Production of L(+)-lactic acid with Rhizopus oryzae immobilized in polyurethane foam cubes

Summary Polyurethane foam cubes were employed as carriers to immobilize Rhizopus oryzae for L(+)-lactic acid production. The immobilizing capacity reached 450 g-fresh cell/l-cube. The production rate of L(+)-lactic acid could be threefold increased by using the immobilized R. oryzae. The immobilized cells could be steadily used in repetitive fermentations for more than 10 batches.     

Improved oxygen transfer and increased l-lactic acid production by morphology control of Rhizopus oryzae in a static bed bioreactor

Rhizopus oryzae was immobilized on a cotton matrix in a static bed bioreactor. Compared with free cells in a stirred tank bioreactor, immobilized R. oryzae in this bioreactor gave higher lactic acid production but lower ethanol production. The highest lactic acid production rate (2.09 g/L h) with the final concentration of 37.83 g/L from 70 g/L glucose was achieved when operating the bioreactor at 700 rpm and 0.5 vvm air. To better understand the relationship between shear effects (agitation and aeration) and R. oryzae morphology and metabolism, oxygen transfer rate, fermentation kinetics, and lactate dehydrogenase activity were determined. In immobilized cell culture, higher oxygen transfer rate and lactic acid production were achieved but lower lactate dehydrogenase activity was found as compared with those in free cell culture operated at the same conditions. These results clearly imply that mass transport was the rate controlling step in lactic acid fermentation by R. oryzae.     

Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis

A novel and simple method was developed for the preparation of magnetic Fe₃O₄ nanoparticles by chemical co-precipitation method and subsequent coating with 3-aminopropyltrimethoxysilane (APTMS) through silanization process. Magnetic Fe₃O₄-chitosan particles were prepared by the suspension cross-linking and covalent technique to be used in the application of magnetic carrier technology. The synthesized immobilization supports were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Using glutaraldehyde as the coupling agent, the lipase from R. oryzae was successfully immobilized onto the functionalized magnetic Fe₃O₄-chitosan beads. The results showed that 86.60% of R. oryzae lipase was bound on the synthesized immobilization support. This immobilized lipase was successfully used for the esterification of phenolic acid which resulted in esterification of phenolic acid in isooctane solvent reaction system for 8 consecutive cycles (totally 384 h), 72.6% of its initial activity was retained, indicating a high stability in pharmaceutical and industrial applications.     

Transesterification of phosphatidylcholine in <Emphasis Type="Italic">sn</Emphasis>-1 position through direct use of lipase-producing <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> cells as whole-cell biocatalyst

The enzymatic process presents an advantage of producing specified phospholipids that rarely exist in nature. In this study, we investigated the regiospecific modification of phosphatidylcholine (PC) in the sn-1 position using immobilized Rhizopus oryzae. In a reaction mixture containing egg yolk PC and exogenous lauric acid (LA) in n-hexane, lipase-producing R. oryzae cells immobilized within biomass support particles (BSPs) showed a much higher transesterification activity than lipase powders. To improve the product yield, several parameters including substrate ratio and reaction time were investigated, resulting in the incorporation of 44.2% LA into the product PC after a 48-h reaction. The analysis of the molecular structure showed that a large proportion of exogenous LA (>90%) was incorporated in the sn-1 position of the enzymatically modified PC. Moreover, the BSP-immobilized R. oryzae maintained its activity for more than 12 batch cycles. The presented results, therefore, suggest the applicability of BSP-immobilized R. oryzae as a whole-cell biocatalyst for the regiospecific modification of phospholipids.     

Immobilization of Rhizopus oryzae in a modified polyvinyl alcohol gel for L(+)-lactic acid production

The production of L(+)-lactic acid (LA) by Rhizopus oryzae immobilized in polyvinyl alcohol (PVA) was investigated. To decrease diffusional resistance, we modified the PVA gel through the addition of sodium alginate and phosphate esterification. The production of L(+)-LA improved notably in the immobilized Rhizopus oryzae. Maximum L(+)-LA production (106.27 g/L), with a yield of 73.1 % and rate of 2.95 g/L·h, was obtained at a temperature of 38 °C, 6 % PVA, and 0.8 % sodium alginate. The immobilized R. oryzae was stable in 14 serial-batch cultures using non-growth medium. The immobilized beads also displayed good tolerance to low temperature and long-term storage at 4 °C with the preservation of biochemical properties.     

Investigations on alkaline protease production with B. subtilis PE-11 immobilized in calcium alginate gel beads

Bacillus subtilis PE-11 cells were immobilized in calcium alginate and used for the production of alkaline protease. The influence of alginate concentration, different cations, concentration of cation, curing time, bead diameter and nutrient strength on alkaline protease production and stability of biocatalyst were investigated. Repeated batch fermentations of immobilized cells in shake flasks were carried out with the optimized parameters such as; 3% alginate, 0.25 M calcium chloride with 1 h curing time, 3.24 mm bead diameter and 0.75% glucose and 0.75% peptone as nutrients. The results indicated that, a good level of enzyme was maintained for a period of about 9 days. The immobilized cells of B. subtilis PE-11 in calcium alginate are more efficient for the production of alkaline protease with repeated batch fermentation.     

Removal of malachite green from aqueous solution by immobilized Pseudomonas sp. DY1 with Aspergillus oryzae

Triphenylmethane dyes are considered to be one of the most recalcitrant pollutants in the environment. Malachite Green (MG) was successfully removed from aqueous solution by Pseudomonas sp. DY1 immobilization with Aspergillus oryzae. Inhibition test in the presence of sodium azide and nystatin indicated that A. oryzae was a natural immobilization reagent, and removal of MG by the immobilized cell pellets was attributed to the biodegradation by Pseudomonas sp. DY1. Optimum conditions of immobilization for maximum biodegradation were obtained using Taguchi design at 37 °C, inoculation size of Pseudomonas sp. DY1 (dry cell mass) 0.01 g, of A. oryzae (spore number) 1.0 × 10⁹, initial pH 6.5. Decolorization and biodegradation of MG by immobilized pellets under optimum conditions were 99.5% and 93.3%, respectively. Immobilized pellets exhibited more than 96% decolorization after 16 days in batch condition, indicating it had stable and high biodegradation capabilities when immobilized for long-term operation.     

Fumaric acid production from xylose by immobilized Rhizopus arrhizus cells

Summary The production of fumaric acid by immobilized Rhizopus arrhizus TKK 204-1-1a mycelium was optimized in batch fermentations using statistical experimental design and empiric modelling. The maximum fumaric acid concentration was obtained at a xylose concentration of about 6% and a carbon:nitrogen ratio of about 160. In repeated batch fermentations with immobilized cells the highest volumetric productivity of fumaric acid reached was 87 mg/l per hour when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 1.75 days. The maximum product concentration was 16.4 g/l when the initial xylose concentration was 10%, the C:N ratio 160 and the residence time 10.25 days. The maximum yield from initial xylose (6.47%) was 23.7% with a product concentration of 15.3 g/l and volumetric productivity of 71 mg/l per hour at a residence time of 9 days and a C:N ratio of 188.3. Immobilization could increase the fumaric acid concentration to a level 3.4 times higher than that produced by free cells.     

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%).     

Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

Modeling of continuous L(+)-lactic acid production with immobilized R. oryzae in an airlift bioreactor

Continuous L(+)-lactic acid production was carried out in an airlift bioreactor with immobilized R. oryzae in polyurethane foam cubes. In a pseudo-steady state, the productivity of lactic acid increased with increasing dilution rate or feeding glucose concentration. A double-layer reaction-diffusion model for the pseudo-steady state process was developed to describe the bioreaction system. Using independently determined model parameters, the model prediction agreed well with the experimental results. Therefore, the model can be employed to understand the fermentation behavior, and for the process design and optimization.     

Co-production of lactic acid and chitin using a pelletized filamentous fungus Rhizopus oryzae cultured on cull potatoes and glucose

Aims: This paper developed a novel process for lactic acid and chitin co-production of the pelletized Rhzious oryzae NRRL 395 fermentation using underutilized cull potatoes and glucose as nutrient source. Methods and Results: Whole potato hydrolysate medium was first used to produce the highest pelletized biomass yield accompanying the highest chitin content in biomass. An enhanced lactic acid production then followed up using batch, repeated batch and fed batch culture with glucose as carbon source and mixture of ammonia and sodium hydroxide as neutralizer. The lactic acid productivity peaked at 2·8 and 3 g l⁻¹ h⁻¹ in repeated batch culture and batch culture, respectively. The fed batch culture had the highest lactate concentration of 140 g l⁻¹. Conclusions: Separation of the biomass cultivation and the lactic acid production is able to not only improve lactic acid production, but also enhance the chitin content. Cull potato hydrolysate used as a nutrient source for biomass cultivation can significantly increase both biomass yield and chitin content. Significance and Impact of the Study: The three-step process using pelletized R. oryzae fermentation innovatively integrates utilization of agricultural residues into the process of co-producing lactic acid and chitin, so as to improve the efficiency, revenues and cost of fungal lactic acid production.     

Growth and bacteriocin production by Enterococcus faecium DPC1146 in batch and continuous culture

Production of the bacteriocin enterocin 1146 (E1146) by Enterococcus faecium DPC1146 was studied in batch and continuous fermentation. Growth was strongly inhibited by lactic acid. In batch fermentations maximum E1146 activity (2.8 MBU L⁻¹) was obtained in 9 h with 20 g L⁻¹ glucose. Increase in initial glucose concentration did not lead to a proportional increase in E1146 activity. A simple linear model was found to be adequate to explain the relationship between specific bacteriocin production rate and specific growth rate in batch fermentations with initial glucose concentration higher than 20 g L⁻¹. Maximum bacteriocin activity (2.9–3.2 MBU L⁻¹) was obtained in continuous fermentations at dilution rates between 0.12 and 0.17 h⁻¹ and specific bacteriocin production rate increased linearly with dilution rate. Received 31 July 1996/ Accepted in revised form 01 November 1996     

Propionic acid fermentation by Propionibacterium acidipropionici: effect of growth substrate

Summary Batch propionic acid fermentations by Propionibacterium acidipropionici with lactose, glucose, and lactate as the carbon source were studied. In addition to propionic acid, acetic acid, succinic acid and CO₂ were also formed from lactose or glucose. However, succinic acid was not produced in a significant amount when lactate was the growth substrate. Compared to fermentations with lactose or glucose at the same pH, lactate gave a higher propionic acid yield, lower cell yield, and lower specific growth rate. The specific fermentation or propionic acid production rate from lactate was, however, higher than that from lactose. Since about equimolar acid products would be formed from lactate, the reactor pH remained relatively unchanged throughout the fermentation and would be easier to control when lactate was the growth substrate. Therefore, lactate would be a preferred substrate over lactose and glucose for propionic acid production using continuous, immobilized cell bioreactors. Correspondence to: S. T. Yang     

Production of glycerol by immobilizedPichia farinosa

Summary Cells of the osmophilic yeastPichia farinosa were immobilized in sintered glass Raschig rings for the production of glycerol. The kinetics of production were observed under different conditions in batch, fed-batch and semicontinuous fermentations in fixed-bed column reactors and compared with those of free cells. 2.6 × 10⁹ cells/g sintered glass were adsorbed. The glycerol productivity amounted to 8.1 g/l per day. The highest concentration reached in batch culture was 86 g/l with immobilized cells. Fermentations using immobilized cells were accelerated compared to fermentations using free cells and maximum yield and productivity were reached at lower initial sugar concentrations. Using scanning electron microscopy it was observed that the shape of the cells was related to the sugar concentration in the medium. The experiments show thatP. farinosa produces glycerol with a high and constant productivity over long periods of time.     

Enhancement and modeling of microparticle-added <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> lactic acid production

Lactic acid has a wide industrial application area and can be produced by fungal strains. However, excessive bulk growth form of fungi during the fermentations is a major problem, which limits the fermentation performance. Microparticles are excellent tools to prevent bulk fungal growth and provide homogenized fermentation broth to increase uniformity and the prediction performance of the models. Therefore, in this study, addition of aluminum oxide and talcum microparticles into fermentations was evaluated to enhance the production of lactic acid by Rhizopus oryzae. The results showed that the bulk fungal growth was prevented and the lactic acid concentration increased from 6.02 to 13.88 and 24.01 g/L, when 15 g/L of aluminum oxide or 10 g/L of talcum was used, respectively, in the shake-flask fermentations. Additionally, substrate concentration, pH, and agitation were optimized in the bioreactors using response surface methodology, and optimum values were determined as 126 g/L of glucose, 6.22 pH, and 387 rpm, respectively. Under these conditions, lactic acid production further increased to 75.1 ± 1.5 g/L with 10 g/L of talcum addition. Also, lactic acid production and glucose consumption in the batch fermentation were successfully modeled with modified Gompertz model and modified logistic model. RMSE and MAE values for lactic acid production were calculated as 2.279 and 1.498 for the modified Gompertz model; 3.6 and 4.056 for the modified logistic model. Additionally, modified logistic model predicted glucose consumption with ?2.088 MAE and 2.868 RMSE, whereas these values were calculated as 2.035 and 3.946 for the modified Gompertz model.     

Organic and inorganic nitrogen source effects on the metabolism of Clostridium acetobytulicum

Summary The effects of organic and inorganic nitrogen combinations on cell growth, solvent production and nitrogen utilization by Clostridium acetobutylicum ATCC 824 was studied in batch fermentations. Fermentations in media with 10 mM glutamic acid, as the organic nitrogen source, and 0 mM to 10 mM ammonium chloride, as the inorganic nitrogen source had a solvent yield of 0.8 to 1.08 mmol solvent/mmol glucose used, with a slow fermentation rate (2 mmol solvent/l h^-1). When media contained 20 mM or 30 mM glutamic acid as well as 2.5 to 7.5 mM ammonium chloride the fermentation rate increased (5.5 mmol/l h^-1) while the solvent yield remained constant (0.86 to 0.96 mmol solvent/mmol glucose used). Total solvent production was higher in media containing 20 mM or 30 mM glutamic acid than with 10 mM glutamic acid.     

Characteristics of immobilized Rhizopus oryzae in polyurethane foam cubes

Summary Rhizopus oryzae was immobilized in polyurethane foam cubes. The effects of the cube size on cell immobilization, cell growth and L(+)-lactic acid production were studied. By the natural attachment method, R. oryzae could be easily immobilized in the polyurethane foam cubes larger than 2.5 × 5 × 5 mm³. The use of small cubes for R. oryzae immobilization was very effective to increase the productivity of L(+)-lactic acid by the immobilized cells. Although it was difficult for smaller cubes to be completely full of the mycelia, increasing the inoculum size in immobilizations was effective to increase the immobilization ratio (a ratio of the number of the cubes containing cells to the total number of cubes).     

Propionic acid production by immobilized cells of a propionate-tolerant strain of Propionibacterium acidipropionici

Cells of the propionate-tolerant strain Propionibacterium acidipropionici P200910, immobilized in calcium alginate beads, were tested for propionic and acetic acid production both in a semidefined laboratory medium and in corn steep liquor in batch, fed-batch, and continuous fermentation. Cell density was about 9.8 × 10⁹ cells/g (wet weight) of beads, and beads were added to the medium at 0.1 g (wet weight) beads/ml. Beads could be reused for several consecutive batch fermentations; propionic acid production in the tenth cycle was about 50%–70% of that in the first cycle. In batch culture complete substrate consumption (glucose in semidefined medium, lactate in corn steep liquor) and maximum acid production were seen within 36 h, and acid yields from the substrate were higher than in free-cell fermentations. Fed-batch fermentations were incubated up to 250 h. Maximum propionic acid concentrations obtained were 45.6 g/l in corn steep liquor and 57 g/l in semidefined medium; this is the highest concentration achieved to date in our laboratory. Maximum acetic acid concentrations were 17 g/l and 12 g/l, respectively. In continuous fermentation of semidefined medium, dilution rates up to 0.31 h^–1 could be used, which gave higher volumetric productivities (0.96 g l^–1 h^–1 for propionic acid and 0.26 g l^–1 h^–1 for acetic acid) than we have obtained with free cells. Corn steep liquor shows promise as an inexpensive medium for production of both acids by immobilized cells of propionibacteria.Journal paper no. J- 15614 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project no. 3122     

Metabolic engineering of Rhizopus oryzae: Effects of overexpressing fumR gene on cell growth and fumaric acid biosynthesis from glucose

Fumaric acid is a dicarboxylic acid used extensively in synthetic resins, food acidulants, and other applications, including oil field fluids and esters. The filamentous fungus Rhizopus oryzae is known for its ability to produce and accumulate high levels of fumaric acid under aerobic conditions. In this work, the overexpression of native fumarase encoded by fumR and its effect on fumaric acid production in R. oryzae were investigated. Three plasmids containing the endogenous fumR gene were constructed and used to transform R. oryzae, and all transformants showed significantly increased fumarase activity during both the seed culture (growth) and fermentation (fumaric acid production) stages. However, fumarase overexpression in R. oryzae yielded more malic acid, instead of fumaric acid, in the fermentation because the overexpressed fumarase also catalyzed the hydration of fumaric acid to malic acid. The results suggested that the overexpressed fumarase, encoded by fumR, by itself was not responsible for the over-production of fumaric acid in R. oryzae.