Dissolved carbon dioxide driven repeated batch fermentation has been performed under four glucose concentrations: ~150, ~200, ~250 and ~300 g glucose l-1, with three dissolved carbon dioxide (DCO2) control conditions: without DCO2 control, with DCO2 control at 750 and 1000 mg l-1 levels. No residual glucose was observed under all performed fermentation conditions, and the repeated batch fermentation system could be operated by a computer as self-cycling system. The collected fermentation results presented that, under the same feeding concentration, ethanol concentration in the presence of DCO2 control was significantly lower than that in the absence of DCO2 control; and a higher biomass concentration in the presence of control was observed in this comparison as well. A higher biomass concentration resulted in a shorter fermentation time, which contributed to a higher ethanol production rate. The highest final ethanol concentration was observed as 113.5 g l-1 at 1000 mg DCO2 l-1 control level under ~300 g glucose l-1 condition, where the lowest ethanol production rate of 1.18 g l-1 h-1 was observed. The highest ethanol production rate was 4.57 g l-1 h-1 and its corresponding ethanol concentration was 66.7 g ethanol l-1 at 1000 mg l-1 DCO¬2 control level under ~200 g glucose l-1 condition. For all fermentation conditions, the viabilities of yeast at the end of fermentation were maintained at near 90% where their corresponding final ethanol concentrations were lower than 100 g l-1. As soon as the final ethanol concentration at the end of each cycle was greater than 110 g l-1, its corresponding viability decreased to ~70%. The ethanol conversion efficiency was maintained at ~90% and ~65% in the absence and presence of DCO2 control, respectively. Based on the changing of biomass concentration profiles in the stabilized cycles, two cell growth phases could be identified in the absence of DCO2 control, and only one cell growth phase was noticeable in the presence of DCO2 control cases. Meanwhile, a sudden decline of DCO2 readings at the end of fermentation was constantly observed in both of in the absence and in the presence of DCO2 control cases, which resulted in developing two control algorithms to determine self-cycling time. Comparison of carbon balance analysis between in the absence and in the presence of DCO2 control suggested that the availability of DCO2 control might alter the metabolic flow during fermentation; and the figure of ethanol concentration against fermentation time illustrated that the changing of DCO2 control level did not affect fermentation results, significantly. Moreover, comparisons of ethanol production rate between different processes and different initial glucose concentrations concluded that the ethanol production rate in the presence of DCO2 control was generally higher than that in the absence of DCO2 control under the same glucose concentration; and the ethanol production rate was decreased with the increasing of glucose concentration under the same DCO2 control condition. The experiment results were scaled up to 106 L as a sample analysis in production scale, which suggested that the fermentation with ~200 g glucose l-1 feeding concentration in the absence of DCO2 controlled would provide best profits in the all fermentation conditions.

Recombinant Pichia pastoris is one of the important methylotropic yeast due to its robustness and ability to produce hormones like human chorionic gonadotropin (hCG), luteinizing hormone (LH) extracellularly. High growth on glycerol and strong protein expression on methanol by insertion of alcohol oxidase (AOX) promoter demand the fermentation to be a multistage operation. Methylotropic pathway demands more oxygen as methanol has to be converted to formaldehyde with half mole of oxygen. Moreover as fermentation progresses cell density in the reactor also increases. In case of Pichia pastoris fermentation cell density usually reaches very high (above 100 gm/lit) at the end of fermentation. Both these contribute in the increased oxygen demand in the fermentation and oxygen transfer turns out to be a limiting step. The present study focuses on the oxygen transfer process and its improvement in the fermentation. Oxygen transfer in bioreactor is a multistep process and involves different kinetic as well as mass transfer steps. In case of fermentation especially at high cell densities, oxygen transfer from bubbles to the broth becomes limiting step. The interface transport is governed by many physical as well as kinetic parameters. It is essential to screen these parameters from the whole set to identify the key parameters. Sensitivity analysis is carried out by using Metabolic Control Analysis (MCA) to quantify the effects of different parameters. It is found that bubble size and oxygen partial pressure are two such key parameters which can be manipulated. Use of pure oxygen to increase partial pressure and thereby solubility of oxygen in broth is a common approach. This work focuses on bubble size manipulation to increase the oxygen transfer rates.The idea behind this work is on to generate micron sized bubbles and utilize them effectively in the fermentation. There are many techniques reported to generate microbubble dispersions. In this work ’Spinning Disc microbubble Generator’ is fabricated to generate microbubbles. A flat disc surrounded by baffles with 5 mm gap in between, when subjected to 5000 rpm generates microbubbles. Some modifications are done to the set up to achieve desired properties of the bubbles. The bubbles generated fall in the range of 30-300 micron with mean size of about 60 micron. Use of Tween-20 surfactant stabilize the bubbles and hence offer a good resistance to coalescence and breakage. The liquid fraction in the bubbles can be as high as 40%. Contineous addition of this dispersion unnecessarily can dilute the fermentation broth. To overcome this volume constrain, a recirculation system is designed. Microbubble dispersion is added contineously to the reactor and equivalent fermentation broth is pumped back to the microbubble generator to achieve steady state to the liquid volume in both the vessels. Mass transfer studies with microbubbles show the potential of microbubble dispersion (MBD) to enhance mass transfer significantly. Decrease in volumetric mass transfer coefficient (KLa) due to surfactant is overcompensated by the increase in the interfacial area and net effect is, potential enhancement in KLa. The enhance- ment factor, that is, ratio of mass transfer coefficient with MBD to mass transfer coefficient with conventional sparging, is obtained to be about 4 to 5. Prior to utilization of bubbles in the recirculation system, cells are checked for the shear sensitiveness. Negligible lysis losses and almost no effect on growth patterns in shake flask culture confirm that the cells used are mechanically stable at operating conditions. Better growth patterns in shake flask are observed when microbubbles are pumped for predetermined duration in the broth. It shows possible use of MBD as oxygen carriers. Glycerol batch phase with MBD and conventional sparging is studied at different initial cell densities. Conventional sparging fails to grow the cells and Dissolved Oxygen (DO) levels close to zero suggest high oxygen demands which can not be sustained by conventional sparging. The same batch is run using MBD. Reasonably good growth patterns are observed. DO levels are well above 70% for most of the time during operation. High oxygen demand which can not be sustained by conventional sparging alone can be sustained by MBD. In this way in high den- sity cultures utilization of MBD can be a good alternative to fulfill required oxygen demand in fermentation.

Call number: LD2668 .T4 1956 C37 / Master of Science

Call number: LD2668 .T4 1978 O72 / Master of Science

Thesis submitted in partial fulfillment for the Degree of Master of Technology: Chemical Engineering, Durban University of Technology, 2010. / THE OBJECTIVE of this study was to devise a purification process, using ultrafiltration membranes as the core technology, for the preparation of corn steep liquor (CSL) as a fermentation feedstock. This process inherently required the development of a pretreatment system for the ultrafiltration membranes for the removal of suspended solids and high fouling material from corn steep liquor. The ultrafiltration membrane system was required for the separation and removal of colloidal solids from corn steep liquor, and to fractionate and separate out unwanted proteins, to render the feedstock suitable for sterilisation and subsequent fermentation. THE CONCEPT of membrane technology was investigated in order to find a more practical alternative for what was deemed to be a difficult process problem. In particular, various pretreatment technologies were investigated to form a compact and robust process package. THE CORN STEEP LIQUOR, a by-product of the corn wet milling process, was obtained from African Products, Germiston, in the form of a concentrated slurry directly from an evaporator system. A diagnostic of the feedstock was carried out and from this information, it was decided that three pretreatment options would be investigated. The first option was the pH treatment of the corn liquor, by the addition of ammonia which induced the precipitation of solids. This was followed by liquid-solid separation, and the clarified liquor was fed to the membrane system. The second option looked at the separation of suspended solids from the liquor by the use of broth conditioning additives and separation of the solids by a decanter centrifuge. The third option investigated was the use of a gyratory screening system for the removal of all solids greater than 100 μ in size. IN THE pH TREATMENT of CSL, the process is effected by the addition of base to pH 7. The technology involves neutralisation of CSL in a mixing system, under predetermined conditions of temperature, agitation and rate of addition, followed by subsequent liquid-solid separation. Trials were conducted on a pilot plant to test the process. Initial trials, conducted on a small scale pilot filter press, proved to be successful for this application. A suspended solids removal of up to 98% was achieved. The average suspended solids in the filtrate was found to range between 0.1 to 0.25 %. Tests were also conducted on a hired “state of art” filtration plant under various conditions. A diaphragm membrane press was found to provide the best performance. Protein recoveries of above 95 % at fluxes of 35 L/m2h at temperatures above 50 °C, and an incremental application of feed pressure was most suited for the process. The removal of the colloidal solids by the above-mentioned process was found to improve the quality of sterilisation. A reduction of more than 90 % in coagulated solids was achieved. v i IT WAS OBSERVED that the separation of suspended solids from CSL is enhanced by the use of coagulation and flocculation. Although not commonly used for this purpose, it was felt that a decanter centrifuge was well suited for the subsequent separation of the flocs from the clarified liquor. This work describes the results of the trials with such a device and the impact of broth conditioning on the efficiency of the separation. Trials have been conducted using an Alfa-Laval Model NX210 decanter, which was not specifically built for the work and therefore imposed several limitations on its performance. Despite these shortcomings, preliminary trials proved to be successful in achieving the separation objective. Tests were conducted using five different batches of CSL. With a maximum suspended solids loading of 4.3 % and a feed rate of 700 L/h, a solids recovery of 90 % was achieved. The clarified liquor contained residual solids between 0.5 and 0.8 %. The sludge had a solids concentration that ranged between 43 % and 65 %. COAGULATION AND FLOCCULATION dosages were kept within the limits of the laboratory evaluations. Flocculant dosages were controlled between 100 and 200 ppm, with the coagulant operating at higher dosages of between 400 and 2000 ppm. The only controllable parameter on the machine itself was the scroll differential speed. The best performance in terms of the cake dryness and centrate clarity was obtained at the lowest scroll differential speed of 4 rpm. THE USE OF GYRATORY SCREENS entailed passing the raw liquor through a set of two screens. The technology involves the use of a gyratory mechanism, which aids in the cleaning of the screens during continuous operation. Trials have been conducted on a pilot plant to test the system. Since the unit used was designed specifically for quick on-site screening exercises, it did not possess the added flexibility and robustness of a properly designed full scale unit. This imposed some limitations on its performance. However, despite these shortcomings, the trials conducted on the pilot plant proved to be successful in meeting the outlined objectives. A NUMBER OF TRIALS were performed on various batches of CSL. There was considerable batch to batch variation in the suspended solids content of the CSL and this was found to ultimately affect the throughput of the screening process. The feed suspended solids varied between 10 and 18 %. The highest throughput achieved was 400 L/h at a feed suspended solids loading of 14.5 %. It was found that temperature made a significant impact on the separation. The loss of heat in the feed stream caused excessive coagulation to occur thus increasing the suspended solids loading and lowering the throughput. The total solids in the sludge stream varied between 45 and 77 %. Protein loss in the sludge stream was around 1 %. Careful attention had to be given to the handling of the sludge stream. This stream displayed rheological characteristics typical of a non-Newtonian thixotropic fluid. The 100 μm screen operated best vi i when prior separation was done using a 180 to 200 μm screen. This reduced the solids loading on the tighter screen and increased the throughput by 10 to 15 %. The self cleaning mechanism also performed more efficiently under these conditions. THE SELECTED OPTION was then based on the influence the operation had on the ultrafiltration membranes, sterilisation of the product prior to fermentation and ultimately the fermentation performance. Subsequent testing of the pretreatment options were performed on an ultrafiltration membrane test cell. The product from the gyratory screens were found to produce the best overall results, where the highest fluxes and least amount of fouling occurred on the membranes tested. ONCE THE PRETREATMENT OPTION was decided, the development of the membane ultrafiltration system was then pursued. Trials were conducted on a laboratory scale, in a membrane test cell, to determine the preliminary screening of the membrane type, fouling effects and fluxes. It was found that polyvinylidene and polyacrylonitrile membranes produced the best overall fluxes of 11.25 and 10.96 L/m2h respectively. These membranes produced permeate protein concentrations of 121 and 115 g/L respectively. Sterilisation tests conducted on the permeate streams produced also showed that these two membranes had the lowest suspended solids concentrations. FERMENTABILITY tests conducted, showed that the ultrafiltered CSL, from these two membranes, produced increased cell counts and protein utilisation along with an increased product yield. Approximately 42 g/L of biomass was generated with lysine yields of 46 g/L. Further testwork revealed the non-Newtonian nature of CSL and its inherent viscosity effects. BENCH-SCALE testwork was conducted for various membrane configurations. With tubular membranes and hollow fibre membranes, average fluxes of 6.23 and 4.5 L/m2h were achieved respectively. Spiral wound membranes were found to be more consistent in their performance, with average fluxes of around 6.25 L/m2h. For the spiral wound membranes, it was found that the Desal-2 mesh spacer with a 80 mil thickness was most appropriate for the duty. PILOT PLANT testwork was conducted to scale-up the membrane system and to eliminate possible risks associated with the technology. The pilot plant studies showed up a number of principle design variables which needed careful attention. The flaws in the piloting system were subsequently rectified and this helped to improve the overall performance of the system.

published_or_final_version / abstract / Civil Engineering / Doctoral / Doctor of Philosophy

The purposeful induction of malolactic fermentation (MLF) in wines such as Pinot Noir and Chardonnay is an established commercial wine making practice in Oregon. This induction is not always successful, especially with white wines, such as Chardonnay. A study was initiated to examine the compatibility of yeasts commonly used in Oregon winemaking with various strains of malolactic bacteria. In preliminary and pilot plant scale experiments, the yeast strain found to be most conducive to malolactic fermentation by lactic acid bacteria was Montrachet (Red Star). The malolactic bacterial strains that were best able to complete malolactic fermentation in various wines, fermented by different yeast strains, were the two Oregon commercial strains, ER1A and Ey2d, and the Pinot Noir juice isolate, DAPN85A. Sensory analysis of aroma by difference from control test was done on Chardonnay wine fermented by 4 different yeast strains and 3 different malolactic bacterial strains. In all cases, there was an overall significant difference in malolactic fermented wine aroma when compared to control wines. Organic acid analyses by high pressure liquid chromatography (HPLC) and analyses of volatile compounds by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) were done on selected Chardonnay wines. Propionic acid was found to diminish in malolactic fermented wines while acetic acid content increased. Isobutanol and isobutyraldehyde increased significantly in MLF wines, compared to the controls. Chemical analyses of MLF and control wines suggested two possible chemical reactions resulting from the MLF. The first was the reduction of isobutyraldehyde to isobutanol, and the second was the hydrolysis of isobutyl acetate to isobutyraldehyde and acetate. On all GC chromatograms of wines, where MLF had occurred, there was an unidentified peak close to the retention time of isoamyl acetate. This peak was not evident in wines where MLF had not occurred. Eight compounds were tentatively identified by GC-MS in malolactic fermented wines which were not found in the control wines. These were 4-methyl-3-pentanoic acid, methyl acetate, ethyl hexanoate, hexyl acetate, 1,12-tridecadiene, hexadecanoic acid, and a compound which was tentatively identified as farnesol, or 1,2-benzenedicarboxylic acid. The latter four compounds had identity fits of less than 900 from the mass spectral analysis. Whether any of these eight compounds match the unknown "ML peak" found in the GC chromatograms is unknown. / Graduation date: 1989

The fields of biodiesel and bioethanol research and development have largely developed independently of one another. Opportunities exist for greater integration of these processes that may result in decreased costs of production for both fuels.To that end, this work addresses the use of the starches and glycerol from processed algal biomass as substrates for fermentation by the yeasts <ital>Saccharomyces cerevisiae</ital> and <ital>Pachysolen tannophilus,</ital> respectively. Ethanol producers commonly employ the former yeast for ethanol production and include the latter yeast among candidate species for cellulosic ethanol production.A simple 95% ethanol extraction at 70°C followed by sulfuric acid hydrolysis at 121°C and 2 atm proved a sufficient pretreatment for <ital>S. cerevisiae</ital> fermentation of starch from <ital>Chlamydomonas reinhardtii</ital> mutant <ital>cw15.</ital> The maximum rate of ethanol production was observed as 14 mL/g-h and a maximum concentration of 0.9±0.01% (m/v) was observed by 28 hours. Some starch appeared invulnerable to hydrolysis.<ital>P. tannophilus</ital> fermentation of glycerol, both independently and among mixed substrates, was likewise demonstrated. It was found that glucose consumption preceded that of glycerol and xylose, but that the latter two substrates were consumed concurrently. Under aerobic, batch conditions, the maximum specific growth rate of the species on a 2% glycerol substrate was observed as 0.04/hr and the yield coefficient for conversion of glycerol to ethanol was 0.07 g/g. While the maximum observed concentration of ethanol in the glycerol-only fermentation was 0.1% m/v, that in mixed media containing 2% each glucose, xylose, and glycerol was 1.5%.Also investigated here was the flocculation of a mutant species of the algae <ital>C. reinhardtii</ital> by a combination of methanol and calcium. Algae harvest is typically an energy-intensive process, but the technique demonstrated here is not. Complete flocculation of cells was observed with only 5 minutes of mixing and less than 10 minutes of settling using 12 mM CaCl₂ and 4.6% methanol. Ethanol was observed to operate in the same capacity, intimating another area in which yeast bioethanol and algal biodiesel processes might enable one another. During growth, either an inhibitor of flocculation was produced or a facilitator was consumed.

A series of studies was conducted using SEM in conjunction with chemical analysis, in situ and in vitro digestion techniques, to characterize the anatomical components from barley, corn, sorghum and wheat grains which constitute "fiber" and investigate their susceptibility to rumen microbial digestion. Fractured grains were used to identify anatomical features and cell types prior to and after extraction or digestion. Certain anatomical features, including pericarp tissue, aleurone cells, endosperm cell walls, corneous and floury endosperm tissue and lemma and palea from barley, were easily identifiable in fractured and ground grains, and in neutral detergent extracted or digested residues. In situ and in vitro incubation conditions were varied to assess the effect of concentrate and/or reduction of pH on the disappearance of identifiable grain fractions. In situ incubations were conducted using steers adapted to 0-, 30- and 90% concentrate diets. In vitro inoculum buffered at pH 7 or 6 was provided by a steer fed 0- or 90% concentrate. Tissues resistant to rumen microbial digestion during extended (144-h) in situ incubations and shorter term (12- to 48-h) in vitro incubations were primarily those identified in NDF, and included pericarp, lemma and palea, and small amounts of corneous endosperm. Remaining tissues identified included barley lemma, palea and pericarp; corn pericarp, tip cap and small amounts of corneous endosperm; sorghum pericarp and corneous endosperm with matrix and protein bodies; and wheat pericarp. In vitro disappearance of isolated NDF after 48-h ranged from 43% for barley to 89% for corn. Labile structures included embryonic tissue and portions of endosperm cell walls, protein matrix and residual starch. Resistant tissues included pericarp, aleurone cell walls, tip cap and portions of the corneous endosperm. Relative rankings of NDF digestibility under all conditions studied were similar (corn > sorghum > wheat > barley) whether determined using isolated NDF or calculated from TIVDMD residues. Neither concentrate level fed to the host animal nor pH of the in vitro incubation flask affected rankings among grains, although increasing concentrate level and/or reducing pH appeared to reduce in vitro NDF disappearance. Evaluation of electron micrographs of fractured grains suggested that similar anatomical structures in the various grains differed in their resistance to microbial digestion. For example, pericarp from barley and wheat appeared to be more resistant than that from corn or sorghum. Endosperm of barley was less resistant than that of sorghum.

No description available.