Selection method of pH conditions to establish Pseudomonas taetrolens physiological states and lactobionic acid production

Microbial physiological responses resulting from inappropriate bioprocessing conditions may have a marked impact on process performance within any fermentation system. The influence of different pH-control strategies on physiological status, microbial growth and lactobionic acid production from whey by Pseudomonas taetrolens during bioreactor cultivations has been investigated for the first time in this work. Both cellular behaviour and bioconversion efficiency from P. taetrolens were found to be negatively influenced by pH-control modes carried out at values lower than 6.0 and higher than 7.0. Production schemes were also influenced by the operational pH employed, with asynchronous production from damaged and metabolically active subpopulations at pH values lower than 6.0. Moreover, P. taetrolens showed reduced cellular proliferation and a subsequent delay in the onset of the production phase under acidic conditions (pH?<?6.0). Unlike cultivations performed at 6.5, both pH-shift and pH-stat cultivation strategies performed at pH values lower than 6.0 resulted in decreased lactobionic acid production. Whereas the cellular response showed a stress-induced physiological response under acidic conditions, healthy functional cells were predominant at medium operational pH values (6.5–7.0). P. taetrolens thus displayed a robust physiological status at initial pH value of 6.5, resulting in an enhanced bioconversion yield and lactobionic acid productivity (7- and 4-fold higher compared to those attained at initial pH values of 4.5 and 5.0, respectively). These results have shown that pH-control modes strongly affected both the physiological response of cells and the biological performance of P. taetrolens, providing key information for bio-production of lactobionic acid on an industrial scale.     

Physiological heterogeneity of Pseudomonas taetrolens during lactobionic acid production

Physiological heterogeneity constitutes a critical parameter in biotechnological systems since both metabolite yield and productivity are often hampered by the presence of undesired physiological cell subpopulations. In the present study, the physiological status and functionality of Pseudomonas taetrolens cells were monitored by multiparameter flow cytometry during fermentative lactobionic acid production at the shake-flask and bioreactor scale. In shake-flask fermentation, the onset of the lactobionic acid production phase was accompanied by a progressive loss of cellular metabolic activity, membrane polarization, and membrane integrity concomitantly to acidification. In fact, population dynamics has shown the prevalence of damaged and dead subpopulations when submitted to a pH?<?4 from 16?h onwards. Furthermore, fluorescence-activated cell sorting revealed that these sublethally injured cells were nonculturable. In contrast, P. taetrolens cells exhibited a robust physiological status during bioreactor cultivations performed with a pH-shifted strategy at 6.5, remaining predominantly healthy and metabolically active (>96?%) as well as maintaining bioconversion efficiency throughout the course of the fermentation. Additionally, an assessment of the seed culture’s physiological robustness was carried out in order to determine the best seed culture age. Results showed that bioreactor culture performance, growth, and lactobionic acid production efficiency were strongly dependent on the physiological heterogeneity displayed by the seed culture. This study provides the most suitable criteria for optimizing lactobionic acid production efficiency through a novel flow cytometric-based approach based on the physiological status of P. taetrolens. It also constitutes a valuable, broad-ranging methodology for the enhancement of microbial bioprocesses involved in the production of secondary metabolites.     

Biosynthesis of Lactobionic Acid in Whey-Containing Medium by Microencapsulated and Free Bacteria of Pseudomonas taetrolens

The aim of this research was to develop a method of its production from whey using bacteria of the species Pseudomonas taetrolens. Analyses of the lactobionic acid production method from whey showed that the following factors have a significant effect on its efficiency: the frequency of whey batch feeding, pH and the type of bacteria application, i.e. microencapsulated vs. free. Lactose and lactobionic acid were assayed using high performance liquid chromatography (HPLC) and liquid chromatography-electrospray ionization-tandem mass spectrometry (LC–ESI–MS). The highest concentration of lactobionic acid of 22.03 mg/cm³ was obtained when whey was batch fed at 72-h intervals, pH was maintained at 6.25 and bacteria were enclosed in alginate microcapsules.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12088-021-00944-4.     

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.     

Production of lactobionic acid from whey by Pseudomonas sp. LS13-1

Pseudomonas sp. LS13-1 was isolated as a producer of lactobionic acid from whey and when grown with 207 g whey l^-1 (150 g lactose l^-1 equivalent) and three intermittent additions of 69 g whey l^-1 (50 g lactose l^-1 equivalent) in a fed-batch culture at pH 5.5 in a 2-l jar fermenter, it produced 175 g lactobionic acid l^-1 after 180 h. In a lactose medium it produced 240 lactobionic acid l^-1 from a total of 300 g lactose l^-1 after 155 h. With the addition of 20 CaCO₃ l^-1 instead of pH control, 290 g lactobionic acid l^-1 was produced in the lactose medium after 155 h with a yield of higher than 90% (mon mol^-1).     

Microbial amensalism in Lactobacillus casei and Pseudomonas taetrolens mixed culture

Pseudomonas taetrolens has recently been revealed as an effective microbial producer of lactobionic acid from carbohydrates contained in dairy byproducts. In terms of food industrial applications, the implementation of lactobionic acid biosynthesis coupled with the classic bacterial production of lactic acid appears an important goal. This research paper studies the simultaneous fermentation of residual cheese whey by P. taetrolens and Lactobacillus casei to co-produce lactic and lactobionic acids. Experimental data showed the importance of the interactions established between the two microorganisms. Changes in physiology, viability, growth, and productive capacity were tested experimentally. Lactobacillus was not seen to suffer any appreciable stress, but considerable variations were observed in the Pseudomonas behavior presumably owing to inhibitory lactic metabolites, interaction that can be classified as microbial amensalism. As to production, lactic acid remained without significant changes in mixed fermentations, whereas the production of lactobionic acid decreased sharply due to the competitive exclusion of Pseudomonas.

     

Production of lactobionic acid and sorbitol from lactose/fructose substrate using GFOR/GL enzymes from Zymomonas mobilis cells: a kinetic study

In this work, we have investigated the kinetics of the biotechnological production of lactobionic acid (LBA) and sorbitol by the catalytic action of glucose-fructose oxidoreductase (GFOR) and glucono-δ-lactonase (GL) enzymes. The cells of bacterium Zymomonas mobilis ATCC 29191 containing this enzymatic complex were submitted to permeabilization and reticulation procedures. The effect of the concentration of substrates on the rate of product formation using a mobilized cell system was investigated. The application of higher fructose concentration seems to not affect the initial rate of formation of the bionic acid. Under conditions of low initial concentration of lactose, the experimental kinetic data of the bi-substrate reaction were modelled by assuming a rate equation of the classical ping-pong mechanism. The found kinetic parameters displayed a low affinity of the GFOR enzyme for both substrates. The enzymatic system did not exhibit normal Michaelis-Menten kinetics in response to a change of concentration of lactose, when fructose was held constant, presenting a sigmoid relationship between initial velocity and substrate concentration. A rate equation based on Hill kinetics was used to describe the kinetic behaviour of this enzyme-substituted reaction at higher lactose concentrations. The results from batch experiments using immobilized cells within Ca-alginate beads revealed that there is no pronounced occurrence of mass transfer limitations on LBA production for beads with 1.2 mm in average diameter. This discussion aids for defining the best operating conditions to maximize the productivity for LBA and sorbitol in this bioconversion and also for reducing the complexity of downstream separation processes.     

Production of organic acids by periplasmic enzymes present in free and immobilized cells of Zymomonas mobilis

In this work the periplasmic enzymatic complex glucose-fructose oxidoreductase (GFOR)/glucono-δ-lactonase (GL) of permeabilized free or immobilized cells of Zymomonas mobilis was evaluated for the bioconversion of mixtures of fructose and different aldoses into organic acids. For all tested pairs of substrates with permeabilized free-cells, the best enzymatic activities were obtained in reactions with pH around 6.4 and temperatures ranging from 39 to 45 °C. Decreasing enzyme/substrate affinities were observed when fructose was in the mixture with glucose, maltose, galactose, and lactose, in this order. In bioconversion runs with 0.7 mol l^?1 of fructose and with aldose, with permeabilized free-cells of Z. mobilis, maximal concentrations of the respective aldonic acids of 0.64, 0.57, 0.51, and 0.51 mol l^?1 were achieved, with conversion yields of 95, 88, 78, and 78 %, respectively. Due to the important applications of lactobionic acid, the formation of this substance by the enzymatic GFOR/GL complex in Ca-alginate-immobilized cells was assessed. The highest GFOR/GL activities were found at pH 7.0–8.0 and temperatures of 47–50 °C. However, when a 24 h bioconversion run was carried out, it was observed that a combination of pH 6.4 and temperature of 47 °C led to the best results. In this case, despite the fact that Ca-alginate acts as a barrier for the diffusion of substrates and products, maximal lactobionic acid concentration, conversion yields and specific productivity similar to those obtained with permeabilized free-cells were achieved.     

Homo-fermentative production of d-lactic acid by Lactobacillus sp. employing casein whey permeate as a raw feed-stock

Casein whey permeate (CWP), a lactose-enriched dairy waste effluent, is a viable feed stock for the production of value-added products. Two lactic acid bacteria were cultivated in a synthetic casein whey permeate medium with or without pH control. Lactobacillus lactis ATCC 4797 produced d-lactic acid (DLA) at 12.5 g l^?1 in a bioreactor. The values of Leudking–Piret model parameters suggested that lactate was a growth-associated product. Batch fermentation was also performed employing CWP (35 g lactose l^?1) with casein hydrolysate as a nitrogen supplement in a bioreactor. After 40 h, L. lactis produced 24.3 g lactic acid l^?1 with an optical purity >98 %. Thus CWP may be regarded as a potential feed-stock for DLA production.     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

Xanthan production by Xanthomonas campestris using whey permeate medium

Xanthan gum is a polysaccharide that is widely used as stabilizer and thickener with many industrial applications in food industry. Our aim was to estimate the ability of Xanthomonas campestris ATCC 13951 for the production of xanthan gum by using whey as a growth medium, a by-product of dairy industry. X. campestris ATCC 13951 has been studied in batch cultures using a complex medium for the determination of the optimal concentration of glucose, galactose and lactose. In addition, whey was used under various treatment procedures (de-proteinated, partially hydrolyzed by β-lactamase and partially hydrolyzed and de-proteinated) as culture medium, to study the production of xanthan in a 2 l bioreactor with constant stirring and aeration. A production of 28 g/l was obtained when partially hydrolysed β-lactamase was used, which proved to be one of the highest xanthan gum production reported so far. At the same time, an effort has been made for the control and selection of the most appropriate procedure for the preservation of the strain and its use as inoculant in batch cultures, without loss of its viability and its capability of xanthan gum production. The pre-treatment of whey (whey permeate medium hydrolyzed, WPH) was very important for the production of xanthan by the strain X. campestris ATCC 13951 during batch culture conditions in a 2 l bioreactor. Preservation methods such as lyophilization, cryopreservation at various glycerol solution and temperatures have been examined. The results indicated that the best preservation method for the producing strain X. campestris ATCC 13951 was the lyophilization. Taking into account that whey permeate is a low cost by-product of the dairy industry, the production of xanthan achieved under the studied conditions was considered very promising for industrial application.     

Fermentative bioconversion in a horizontal rotating tubular bioreactor

The performance of a horizontal rotating tubular bioreactor (HRTB) was investigated with a biological system under non-sterile conditions. A spontaneously developed microbial culture was cultivated in a simple glucose/yeast extract medium. A fermentative bioconversion was examined by different combinations of process parameters (bioreactor rotation speed 5–30 min⁻¹ and medium inflow rate 1–10 l h⁻¹). Bioconversion dynamics in HRTB was monitored by withdrawing the samples from five positions along the bioreactor. Investigation in HRTB showed a rapid and an efficient glucose conversion into different products of metabolism. Glucose consumption rate along the HRTB depended on medium inflow rate, while bioreactor rotation speed did not have a significant influence. Complete glucose conversion in HRTB was observed at inflow rates of up to 6.5 l h⁻¹. The pH gradient along the HRTB was detected at higher medium inflow rates (6.5 and 10 l h⁻¹), but did not significantly influence substrate conversion efficiency. A discussion of its potential use and a comparison of HRTB with other bioreactors are also presented.     

Semicontinuous production of lactic acid in a bioreactor coupled with nanofiltration membranes

The advantages of nanofiltration membranes coupled with a CSTR were demonstrated for the semicontinuous production of lactic acid from whey permeate. Lactic acid was removed from the growth medium while lactose was kept in the bioreactor with the bacterial cells; moreover, Mg²⁺ ions were also recycled in the bioreactor at 96% and the nanofiltrate color was greatly reduced. The highest volumetric productivity achieved with this device was 7.1 g l⁻¹ h⁻¹ and the lactate concentration was 55 g l⁻¹. The specific productivity was 3.54 h⁻¹. More than 99% of the membrane fouling after 44 h of fermentation was reversible. The initial permeate flux was restored easily by a water rinse. The performance of this type of membrane bioreactor was discussed.     

Lactobionic and cellobionic acid production profiles of the resting cells of acetic acid bacteria

Lactobionic acid was produced by acetic acid bacteria to oxidize lactose. Gluconobacter spp. and Gluconacetobacter spp. showed higher lactose-oxidizing activities than Acetobacter spp. Gluconobacter frateurii NBRC3285 produced the highest amount of lactobionic acid per cell, among the strains tested. This bacterium assimilated neither lactose nor lactobionic acid. At high lactose concentration (30%), resting cells of the bacterium showed sufficient oxidizing activity for efficient production of lactobionic acid. These properties may contribute to industrial production of lactobionic acid by the bacterium. The bacterium showed higher oxidizing activity on cellobiose than that on lactose and produced cellobionic acid.     

Production of free fatty acids from switchgrass using recombinant Escherichia coli

Switchgrass is a promising feedstock to generate fermentable sugars required for the sustainable operation of biorefineries because of their abundant availability, easy cropping system, and high cellulosic content. The objective of this study was to investigate the potentiality of switchgrass as an alternative sugar supplier for free fatty acid (FFA) production using engineered Escherichia coli strains. Recombinant E. coli strains successfully produced FFAs using switchgrass hydrolysates. A total of about 3 g/L FFAs were attained from switchgrass hydrolysates by engineered E. coli strains. Furthermore, overall yield assessments of our bioconversion process showed that 88 and 46% of the theoretical maximal yields of glucose and xylose were attained from raw switchgrass during sugar generation. Additionally, 72% of the theoretical maximum yield of FFAs were achieved from switchgrass hydrolysates by recombinant E. coli during fermentation. These shake‐flask results were successfully scaled up to a laboratory scale bioreactor with a 4 L working volume. This study demonstrated an efficient bioconversion process of switchgrass‐based FFAs using an engineered microbial system for targeting fatty acid production that are secreted into the fermentation broth with associated lower downstream processing costs, which is pertinent to develop an integrated bioconversion process using lignocellulosic biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:91–98, 2018     

A model‐driven approach towards rational microbial bioprocess optimization

Due to sustainability concerns, bio‐based production capitalizing on microbes as cell factories is in demand to synthesize valuable products. Nevertheless, the nonhomogenous variations of the extracellular environment in bioprocesses often challenge the biomass growth and the bioproduction yield. To enable a more rational bioprocess optimization, we have established a model‐driven approach that systematically integrates experiments with modeling, executed from flask to bioreactor scale, and using ferulic acid to vanillin bioconversion as a case study. The impacts of mass transfer and aeration on the biomass growth and bioproduction performances were examined using minimal small‐scale experiments. An integrated model coupling the cell factory kinetics with the three‐dimensional computational hydrodynamics of bioreactor was developed to better capture the spatiotemporal distributions of bioproduction. Full‐factorial predictions were then performed to identify the desired operating conditions. A bioconversion yield of 94% was achieved, which is one of the highest for recombinant Escherichia coli using ferulic acid as the precursor.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Anaerobic fermentative production of lactic acid using cheese whey and corn steep liquor

Cheese whey was the most suitable substrate for production of lactic acid under anaerobic conditions by Entercoccus flavescens which, on supplementating with corn steep liquor (5% v/v) and 10 mM CaCO₃ at pH 5.5, 37°C, yielded 12.6 g lactic acid/l in 36 h. Production was scaled up to a 10 l bioreactor under controlled pH and continuous CO₂ supply and gave 28 g lactic acid/l in 30 h resulting in a net 8.7-fold increase in production as compared to unoptimized conditions.     

Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor

Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis. The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor. Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey. Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation. The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h). Both lactate and acetate at high concentrations inhibited the homoacetic fermentation. To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate. The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C. formicoaceticum. Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume. The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred. This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey.     

Bio-production of lactobionic acid: Current status,applications and future prospects

Lactobionic acid has appeared on the commercial scene as a versatile polyhydroxy acid with numerous promising applications in the food, medicine, pharmaceutical, cosmetics and chemical industries. This high value-added bio-product has recently received growing attention as a bioactive compound, providing an excellent chemical platform for the synthesis of novel potentially biocompatible and biodegradable drug delivery vehicles. Recent advances in tissue engineering and nanomedicine have also underlined the increased importance of this organic acid as a key biofunctionalization agent. The growing commercial relevance of lactobionic acid has therefore prompted the development of novel systems for its biotechnological production that are both sustainable and efficient. The present review explores recent advances and studies related to lactobionic acid bio-production, whether through microbial or enzymatic approaches, highlighting the key bioprocessing conditions for enhanced bio-production. Detailed overviews of the current microbial cell factories as well as downstream processing methodologies for lactobionic acid production are also presented. Furthermore, the potential prospects and current applications of this polyhydroxy acid are also discussed, with an emphasis on the role of lactobionic acid as a key platform in the development of novel drugs, biomaterials, nanoparticles and biopolymer systems.