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).     

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.     

Efficient lactobionic acid production from whey by Pseudomonas taetrolens under pH-shift conditions

Lactobionic acid finds applications in the fields of pharmaceuticals, cosmetics and medicine. The production of lactobionic acid from whey by Pseudomonas taetrolens was studied in shake-flasks and in a bioreactor. Shake-flask experiments showed that lactobionic acid was a non-growth associated product. A two-stage pH-shift bioconversion strategy with a pH-uncontrolled above 6.5 during the growth phase and maintained at 6.5 during cumulative production was adopted in bioreactor batch cultures. An inoculation level of 30% promoted high cell culture densities that triggered lactobionic acid production at a rate of 1.12 g/Lh. This methodology displayed efficient bioconversion with cheese whey as an inexpensive substrate for lactobionic acid production.     

Glucose metabolic flux distribution of Lactobacillus amylophilus during lactic acid production using kitchen waste saccharified solution

The ¹³C isotope tracer method was used to investigate the glucose metabolic flux distribution and regulation in Lactobacillus amylophilus to improve lactic acid production using kitchen waste saccharified solution (KWSS). The results demonstrate that L. amylophilus is a homofermentative bacterium. In synthetic medium, 60.6% of the glucose entered the Embden–Meyerhof–Parnas (EMP) to produce lactic acid, whereas 36.4% of the glucose entered the pentose phosphate metabolic pathway (HMP). After solid–liquid separation of the KWSS, the addition of Fe³⁺ during fermentation enhanced the NADPH production efficiency and increased the NADH content. The flux to the EMP was also effectively increased. Compared with the control (60.6% flux to EMP without Fe³⁺ addition), the flux to the EMP with the addition of Fe³⁺ (74.3%) increased by 23.8%. In the subsequent pyruvate metabolism, Fe³⁺ also increased lactate dehydrogenase activity, and inhibited alcohol dehydrogenase, pyruvate dehydrogenase and pyruvate carboxylase, thereby increasing the lactic acid production to 9.03 g l⁻¹, an increase of 8% compared with the control. All other organic acid by-products were lower than in the control. However, the addition of Zn²⁺ showed an opposite effect, decreasing the lactic acid production. In conclusion it is feasible and effective means using GC-MS, isotope experiment and MATLAB software to integrate research the metabolic flux distribution of lactic acid bacteria, and the results provide the theoretical foundation for similar metabolic flux distribution.     

Production of extracellular lactase fromFusarium moniliforme

Summary A strain ofFusarium moniliforme, previously used for microbial protein production, excreted lactase (-D-galactosidase, EC.3.2.1 23) when cultivated either in a whey liquid medium or on a wheat bran solid medium. The enzyme produced in both media had pH and temperature optima of 4–5 and 50–60°C respectively and was particularly suitable for processing acid whey.In the whey culture, maximum lactase yield was observed after 95 h of growth at 30°C and whey lactose concentration of 9%. The addition of ammonium, potassium and sodium ions to the growth medium considerably enhanced lactase production. A maximum enzyme yield corresponding to hydrolysis of 3 nmoles o-nitrophenyl--D-galactopyranoside sec^–1 ml^–1 of growth medium, at pH 5 and 60°C, was obtained.In the wheat bran culture, the maximum enzyme yield was obtained after 140 h of growth at 28–30°C. A marked increase in the enzyme production was observed when nitrate or phosphate was added to the growth medium. Also, the addition of certain agricultural by-products (molasses, whey) enhanced lactase production. The observed maximum yield corresponding to the hydrolysis of 182 nmoles of ONPG sec^–1 g^–1 of wheat bran, at pH 5 and 60°C, is comparable to that reported for certain microorganisms used commercially for lactase production.     

Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

Background

Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H₂ production involves consumption of 2H⁺, hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms.

Methods and Principal Findings

We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H₂ to 2H⁺. Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H₂ production and consumption was tested using a H₂-specific Clark-type electrode. Hyd-3-dependent H₂ production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H₂ consumption was maximal at alkaline pH. H₂ production, was unaffected by a shift in external or internal pH. H₂ production was associated with hycE expression levels as a function of external pH.

Conclusions

Anaerobic growing cultures of E. coli generate H₂ via Hyd-3 at low external pH, and consume H₂ via Hyd-2 at high external pH. Hyd-3 proton conversion to H₂ is required for acid resistance in anaerobic cultures of E. coli.     

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.     

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.     

Ethanol production from whey permeate by immobilized yeast cells

The ability of two yeast strains to utilize the lactose in whey permeate has been studied. Kluyveromyces marxianus NCYC 179 completely utilized the lactose (9.8%), whereas Saccharomyces cerevisiae NCYC 240 displayed an inability to metabolize whey lactose for ethanol production. Of the two gel matrices tested for immobilizing K. marxianus NCYC 179 cells, sodium alginate at 2% (w/v) concentration proved to be the optimum gel for entrapping the yeast cells effectively. The data on optimization of physiological conditions of fermentation (temperature, pH, ethanol concentration and substrate concentration) showed similar effects on immobilized and free cell suspensions of K. marxianus NCYC 179, in batch fermentation. A maximum yield of 42.6 g ethanol l^?1 (82% of theoretical) was obtained from 98 g lactose l^?1 when fermentation was carried at pH 5.5 and 30°C using 120 g dry weight l^?1 cell load of yeast cells. These results suggest that whey lactose can be metabolized effectively for ethanol production using immobilized K. marxianus NCYC 179 cells.     

Kinetic study of Lactococcus lactis strains (SLT6 and SLT10) growth on papain-hydrolysed whey

The effect of controlled whey hydrolysis by papain on growth of two lactic acid bacteria isolated from artisanal Leben: Lactococcus lactis var. diacetylactis (SLT6 and SLT10) was investigated. The higher biomass and maximum specific growth rate (μ _max) were obtained after 30 min of hydrolysis. HPLC analysis of peptides showed that whey hydrolysis reduced the amount of peptides of MW > 400 Da and increased those peptides of MW < 400 Da. The two studied strains exhibited different peptide requirements. The pH-controlled batch cultures in 30 min hydrolysed whey followed the Monod kinetic for growth and for lactate production. The values of the key kinetic constants were: maximum specific growth rate (μ _max), 1.08 and 0.56 h^?1; yield biomass on lactose (Y _x/s), 0.20 and 0.18 g g^?1 and saturation constant K _s, 4.2 and 2.8 g L^?1 for SLT6 and SLT10, respectively. When compared with batch experimental data, the model provided good predictions for growth, lactose utilisation and lactate production profiles.     

Acetic acid production from whey lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum

Summary Acetic acid was produced from anaerobic fermentation of lactose by the co-culture ofStreptococcus lactis andClostridium formicoaceticum at 35° C and pHs between 7.0 and 7.6. Lactose was converted to lactic acid, and then to acetic acid in this mixed culture fermentation. The overall acetic acid yield from lactose was about 95% at pH 7.6 and 90% at pH 7.0. The fermentation rate was also higher at pH 7.6 than at pH 7.0. In batch fermentation of whey permeate containing about 5% lactose at pH 7.6, the concentration of acetic acid reached 20 g/l within 20 h. The production rate then became very slow due to end-product inhibition and high Na⁺ concentration. About 30 g/l acetate and 20 g/l lactate were obtained at a fermentation time of 80 h. However, when diluted whey permeate containing 2.5% lactose was used, all the whey lactose was converted to acetic acid within 30 h by this mixed culture.     

The production of lactic acid from whey permeate byLactobacillus helveticus

Summary The effect of pH on growth and lactic acid production ofLactobacillus helveticus was investigated in a continuous culture using supplemented whey ultrafiltrate. Maximum lactate productivity of 5 gl^–1h^–1 occurred at pH 5.5. Whey permeates concentrated up to four times were fermented using batch cultures. Maximum lactic acid concentration of 95 gl^–1 was attained, but residual sugars indicated a possible limitation in growth factors.Nomenclature D Dilution rate [h^–1] - X Biomass [gl^–1] - Glu Glucose consentration [gl^–1] - Gal Galactose consentration [gl^–1] - S Substrate, Lactose consentration [gl^–1] - P Product, Lactate consentration [gl^–1] - Y_p/s Yield, defined as P/S [gg^–1] - r_i Rate of synthesis or consumption of i [gl^–1h^–1]     

Optimization of S-adenosyl-L-methionine production by Kluyveromyces lactis on whey in batch culture using a mathematical model

Growth, lactose utilization and S-adenosyl-l-methionine (AdoMet) production by Kluyveromyces lactis AM-65 on whey in batch fermentation were investigated and an unstructured model of the process has been derived. The optimal set of parameters was estimated by fitting the model to experimental results. After incubation for 20 h the optimal fermentation conditions (28.5 °C, pH 5.3, agitation at 270 rpm) resulted in AdoMet production at 1.55 g l^–1.     

Production of bioethanol from organic whey using <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis>

Ethanol production by K. marxianus in whey from organic cheese production was examined in batch and continuous mode. The results showed that no pasteurization or freezing of the whey was necessary and that K. marxianus was able to compete with the lactic acid bacteria added during cheese production. The results also showed that, even though some lactic acid fermentation had taken place prior to ethanol fermentation, K. marxianus was able to take over and produce ethanol from the remaining lactose, since a significant amount of lactic acid was not produced (1–2 g/l). Batch fermentations showed high ethanol yield (~0.50 g ethanol/g lactose) at both 30°C and 40°C using low pH (4.5) or no pH control. Continuous fermentation of nonsterilized whey was performed using Ca-alginate-immobilized K. marxianus. High ethanol productivity (2.5–4.5 g/l/h) was achieved at dilution rate of 0.2/h, and it was concluded that K. marxianus is very suitable for industrial ethanol production from whey.     

Proton Electrochemical Gradients in Washed Cells of Nitrosomonas europaea and Nitrobacter agilis

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [³H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, ¹⁴C-benzoic and ¹⁴C-acetyl salicylic, and the weak base [¹⁴C]methylamine. Nitrosomonas europaea accumulated ¹⁴C-benzoic acid and ¹⁴C-acetyl salicylic acid when the external pH was below 7.0 and [¹⁴C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [¹⁴C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Δψ only contributed to Δp. In Nitrosomonas europaea the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Optimization of biogas production from co-digestion of whey and cow manure

Anaerobic co-digestion is effective and environmentally attractive technology for energy recovery from organic waste. Organic, agricultural and industrial wastes are good substrates for anaerobic co-digestion because they contain high levels of easily biodegradable materials. In this paper enhancement of biogas production from codigestion of whey and cow manure was investigated in a series of batch experiments. The influence of whey ratio on specific biogas production in a mixture with cow manure was analyzed at 35 and 55°C, for different initial pH values and for different concentrations of supplemental bicarbonate in experiments carried out over 12 days. Good biogas production (6.6 dm³/dm³), methane content (79.4%) in a biogas mixture and removal efficiencies for total solids (16%) were achieved at optimum process conditions (temperature of 55°C, 10% v/v of whey and 5 g/dm³ NaHCO₃ in the initial mixture). In order to validate optimized conditions for co-digestion of whey and cow manure in the one-stage batch process, the experiments were performed within 45 days. The high biogas production (21.8 dm³/dm³), a good methane content (78.7%) in a biogas mixture as well as maximum removal efficiencies for total solids (32.3%), and chemical oxygen demand (56.3%), respectively indicate that whey could be efficiently degraded to biogas in a onestage batch process when co-digested with cow manure.     

Oxalic acid production by Aspergillus niger

Oxalic acid is formed by Aspergillus niger at nearly neutral pH values. In this study the applicability of milk whey as a carbon source was investigated, both in shaking flask experiments and in a stirred tank reactor. The influence of pH on oxalic acid formation showed that the maximum production rate and higher concentration of the product are observed at pH 6. At pH 7 the same production rate was obtained although at a lower oxalic acid concentration. The process was shown to be inhibited by product from an oxalic acid concentration of about 10?kg/m³ and its behaviour was fitted by Luong's equation. In a 10-dm³ strirred tank ferment the stirrer speed was varied in a range from 100 to 600 rpm. At values between 200 and 400 rpm, maximum production rates of oxalic acid of 6.8?kg/m³·d and 6.5?kg/m³·d were reached, respec-tively. A final concentration of 41.4?kg oxalic acid/m³ was reached operating at 400 rpm.     

Detection of Melamine in Feed Using Liquid-Liquid Extraction Treatment Combined with Surface-Enhanced Raman Scattering Spectroscopy

A rapid, selective, and sensitive method to determine the melamine content in animal feeds was developed using surface-enhanced Raman scattering spectroscopy on aggregated 55 nm Au nanoparticles with liquid–liquid extraction sample preparation. Butyl alcohol was used as the initial extraction solvent, and liquid–liquid extraction was performed twice using HCl (pH 3–4) and 6∶1 (v/v) n-butyl alcohol/ethyl acetate. The intensity of the matrix-based peak at 731 cm⁻¹ was set at 100 as a basis for the feeds, and the peak at 707 cm⁻¹ was the characteristic peak of melamine used in the calculations. Sufficient linearity was obtained in the range 2–10 µg·g⁻¹ (R ² = 0.991). Limits of detection and quantification in the feeds were 0.5 and 2 µg·g⁻¹, respectively. The recovery rates were 82.5–90.2% with coefficients of variation below 4.02%. This new protocol could be easily developed for the routine monitoring of on-site feed quality and market surveillance.     

Synbiotic Fermentation for the Co‐Production of Lactic and Lactobionic Acids from Residual Dairy Whey

Besides its properties as an antioxidant, stabilizer, or acidifier, lactobionic acid has emerged as a potential prebiotic compound, raising the possibility of being included together with the probiotic microorganism Lactobacillus casei in novel functional fermented foods with synbiotic characteristics. Their manufacturing strategy could benefit from the recently implemented microbial synthesis of lactobionic acid by the strong producer Pseudomonas taetrolens, employing residual dairy whey as raw material. The phenomenon of amensalism established between Pseudomonas and Lactobacillus makes simultaneous fermentation unfeasible. A novel sequential process has been developed in which L. casei is inoculated in a second step. Its ability to utilize lactobionic acid as a carbon and energy source was previously tested. Experimental results showed the capacity of L. casei to work efficiently on the residual substrate fermented by P. taetrolens, producing lactic acid by degrading the remaining lactose, with a lactic acid yield on substrate and productivity of 0.95 g g^?1 and 0.20 g L^?1 h^?1, respectively. Lactobionic acid was barely consumed in this complex growth medium, thus ensuring its presence in the resulting fermented product. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1250–1256, 2017