbonate and organic phase [91% (v/v) organic solvent, 5% (v/v or wt/v) carrier compound, and 4% (v/v) surfactant Span 80]. Alamine 336, or tri-n-octylamine in n-heptane; Alamine 336, Alamine 304, or tributyl phosphate in hexane; and Alamine 304 or tributyl phosphate in iso-octane; Alamine 304 or Amberlite in xylene demonstrated high lactic acid extraction. For determination of bacterial toxicity of selected solvent and carrier compounds, Lactobacillus casei subsp. rhamnosus (ATCC 11443) was grown in LAF medium containing one of the selected organic solvent, carrier compound, and Span 80 in 250 ml flask at 37 • C and 125 rpm. Samples were collected regularly during 48 hour incubation, and measured for changes in cell density by absorbance at 620 nm, cell count using a fluorescent dye with flow cytometry, and lactic acid, and glucose concentrations by HPLC. Hexadecane:tributyl phosphate, n-dodecane:tri-n-octylamine, and kerosene:tri-n-octylphosphine oxide demonstrated the least microbial toxicity among the tested blends with excess solvent media. Whereas, hexanes:Alamine 304 and xylenes:Alamine 304 were nontoxic in solvent saturated media.

Biofilm is a natural form of cell immobilization in which microorganisms attach onto solid support. In this study, a pigment-reduced pullulan-producing strain, Aureobasidium pullulans (ATCC 201253), was used for continuous pullulan fermentation in a plastic composite support (PCS) biofilm reactor. Optimal conditions for the continuous pullulan production were determined by evaluating the effects of the feeding medium with various concentrations of ammonium sulfate and sucrose and dilution rate. Pullulan concentration and production rate reached maximum (8.3 g/l and 1.33 g/l/h) when 15 g/l of sucrose, 0.9 g/l of ammonium sulfate, and 0.4 g/l of yeast extract were applied in the medium, and the dilution rate was at 0.16 h −1. The purity of produced pullulan was 93.0%. The ratio of hyphal cells of A. pullulans increased when it was grown on the PCS shaft. Overall, the increased pullulan productivity can be achieved through biomass retention by using PCS biofilm reactor.

An online removal of nisin by silicic acid coupled with a micro-filter module was proposed as an alternative to reduce detrimental effects caused by adsorption of nisin onto producer, enzymatic degradation by protease, and product inhibition during fermentation. In this study, silicic acid was successfully used to recover nisin from the fermentation broth of Lactococcus lactis subsp. lactis NIZO 22186. The effect of pH (at 6.8 and 3.0) during adsorption process and several eluents (deionized water, 20% ethanol, 1 M NaCl, and 1 M NaCl+20% ethanol) for desorption were evaluated in a small batch scale. Higher nisin adsorption onto silicic acid was achieved when the adsorption was carried out at pH 6.8 (67% adsorption) than at pH 3.0 (54% adsorption). The maximum recovery was achieved (47% of nisin was harvested) when the adsorption was carried out at pH 6.8 and 1 M NaCl+20% ethanol was used as an eluent for desorption. Most importantly, nisin production was significantly enhanced (7,445 IU/ml) when compared with the batch fermentation without the online recovery (1,897 IU/ ml). This may possibly be attributed to preventing the loss of nisin due the detrimental effects and a higher biomass density achieved during online recovery process, which stimulated production of nisin during fermentation.

Apart from its widely accepted commercial applications as a food preservative, nisin emerges as a promising alternative in medical applications for bacterial infection in both humans and livestock. Improving nisin production through optimization of fermentation parameters would make nisin more cost-effective for various applications. Since nisin production by Lactococcus lactis NIZO 22186 was highly influenced by the pH profile employed during fermentation, three different pH profiles were evaluated in this study: (1) a constant pH profile at 6.8 (profile 1), (2) a constant pH profile with autoacidification at 4 h (profile 2), and (3) a stepwise pH profile with pH adjustment every 2 h (profile 3). The results demonstrated that the low-pH stress exerted during the first 4 h of fermentation in profile 3 detrimentally affected nisin production, resulting in a very low maximum nisin concentration (593 IU ml −1). On the other hand, growth and lactic acid production were only slightly delayed, indicating that the loss in nisin production was not a result of lower growth or shifting of metabolic activity toward lactic acid production. Profile 2, in which pH was allowed to drop freely via autoacidification after 4 h of fermentation, was found to yield almost 1.9 times higher nisin (3,553 IU ml −1) than profile 1 (1,898 IU ml −1), possibly as a result of less adsorption of nisin onto producer cells. Therefore, a combination of constant pH and autoacidification period (profile 2) was recommended as the pH profile during nisin production in a biofilm reactor.

A biofilm reactor not only shortens the lag phase of nisin production, but also enhances nisin production when combined with an appropriate pH profile. Due to the substrate inhibition that takes place at high levels of carbon source, fed-batch fermentation was proposed as a better alternative for nisin production. In this study, the combined effects of fed-batch fermentation and various pH profiles on nisin production in a biofilm reactor were evaluated. The tested pH profiles include 1) a constant pH profile at 6.8 (profile 1), 2) a constant pH profile with an autoacidification after 4 h (profile 2), and 3) a step-wise pH profile with pH adjustment every 2 h (profile 3). When profile 1 was applied, fed-batch fermentation enhanced nisin production for both suspended-cell (4,188 IU ml −1) and biofilm (4,314 IU ml −1) reactors, yielded 1.8-and 2.3-fold higher nisin titer than their respective batch fermentation. On the other hand, pH profiles that include periods of autoacidification (profiles 2 and 3) resulted in a significantly lower nisin production in fed-batch fermentation (2,494 and 1,861 IU ml −1 for biofilm reactor using profile 2 and 3, respectively) due to toxicity of excess lactic acid produced during the fermentation. Overall, this study suggested that fed-batch fermentation can be successfully used to enhance nisin production for both suspended-cell and biofilm reactors.