Continuous propionate production from whey permeate using a novel fibrous bed bioreactor

Continuous production of propionate from whey lactose by Propionibacterium acidipropionici immobilized in a novel fibrous bed bioreactor was studied. In conventional batch propionic acid fermentation, whey permeate without nutrient supplementation was unable to support cell growth and failed to give satisfactory fermentation results for over 7 days. However, with the fibrous bed bioreactor, a high fermentation rate and high conversion were obtained with plain whey permeate and de-lactose whey permeate. About 2% (wt/vol) propionic acid was obtained from a 4.2% lactose feed at a retention time of 35 to 45 h. The propionic acid yield was approximately 46% (wt/vol) from lactose. The optimal pH for fementation was 6.5, and lower fermentation rates and yields were obtained at lower pH values. The optimal temperature was 30 degrees C, but the temperature effect was not dramatic in the range of 25 to 35 degrees C. Addition of yeast extract and trypticase to whey permeate hastened reactor startup and increased the fermentation rate and product yields, but the addition was not required for long-term reactor performance. The improved fermentation results with the immobilized cell bioreactor can be attributed to the high cell density, approximately 50 g/L, attained in the bioreactor, Cells were immobilized by loose attachement to fiber surfaces and entrapment in the void spaces within the fibrous matrix, thus allowing constant renewal of cells. Consequently, this bioreactor was able to operate continuously for 6 months without encountering any clogging, degeneration, or contamination problems. Compared to conventional batch fermentors, the new bioreactor offers many advantages for industrial fermentation, including a more than 10-fold increase in productivity, acceptance of low-nutrient feedstocks such as whey permeate, and resistance to contamination. (c) 1994 John Wiley & Sons, Inc.     

Continuous propionic acid fermentation by immobilized Propionibacterium acidipropionici in a novel packed-bed bioreactor

Continuous propionic acid fermentations of lactate by Propionibacterium acidipropionici were studied in spiral wound fibrous bed bioreactors. Cells were imobilized by natural attachment to fiber surfaces and entrapment in the void volume within the fibrous matrix. A high cell density of approximately 37 g/L was attained in the reactor and the reactor productivity was approximately 4 times higher than that from a conventional batch fermentation. The bioreactor was able to operate continuously for 4 months without encountering any clogging, degeneration, or contamination problems. Also, the reactor could accept low-nutrient and low-pH feed without sacrificing much in reactor productivity. This new type of immobilized cell bioreactor is scalable and thus is suitable for industrial production of propionate. (c) 1992 John Wiley & Sons, Inc.     

Enhanced propionic acid fermentation by Propionibacterium acidipropionici mutant obtained by adaptation in a fibrous-bed bioreactor

Fed-batch fermentations of glucose by P. acidipropionici ATCC 4875 in free-cell suspension culture and immobilized in a fibrous-bed bioreactor (FBB) were studied. The latter produced a much higher propionic acid concentration (71.8 +/- 0.8 g/L vs. 52.2 +/- 1.1 g/L), indicating enhanced tolerance to propionic acid inhibition by cells adapted in the FBB. Compared to the free-cell fermentation, the FBB culture produced 20-59% more propionate (0.40-0.65 +/- 0.02 g/g vs. 0.41 +/- 0.02 g/g), 17% less acetate (0.10 +/- 0.01 g/g vs. 0.12 +/- 0.02 g/g), and 50% less succinate (0.09 +/- 0.02 g/g vs. 0.18 +/- 0.03 g/g) from glucose. The higher propionate production in the FBB was attributed to mutations in two key enzymes, oxaloacetate transcarboxylase and propionyl CoA: succinyl CoA transferase, leading to the production of propionic acid from pyruvate. Both showed higher specific activity and lower sensitivity to propionic acid inhibition in the mutant than in the wild type. In contrast, the activity of PEP carboxylase, which converts PEP directly to oxaloacetate and leads to the production of succinate from glucose, was generally lower in the mutant than in the wild type. For phosphotransacetylase and acetate kinase in the acetate formation pathway, however, there was no significant difference between the mutant and the wild type. In addition, the mutant had a striking change in its morphology. With a threefold increase in its length and approximately 24% decrease in its diameter, the mutant cell had an approximately 10% higher specific surface area that should have made the mutant more efficient in transporting substrates and metabolites across the cell membrane. A slightly lower membrane-bound ATPase activity found in the mutant also indicated that the mutant might have a more efficient proton pump to allow it to better tolerate propionic acid. In addition, the mutant had more longer-chain saturated fatty acids (C17:0) and less unsaturated fatty acids (C18:1), both of which could decrease membrane fluidity and might have contributed to the increased propionate tolerance. The enhanced propionic acid production from glucose by P. acidipropionici was thus attributed to both a high viable cell density maintained in the reactor and favorable mutations resulted from adaptation by cell immobilization in the FBB.     

Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH

Batch propionic acid fermentation of lactose by Propionibacterium acidipropionici were studied at various pH values ranging from 4.5 to 7.12. The optimum pH range for cell growth was between 6.0 and 7.1, where the specific growth rate was approximately 0.23 h(-1). The specific growth rate decreased with the pH in the acids have been identified as the two major fermentation products from lactose. The production of propionic acid was both growth and nongrowth associated, while acetic acid formation was closely associated with cell growth. The propionic acid yield increased with decreasing pH; It changed from approximately 33% (w/w) at pH 6.1-7.1 to approximately 63% at pH 4.5-5.0. In contrast, the acetic acid yield was not significantly affected by the pH; it remained within the range of 9%-12% at all pH values. Significant amounts of succinic and pyruvic acids were also formed during propionic acid fermentation of lactose. However, pyruvic acid was reconsumed and disappeared toward the end of the fermentation. The succinic acid yield generally decreased with the pH, from a high value of 17% at pH 7.0 to a low 8% at pH 5.0 Effects of growth nutrients present in yeast ex-tract on the fermentation were also studied. In general, the same trend of pH effects was found for fermentations with media containing 5 to 10 g/L yeast extract. However, More growth nutrients would be required for fermentations to be carried out efficienytly at acidic pH levels.     

Engineering Propionibacterium acidipropionici for enhanced propionic acid tolerance and fermentation

Propionibacterium acidipropionici, a Gram‐positive, anaerobic bacterium, has been the most used species for propionic acid production from sugars. In this study, the metabolically engineered mutant ACK‐Tet, which has its acetate kinase gene knocked out from the chromosome, was immobilized and adapted in a fibrous bed bioreactor (FBB) to increase its acid tolerance and ability to produce propionic acid at a high final concentration in fed‐batch fermentation. After about 3 months adaptation in the FBB, the propionic acid concentration in the fermentation broth reached ～100 g/L, which was much higher than the highest concentration of ～71 g/L previously attained with the wild‐type in the FBB. To understand the mechanism and factors contributing to the enhanced acid tolerance, adapted mutant cells were harvested from the FBB and characterized for their morphology, growth inhibition by propionic acid, protein expression profiles as observed in SDS–PAGE, and H⁺‐ATPase activity, which is related to the proton pumping and cell's ability to control its intracellular pH gradient. The adapted mutant obtained from the FBB showed significantly reduced growth sensitivity to propionic acid inhibition, increased H⁺‐ATPase expression and activity, and significantly elongated rod morphology. Biotechnol. Bioeng. 2009; 104: 766–773 © 2009 Wiley Periodicals, Inc.     

Production of propionic acid from whey permeate by sequential fermentation, ultrafiltration, and cell recycling

This article deals with the production by fermentation of a mycostatic and aromatic food additive based on propionic acid. Membrane bioreactors have been used from laboratory scale up to pilot and industrial production plants. Due to the high cell densities achieved by the sequential recycling mode of operation, a mixed acids solution was rapidly produced from whey permeate. The sterile fermented broth obtained was subsequently concentrated at different levels by evaporation and spray drying according to the projected use. Concentrated Propionibacterium cells (200 g . L(-1) DW) were obtained from the process by periodic bleeds and could be used to good effect as cheese starters, silage preservatives, or probiotics. Propionic acid concentrations from 30 to 40 g . L(-1) were easily achieved with no residual lactose. The highest volumetric productivity was 1.6 g . L(-1) . h(-1) for total acid and 1.2 g . L(-1) . h(-1) for propionic acid with a specific productivity of 0.035 h(-1). (c) 1993 John Wiley & Sons, Inc.     

Plant cell bioreactor for the production of protoberberine alkaloids from immobilized Thalictrum rugosum cultures

Cultured Thalictrum rugosum cells were immobilized using a glass fiber substratum previously shown to provide optimum immobilization efficiency based on spontaneous adhesion mechanisms. When cultivated in shake flasks, immobilized cells exhibited decreased growth and protoberberine alkaloid production rates in comparison to freely suspended cells. Since alkaloid production is growth associated in T. rugosum, the decreased specific production rate was a function of the slower growth rate. Cells immobilized on glass fiber mats appear to be amenable for extended culture periods. Maximum biomass and protoberberine alkaloid levels were maintained for at least 14 days in immobilized cultures. In contrast, fresh weight, dry weight, and total alkaloid content decreased in suspension cultures following the linear growth phase.Glass fiber mats were incorporated in to a 4.5-L plant cell bioreactor as horizontal disks supported on a central rod. Mixing in the reactor was provided by the combined actions of a magnetic impeller and a cylindrical sparging colum. fThe magnetic impeller and a cylindrical sparging column. The entire inoculum biomass of T. rougosum, introduced as suspension, was spontaneously immobilized with in 8h. During liner phase, the growth rate of bioreactor cultivated immobilized cells (mu = 0.06 day(-1)) was 50% that immobilized cell viability in both systems was determined to be similar. The increase in specific production of protoberberine alklodis was initially similar in bioreactor-and culture period. The increase in specific production of protoberberine alkaloids was initially similar in bioreactor-and shake-flask-cultivated immobilized cells. However, the maximum specific production of bioreactor grown cultures was lower. The scale up potential of an immobilization strategy based on the spontaneous adhesion of immobilization strategy based on the spontaneous adhesion of cultured plant cells to glass fiber is demonstrated.     

Cephalosporin C production by immobilized Cephalosporium acremonium cells in a repeated batch tower bioreactor

The industrial production of antibiotics with filamentous fungi is usually carried out in conventional aerated and agitated tank fermentors. Highly viscous non-Newtonian broths are produced and a compromise must be found between convenient shear stress and adequate oxygen transfer. In this work, cephalosporin C production by bioparticles of immobilized cells of Cephalosporium acremonium ATCC 48272 was studied in a repeated batch tower bioreactor as an alternative to the conventional process. Also, gas-liquid oxygen transfer volumetric coefficients, k(L)a, were determined at various air flow-rates and alumina contents in the bioparticle. The bioparticles were composed of calcium alginate (2.0% w/w), alumina ( < 44 micra), cells, and water. A model describing the cell growth, cephalosporin C production, oxygen, glucose, and sucrose consumption was proposed. To describe the radial variation of oxygen concentration within the pellet, the reaction-diffusion model forecasting a dead core bioparticle was adopted. The k(L)a measurements with gel beads prepared with 0.0, 1.0, 1.5, and 2.0% alumina showed that a higher k(L)a value is attained with 1.5 and 2.0%. An expression relating this coefficient to particle density, liquid density, and air velocity was obtained and further utilized in the simulation of the proposed model. Batch, followed by repeated batch experiments, were accomplished by draining the spent medium, washing with saline solution, and pouring fresh medium into the bioreactor. Results showed that glucose is consumed very quickly, within 24 h, followed by sucrose consumption and cephalosporin C production. Higher productivities were attained during the second batch, as cell concentration was already high, resulting in rapid glucose consumption and an early derepression of cephalosporin C synthesizing enzymes. The model incorporated this improvement predicting higher cephalosporin C productivity.     

Continuous propionic acid production from cheese whey usingin situ spin filter

The potential use of spin filter device to retainPropionibacterium acidipropionici in the bioreactor under continuous mode of fermentation and improve propionic acid productivity, was examined. The yield of propionic acid based on lactose concentration was 51% in batch and 54% in continuous (dilution rate=0.05 h⁻¹) operation. The yield in continuous fermentation with cell retention using spin filter of 10 micron size (dilution rate=0.05 h⁻¹) was even higher at 70% (w/w). The volumetric productivity under batch and continuous mode of operation were 0.312 g L⁻¹ h⁻¹ and 0.718 g L⁻¹ h⁻¹ respectively. Continuous fermentation with cell retention demonstrated even higher volumetric productivities at 0.98 g L⁻¹ h⁻¹ with out clogging problems It could be used for utilization of cheese whey to produce propionic acid at higher yield and productivities.     

Construction and characterization of ack knock-out mutants of Propionibacterium acidipropionici for enhanced propionic acid fermentation

Propionibacterium acidipropionici produces propionic acid from glucose with acetic acid, succinic acid, and CO2 as byproducts. In this work, inactivation of ack gene, encoding acetate kinase (AK), by gene disruption and integrational mutagenesis was studied as a method to reduce acetate formation in propionic acid fermentation. The partial ack gene of approximately 750 bp in P. acidipropionici was cloned using a PCR-based method with degenerate primers and sequenced. The deduced amino acid sequence had 88% similarity and 76% identity with the amino acid sequence of AK from Bacillus subtilis. The partial ack gene was used to construct a linear DNA fragment with an inserted tetracycline resistance cassette and a nonreplicative integrational plasmid containing a tetracycline resistance gene cassette. These DNA constructs were then introduced into P. acidipropionici by electroporation, resulting in two mutants, ACK-Tet and TAT-ACK-Tet, respectively. Southern hybridization confirmed that the ack gene in the mutant ACK-Tet was disrupted by the inserted tetracycline resistance gene. As compared to the wild-type, the activities of AK were reduced by 26% and 43% in ACK-Tet and TAT-ACK-Tet mutants, respectively. The specific growth rate of these mutants was reduced by approximately 25% to 0.10/h (0.13/h for the wild-type), probably because of reduced acetate and ATP production. Both mutants produced approximately 14% less acetate from glucose. Although ack disruption alone did not completely eliminate acetate production, the propionate yield was increased by approximately 13%.     

Efficient co‐production of propionic acid and succinic acid by Propionibacterium acidipropionici using membrane separation coupled technology

To improve the fermentation efficiency of Propionibacterium acidipropionici, a semi‐continuous coupled fermentation process was established to achieve co‐production of propionic acid (PA) and succinic acid (SA). First, the optimal proportion of glucose (Glc) and glycerol (Gl) as a mixed carbon source was determined, and the feeding procedure of Gl was optimized to make more energy flow in the direction of product synthesis. Then, ZGD630 anion exchange resin was used for efficient adsorption of PA, thereby eliminating the feedback inhibition effect of PA. Finally, an efficient, coupled fermentation process of P. acidipropionici characterized by membrane separation and chromatography technology was developed. The concentrations of PA and SA reached 62.22 ± 2.32 and 20.45 ± 1.34 g L⁻¹, with corresponding productivity of 0.43 and 0.14 g L⁻¹ h⁻¹, increased by 65.38% and 48.54%, respectively. Membrane separation coupled fermentation of PA and SA could significantly improve the process economics of P. acidipropionici, and has good prospects for industrial application.     

产酸丙酸杆菌生长特性及5L罐发酵动力学研究

论文在摇瓶水平对产酸丙酸杆菌基本生长特性（温度、pH、摇床转速、接种量、种龄等）、碳源、氮源利用情况、产物抑制及5 L罐发酵动力学进行了研究。结果表明,该菌在32℃,初始pH 6．5,摇床转速150 r/min,接种24 h的种子液,接种量为5％条件下,产酸丙酸杆菌生长及产酸水平达最高值;该菌可利用碳源十分广泛,但对氮源要求比较高,只可利用有机氮源;在不同初始葡萄糖浓度下,产酸丙酸杆菌生长及产酸水平差异不大,无明显底物抑制现象;在2g/L的初始丙酸盐浓度下,该菌生长受到明显抑制;在5L发酵罐中,初始葡萄糖浓度为58．8 g/L,发酵72 h,葡萄糖消耗完全,丙酸终浓度达22．4 g/L,丙酸得率和产率分别达0．381 g/g和0．295 g/（L·h）,丙酸占总酸比例达72．10％。     

Examination of immobilized cells in a rotating packed drum reactor for the production of ethanol from d-glucose

A rotating packed drum reactor has been proposed as an immobilized whole cell reactor and its performance for ethanol production has been studied with yeast cells immobilized in calcium alginate gel. In a continuous operation with synthetic d-glucose medium containing 125 g d-glucose l^?1, ethanol productivity was 20 g l^?1 h^?1 at a space velocity of 0.38 l (l gel)^?1 h^?1. With intermittent aeration the viability of yeast cells after 270 h of operation remained above 65%. CO₂ removal was easy, but d-glucose conversion was low at a high space velocity.     

Simultaneous production of propionic acid and vitamin B12 from corn stalk hydrolysates by Propionibacterium freudenreichii in an expanded bed adsorption bioreactor

Abstract

Vitamin B12 and propionic acid that were simultaneous produced by Propionibacterium freudenreichii are both favorable chemicals widely used in food preservatives, medicine, and nutrition. While the carbon source and propionic acid accumulation reflected fermentation efficiency. In this study, using corn stalk as a carbon source and fed-batch fermentation process in an expanded bed adsorption bioreactor was studied for efficient and economic biosynthesis of acid vitamin B12 and propionic. With liquid hot water pretreated corn stalk hydrolysates as carbon source, 28.65?mg L^?1 of vitamin B12 and 17.05?g L^?1 of propionic acid were attained at 168?h in batch fermentation. In order to optimize the fermentation outcomes, fed-batch fermentation was performed with hydrolyzed corn stalk in expanded bed adsorption bioreactor (EBAB), giving 47.6?mg L^?1 vitamin B12 and 91.4?g L^?1 of propionic acid at 258?h, which correspond to product yields of 0.37?mg g^?1 and 0.75?g g^?1, respectively. The present study provided a promising strategy for economically sustainable production of vitamin B12 and propionic acid by P. freudenreichii fermentation using biomass cornstalk as carbon source and expanded bed adsorption bioreactor.     

Lactic acid production from whey permeate by immobilized Lactobacillus casei

Two matrices have been assessed for their ability to immobilize Lactobacillus casei cells for lactic acid fermentation in whey permeate medium. Agar at 2% concentration was found to be a better gel than polyacrylamide in its effectiveness to entrap the bacterial cells to carry out batch fermentation up to three repeat runs. Of the various physiological parameters studied, temperature and pH were observed to have no significant influence on the fermentation ability of the immobilized organism. A temperature range of 40–50°C and a pH range of 4.5–6.0 rather than specific values, were found to be optimum when fermentation was carried out under stationary conditions. In batch fermentation ～90% conversion of the substrate (lactose) was achieved in 48 h using immobilized cell gel cubes of 4 × 2 × 2 mm size, containing 400 mg dry bacterial cells per flask and 4.5% w/v (initial) whey lactose content as substrate. However, further increase in substrate levels tested (>4.5% w/v) did not improve the process efficiency. Supplementation of Mg²⁺ (1 mM) and agricultural by-products (mustard oil cake, 6%) in the whey permeate medium further improved the acid production ability of the immobilized cells under study.     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

利用纤维床反应器固定化发酵生产丙酸

构建了一种纤维床反应器(FBB), 并将其应用于丙酸的生产。将棉纤维绕成桶状, 固定于反应器中, 即可用于丙酸固定化发酵。以40 g/L的葡萄糖为碳源, 与游离细胞相比, 利用FBB生产丙酸, 丙酸产量由14.58 g/L提高至20.41 g/L, 发酵时间由120 h缩短至60 h。研究了不同糖浓度条件下FBB生产丙酸情况, 并将补料策略应用于丙酸发酵中。结果表明: 补料发酵能够有效改善Propionibacterium freudenreichii CCTCC M207015在高糖条件下丙酸对葡萄糖转化率较低、副产物较多的问题。经补料发酵280 h, 丙酸产量达45.91 g/L, 丙酸质量约占有机酸总质量比例为72.31%。     

A novel three-compartmented electrochemical bioreactor for enrichment of strict anaerobes based on metabolite production

In this study, a novel three-compartmented electrochemical bioreactor (3-CEB) was designed in an effort to overcome the disadvantages of the two-compartmented electrochemical bioreactor (2-CEB) separated with a cation-selective membrane for enrichment of strict anaerobes. The 3-CEB was comprised of an anode, outlet, and a cathode compartment. The outlet compartment was positioned between the anode and cathode compartment, and it was separated with the anode side by a rubber plate and with the cathode side by a porous glass membrane. A platinum wire bridging the anode and outlet compartment operated as a redox passage, however, through which no material could permeate. Butyrate fermentation bacteria were enriched on the basis of the metabolite production. Butyrate generated by strict anaerobes was significantly more abundant in the 3-CEB than in the 2-CEB. Acetic acid and lactic acid generated by facultative anaerobes was relatively higher in the 2-CEB than in the 3-CEB. Meanwhile, butyrate was not generated in the bioreactor utilized for the control test, to which the electrochemical potential was not charged. In a continuous culture using the 3-CEB, the majority of the glucose was fermented to butyrate, and the acetate additionally supplied to the bacterial culture was metabolically reduced to butyrate. More lactate than butyrate was generated from glucose in the 2-CEB.     

Kinetics of biodegradation of p-xylene and naphthalene and oxygen transfer in a novel airlift immobilized bioreactor

The scope of this study included the biodegradation performance and the rate of oxygen transfer in a pilot-scale immobilized soil bioreactor system (ISBR) of 10-L working volume. The ISBR was inoculated with an acclimatized population of contaminant degrading microorganisms. Immobilization of microorganisms on a non-woven polyester textile developed the active biofilm, thereby obtaining biodegradation rates of 81 mg/L x h and 40 mg/L x h for p-xylene and naphthalene, respectively. Monod kinetic model was found to be suitable to correlate the experimental data obtained during the course of batch and continuous operations. Oxygen uptake and transfer rates were determined during the batch biodegradation process. The dynamic gassing-out method was used to determine the oxygen uptake rate (OUR) and volumetric oxygen mass transfer, K(L) a. The maximum volumetric OUR of 255 mg O(2)/L x h occurred approximately at 720-722 h after inoculation, when the dry weight of biomass concentration was 0.67 g/L.