Continuous production of ellagic acid in a packed-bed reactor

Ellagic acid is a high-value bioactive compound that is used in the food, cosmetic and pharmaceutical industries. The aim of this work was to develop a continuous system for ellagic acid production. Ellagitannase produced by solid-state fermentation and attached to polyurethane foam particles was used as a biocatalyst in a continuous bioreactor for the hydrolysis of ellagitannins from pomegranate by-product. A packed-bed reactor containing the biocatalyst (22.22 Units per gram of dry solid, U gds⁻¹) was fed with a pomegranate ellagitannins solution (0.1%, w/v) at a flow rate of 0.27 mL min⁻¹ at 60 °C. The bioreactor completed several biotransformations while maintaining the hydrolysis rate (60%) with a half-life of 10 continuous cycles of ellagic acid production. Volumetric productivity and ellagic acid yield were 1.09 g L⁻¹ h⁻¹ and 235.89 mg g⁻¹ of pomegranate ellagitannins during the first 70 min of hydrolysis, respectively. The developed biocatalyst showed good operational and mechanical stability and may be successfully used for ellagitannin hydrolysis in a continuous system. This is the first report of high-yield continuous production of ellagic acid using an auto-immobilized enzyme.     

Effect of individual and mixed live yeast culture feeding on growth performance,nutrient utilization and microbial crude protein synthesis in lambs

This study investigated effects of feeding three individual, and a mixed, yeast culture (Kluyveromyces marximanus NRRL3234, Saccharomyces cerevisiae NCDC42, Saccharomyces uvarum ATCC9080 all in a 1:1:1, ratio) on growth performance, nutrient utilization and microbial crude protein (CP) synthesis in feedlot lambs during the post-weaning phase of growth. Sixty weaner lambs (90 ± 3.5 d old and 15.9 ± 0.50 kg BW) were fed for 91 d in five equal groups. The control group of lambs received sterilized culture medium while the treatment groups were fed a yeast culture in addition to a ad libitum total mixed ration (TMR). The yeast culture, dosed at 1 ml/kg body weight (BW) had 1.5–2.0 × 10⁹ live cells/ml. Yeast culture supplementation did not influence intake and digestibility of organic matter (OM), CP, neutral detergent fiber (NDF), acid detergent fiber (ADF) and hemicellulose and the metabolizable energy (ME) level of the diets were similar between control and yeast supplemented lambs. Lambs in all groups were in positive N balance, but N intake and N voided in feces and urine, as well as N balance, did not change due to yeast culture supplementation. Urinary allantoin excretion was similar, but purine derivatives absorbed (mM/d) were higher (P<0.05) in yeast culture supplemented lambs. Yeast culture supplementation improved (P<0.05) microbial CP synthesis. Supplementation of SC and mixed yeast improved (P=0.002) BW gain of lambs by 21% and 16% respectively. All yeast culture supplemented lambs had higher feed efficiency in comparison to control lambs. Among the three yeast cultures used, S. cerevisiae had the most potential as a growth promoting feed additive in feedlot lamb production, and it may serve as an alternate to antibiotics and ionophores as a growth promoter of weaner lambs.     

Isolation of hexavalent chromium resistant bacteria from industrial saline effluents and their ability of bioaccumulation

The mixed cultures has been isolated from industrial saline wastewater contaminated with chromium(VI), using enrichment in the presence of 50 mg l⁻¹ chromium(VI) and 4% (w/v) NaCl at pH 8. In this study, the molasses (M) medium was selected a suitable medium for the effective chromium bioaccumulation by the mixed cultures. Eleven pure isolates obtained from mixed cultures and some of them showed high bioaccumulation in the M media containing about 100 mg l⁻¹ chromium(VI) and 4% NaCl. The strain 8 (99.3%) and 10 (99.1%) were able to bioaccumulate more efficient than the mixed culture (98.9%) in this media. But the highest specific Cr uptake was obtained by the mixed cultures followed by strain 8 and 10 with 56.71, 33.14 and 21.7 mg g⁻¹, respectively. Bioaccumulation of chromium(VI) ions by the strain 8 growing in the media with chromium(VI) and NaCl was studied in a batch system as a function of initial chromium(VI) (86.6–547.6 mg l⁻¹) and NaCl (0, 2, 4, 6% w/v) concentrations. During all the experiments, the uptake yield of the strain 8 was highly affected from NaCl concentrations in the medium at high initial chromium(VI) concentrations. But at low chromium(VI) concentration, strain 8 was not affected from NaCl concentrations in the medium. The maximum uptake yield were obtained in the M media with 2% NaCl as 98.8% for 110.0 mg l⁻¹, 98.6% for 217.1 mg l⁻¹, 98.6% for 381.7 mg l⁻¹ and 98.2% for 547.6 mg l⁻¹ initial chromium(VI) concentrations. The strain 8 tolerated a 6% (w/v) NaCl concentration was able to bioaccumulate more than 95% of the applied chromium(VI) at the 97.6–224.4 mg l⁻¹ initial chromium(VI) concentrations. The results presented in this paper was shown that these pure and mixed cultures might be of use for the bioaccumulation of chromium(VI) from saline wastewater.     

Isolation and characterization of ellagitannins as the major polyphenolic components of Longan (Dimocarpus longan Lour) seeds

Longan (Dimocarpus longan Lour, syn. Euphoria longan Lam.) represents an important fruit in Northern Thailand and has significant economic impact. The fruit is either consumed fresh or as commercially prepared dried and canned products. The canning industry in Thailand produces considerable quantities of waste products, in particular Longan seeds. Because these seeds may be an exploitable source of natural phenolic antioxidants, it was of interest to identify, purify and quantitate the major potential antioxidant phenolics contained therein. The polyphenolic fraction from ground Longan seeds was obtained by extraction with methanol after delipidation with hexane. The hexane extract contained predominantly long-chain fatty acids with major contributions from palmitic (35%) and oleic (28%) acids. The polyphenolic fraction (80.90 g/kg dry weight) was dominated by ellagic acid (25.84 g/kg) and the known ellagitannins corilagin (13.31 g/kg), chebulagic acid (13.06 g/kg), ellagic acid 4-O-α-l-arabinofuranoside (9.93 g/kg), isomallotinic acid (8.56 g/kg) and geraniin (5.79 g/kg). Structure elucidation was performed with mass spectrometry and complete assignment of ¹H and ¹³C NMR signals. The methanol extracts exhibited strong antioxidant capacities with an IC₅₀ of 154 μg/ml for reactive oxygen species attack on salicylic acid and 78 μg/ml for inhibition of xanthine oxidase in the hypoxanthine/xanthine oxidase assay. The extracts were less effective in the 2-deoxyguanosine assay (IC₅₀ = 2.46 mg/ml), indicating that gallates along with ellagic acid and its congeners exert their potential antioxidant effects predominantly by precipitation of proteins such as xanthine oxidase. This was confirmed for the pure compounds gallic acid, methyl gallate, ellagic acid and corilagin.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Conversion of cassava flour to fuel ethanol by sequential solid state and submerged cultures

A process for conversion of cassava flour to ethanol was developed. This involved direct inoculation of Aspergillus awamori spores into a cassava flour paste and incubation for some period during which hydrolytic enzymes are produced (solid state culture or koji production) and subsequent addition of water and yeast cells, during which there is simultaneous hydrolysis and ethanol production (submerged culture). When cassava flour alone was used for the solid state phase, the paste was very sticky, making mixing and aeration difficult. However, addition of rice bran improved the texture and enzyme production. The optima rice bran concentration, spore inoculum concentration, and duration of solid state culture before submerged culture were 20%, 6.16 × 10⁶ spores/100 g, and 2 days, respectively. Under these optimum conditions, a high ethanol concentration of 120 g/L and ethanol yield of 0.309 g-ethanol/g-cassava flour were obtained. This ethanol yield corresponds to 0.44 g-ethanol/g-cassava starch.     

Effects of culture redox potential on succinic acid production by Corynebacterium crenatum under anaerobic conditions

During mixed-acid fermentation by Corynebacterium crenatum under anaerobic conditions, two moles of NADH are required to synthesize 1 mol of succinic acid. In this work, four controlled culture redox potentials and different carbon sources with different oxidation states were used to investigate the possibility of enhancing the succinic acid production by increasing the availability of NADH. When the culture redox potential was ?300 mV, the yield of succinic acid was 0.31 g/g, representing a 72% increase compared with the yield when the culture redox potential was ?40 mV. Meanwhile, the molar ratio of succinic acid/lactic acid increased from 0.27 to 0.48. When 0.1% neutral red was added to the acid production medium, the yield of succinic acid was 0.25 g/g, and the molar ratio of succinic acid/lactic acid was 0.38. Both values were higher than those obtained from glucose only (0.19 g/g, 0.26) or gluconate (0.05 g/g, 0.18). A higher NADH/NAD⁺ ratio and increased enzymatic activity could be achieved to enhance the succinic acid production by manipulating the culture to a more reductive environment.     

Improved accumulation of phenylethanoid glycosides by precursor feeding to suspension culture of Cistanche salsa

Effects of some precursors on phenylethanoid glycosides (PeGs) accumulation in Cistanche salsa cell suspension cultures were investigated. Precursors such as tyrosine, phenylalanine, caffeic acid and cucumber juice at proper concentrations could increase the total accumulation of PeGs (echinacoside, acteoside, 2′-acetylacteoside) by 50%, 12%, 12% and 23%, respectively. Under the combined feeding of precursors at proper concentrations, the total production of PeGs in bio-staged culture reached the highest amount of 1358.1 mg l⁻¹ (640.8 mg echinacoside l⁻¹, 689.4 mg acteoside l⁻¹ and 54.9 mg 2′-acetylacteoside l⁻¹), which was about two-fold of that in the control. This study showed promise for obtaining large-scale production of active ingredients in plant cells by the solid–liquid two step culture (SLTSC) technique and also provided for the first time an example for producing PeGs by C. salsa cell culture. The improved production of PeGs was higher than that in previous reports on PeG production by Cistanche deserticola cell culture fed with precursors.     

Improvement in natamycin production by Streptomyces natalensis with the addition of short-chain carboxylic acids

Natamycin is an important tetraene (polyene) antibiotic produced in submerged culture by different strains of Streptomyces sp. In the present work, the effects of the addition of short-chain carboxylic acids (acetic, propionic and butyric) on cell growth and the kinetics of natamycin production were investigated during submerged cultivation of Streptomyces natalensis. The addition of acetic and propionic acids showed stimulatory effects on natamycin production when added to the fermentation medium at concentrations below 2 g L^?1 at the beginning of cultivation. In addition, when acetic and propionic acids were added in a mixture (7:1) at a total concentration of 2 g L^?1, antibiotic production increased significantly, reaching 3.0 g L^?1 (approximately 223% and 250% increases in volumetric and specific antibiotic production, respectively, compared with the control culture). Moreover, the addition of carboxylic acids not only increased the antibiotic yield but also decreased the production time from 96 h to only 84 h in shake-flask cultures. A further enhancement in natamycin production was achieved by cultivation in a 2-L stirred-tank bioreactor under controlled pH conditions. The maximum volumetric production of 3.98 g L^?1 was achieved after 84 h in carboxylic acid-supplemented culture (acetate and propionate in a ratio of 7:1).     

In vitro methane formation and carbohydrate fermentation by rumen microbes as influenced by selected rumen ciliate species

Ciliate protozoa contribute to ruminal digestion and emission of the greenhouse gas methane. Individual species of ciliates co-cultured with mixed prokaryote populations were hypothesized to utilize carbohydrate types differently. In an in vitro batch culture experiment, 0.6 g of pure cellulose or xylan was incubated for 24 h in 40-mL cultures of Entodinium caudatum, Epidinium ecaudatum, and Eudiplodinium maggii with accompanying prokaryotes. Irrespective of ciliate species, gas formation (mL) and short-chain fatty acids (SCFA) concentrations (mmol L^?1) were higher with xylan (71; 156) than with cellulose (52; 105). Methane did not differ (7.9% of total gas). The SCFA profiles resulting from fermentation of the carbohydrates were similar before and after removing the ciliates from the mixed microbial population. However, absolute methane production (mL 24 h^?1) was lower by 50% on average after removing E. caudatum and E. maggii. Methanogen copies were less without E. maggii, but not without E. ecaudatum. Within 3 weeks part of this difference was compensated. Butyrate proportion was higher in cultures with E. maggii and E. ecaudatum than with E. caudatum and only when fermenting xylan. In conclusion, the three ciliate species partly differed in their response to carbohydrate type and in supporting methane formation.     

Effect of chlorate,molybdate, and shikimic acid on Salmonella enterica serovar Typhimurium in aerobic and anaerobic cultures

Experiments were conducted to determine factors that affect sensitivity of Salmonella enterica serovar Typhimurium to sodium chlorate (5 mM). In our first experiment, cultures grown without chlorate grew more rapidly than those with chlorate. An extended lag before logarithmic growth was observed in anaerobic but not aerobic cultures containing chlorate. Chlorate inhibition of growth during aerobic culture began later than that observed in anaerobic cultures but persisted once inhibition was apparent. Conversely, anaerobic cultures appeared to adapt to chlorate after approximately 10 h of incubation, exhibiting rapid compensatory growth. In anaerobic chlorate-containing cultures, 20% of total viable counts were resistant to chlorate by 6 h and had propagated to 100% resistance (>10⁹ CFU mL^?1) by 24 h. In the aerobic chlorate-containing cultures, 12.9% of colonies had detectable resistance to chlorate by 6 h, but only 1% retained detectable resistance at 24 h, likely because these cultures had opportunity to respire on oxygen and were thus not enriched via the selective pressure of chlorate. In another study, treatment with shikimic acid (0.34 mM), molybdate (1 mM) or their combination had little effect on aerobic or anaerobic growth of Salmonella in the absence of added chlorate. As observed in our earlier study, chlorate resistance was not detected in any cultures without added chlorate. Chlorate resistant Salmonella were recovered at equivalent numbers regardless of treatment after 8 h of aerobic or anaerobic culture with added chlorate; however, by 24 h incubation chlorate sensitivity was completely restored to aerobic but not anaerobic cultures treated with shikimic acid or molybdate but not their combination. Results indicate that anaerobic adaptation of S. Typhimurium to sodium chlorate during pure culture is likely due to the selective propagation of low numbers of cells exhibiting spontaneous resistance to chlorate and this resistance is not reversible by molybdenum supplementation.     

From zero to hero – Production of bio-based nylon from renewable resources using engineered Corynebacterium glutamicum

Polyamides are important industrial polymers. Currently, they are produced exclusively from petrochemical monomers. Herein, we report the production of a novel bio-nylon, PA5.10 through an integration of biological and chemical approaches. First, systems metabolic engineering of Corynebacterium glutamicum was used to create an effective microbial cell factory for the production of diaminopentane as the polymer building block. In this way, a hyper-producer, with a high diaminopentane yield of 41% in shake flask culture, was generated. Subsequent fed-batch production of C. glutamicum DAP-16 allowed a molar yield of 50%, a productivity of 2.2 g L⁻¹ h⁻¹, and a final titer of 88 g L⁻¹. The streamlined producer accumulated diaminopentane without generating any by-products. Solvent extraction from alkalized broth and two-step distillation provided highly pure diaminopentane (99.8%), which was then directly accessible for poly-condensation. Chemical polymerization with sebacic acid, a ten-carbon dicarboxylic acid derived from castor plant oil, yielded the bio-nylon, PA5.10. In pure form and reinforced with glass fibers, the novel 100% bio-polyamide achieved an excellent melting temperature and the mechanical strength of the well-established petrochemical polymers, PA6 and PA6.6. It even outperformed the oil-based products in terms of having a 6% lower density. It thus holds high promise for applications in energy-friendly transportation. The demonstration of a novel route for generation of bio-based nylon from renewable sources opens the way to production of sustainable bio-polymers with enhanced material properties and represents a milestone in industrial production.     

Signal transducer and oxidative stress mediated modulation of phenylpropanoid pathway to enhance rosmarinic acid biosynthesis in fungi elicited whole plant culture of Solenostemon scutellarioides

This study aimed to improve rosmarinic acid (RA) production in the whole plant culture of Solenostemon scutellarioides through elicitation with phytopathogenic fungi. Amongst selected fungi, Aternaria alternata caused significant elevation (p < 0.05–0.01) in RA accumulation (∼1.3–1.6-fold) between 25 and 100 μg l⁻¹. However, elicitation at the dose of 50 μg l⁻¹ has been found to be most effective and intracellular RA content reached almost ∼1.6-fold (p < 0.01) higher in day 7. Therefore, A. alternata (50 μg l⁻¹) was selected for mechanism evaluation. A significant elevation of intercellular jasmonic acid was observed up to day 6 after elicitation with A. alternata (50 μg l⁻¹). A significant increase in tissue H₂O₂ and lipid peroxidation coupled with depletion of antioxidant enzymes superoxide dismutase and catalase indicated augmented oxidative stress associated with biotic interaction. Preceding the elicitor-induced RA accumulation, a notable alteration in the specific activities of biosynthetic enzymes namely PAL and TAT was recorded, while, no significant change in the activities of RAS was observed. HPPR activity was slightly improved in elicited plant. Therefore, it could be concluded that A. alternata elicited the biosynthesis of rosmarinic acid via signal transduction through jasmonic acid coupled with elicitor induced oxidative stress and associated mechanism.     

Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid

The production of bio-based succinic acid is receiving great attention, and several predominantly prokaryotic organisms have been evaluated for this purpose. In this study we report on the suitability of the highly acid- and osmotolerant yeast Saccharomyces cerevisiae as a succinic acid production host. We implemented a metabolic engineering strategy for the oxidative production of succinic acid in yeast by deletion of the genes SDH1, SDH2, IDH1 and IDP1. The engineered strains harbor a TCA cycle that is completely interrupted after the intermediates isocitrate and succinate. The strains show no serious growth constraints on glucose. In glucose-grown shake flask cultures, the quadruple deletion strain Δsdh1Δsdh2Δidh1Δidp1 produces succinic acid at a titer of 3.62 g L^?1 (factor 4.8 compared to wild-type) at a yield of 0.11 mol (mol glucose)^?1. Succinic acid is not accumulated intracellularly. This makes the yeast S. cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.     

Simultaneous saccharification and fermentation of kraft pulp by recombinant Escherichia coli for phenyllactic acid production

Simultaneous saccharification and fermentation (SSF) of renewable cellulose for the production of 3-phenyllactic acid (PhLA) by recombinant Escherichia coli was investigated. Kraft pulp recovered from biomass fractionation processes was used as a model cellulosic feedstock and was hydrolyzed using 10–50 filter paper unit (FPU) g⁻¹ kraft pulp of a commercial cellulase mixture, which increased the glucose yield from 21% to 72% in an enzyme dose-dependent manner. PhLA fermentation of the hydrolyzed kraft pulp by a recombinant E. coli strain expressing phenylpyruvate reductase from Wickerhamia fluorescens TK1 produced 1.9 mM PhLA. The PhLA yield obtained using separate hydrolysis and fermentation was enhanced from 5.8% to 42% by process integration into SSF of kraft pulp (20 g L⁻¹) in a complex medium (pH 7.0) at 37 °C. The PhLA yield was negatively correlated with the initial glucose concentration, with a five-fold higher PhLA yield observed in culture medium containing 10 g L⁻¹ glucose compared to 100 g L⁻¹. Taken together, these results suggest that the PhLA yield from cellulose in kraft pulp can be improved by SSF under glucose-limited conditions.     

Enrichment,isolation, and characterization of phenol-degrading Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6

The objectives of this research were to isolate pure phenol-degrading strains from enriched mixed cultures, monitoring the variations of species during the enrichment period. Two strains were isolated from the acclimated mixed culture. They were identified as Pseudomonas resinovorans strain P-1 and Brevibacillus sp. strain P-6. DGGE indicated that strain P. resinovorans appeared at the beginning, and maintained well during the enrichment period. The second strain, Brevibacillus sp., did not appear in the initial stage, but showed up after 2 weeks of enrichment. The optimum growth temperatures for P. resinovorans and Brevibacillus sp. were 31 and 39 °C, respectively. P. resinovorans could degrade phenol completely within 57.5 h, when the initial phenol concentration was lower than 600 mg l⁻¹. If the initial phenol concentration was lower than 200 mg l⁻¹, Brevibacillus sp. could remove phenol completely within 93.1 h. It was obvious that the phenol-degrading ability of P. resinovorans was much better than that of Brevibacillus sp. The metabolic pathway for P. resinovorans phenol degradation was assigned to the meta-cleavage activity of catechol 2,3-dioxygenase.     

Continuous production of selenomethionine-enriched Chlorella sorokiniana biomass in a photobioreactor

This article describes the enrichment of the fresh-water green microalga Chlorella sorokiniana in selenomethionine (SeMet). The microalga was cultivated in a 2.2 L glass-vessel photobioreactor, in a culture medium supplemented with selenate (SeO₄^2?) concentrations ranging from 5 to 50 mg L^?1. Although selenate exposure lowered culture viability, C. sorokiniana grew well at all tested selenate concentrations, however cultures supplemented with 50 mg L^?1 selenate did not remain stable at steady state. A suitable selenate concentration in fresh culture medium for continuous operation was determined, which allowed stable long-term cultivation at steady state and maximal SeMet productivity. In order to do that, the effect of dilution rate on biomass productivity, viability and SeMet content of C. sorokiniana at several selenate concentrations were determined in the photobioreactor. A maximal SeMet productivity of 21 μg L^?1 day^?1 was obtained with 40 mg L^?1 selenate in the culture medium. Then a continuous cultivation process at several dilution rates was performed at 40 mg L^?1 selenate obtaining a maximum of 246 μg L^?1 day^?1 SeMet at a low dilution rate of 0.49 day^?1, calculated on total daily effluent volume. This paper describes for the first time an efficient long-term continuous cultivation of C. sorokiniana for the production of biomass enriched in the high value amino acid SeMet, at laboratory scale.     

Influence on dithiolopyrrolone antibiotic production by organic acids in Saccharothrix algeriensis NRRL B-24137

The influence of organic acids on growth and dithiolopyrrolone antibiotic production by Saccharothrix algeriensis NRRL B-24137 was studied. The production of dithiolopyrrolones depends upon the nature and concentration of the organic acids in the culture medium. Study of the nature of organic acids showed that the most effective organic acids for thiolutin specific production were maleic, 4-hydroxybenzoic, benzentetracarboxylic, pantothenic, pivalic and pyruvic acids (which yielded almost five-fold over the starting medium) and pimelic acid (more than three-fold). 4-Bromobenzoic acid showed the best production of senecioyl-pyrrothine (59 mg g⁻¹ DCW). Tiglic acid showed the best production of tigloyl-pyrrothine (22 mg g⁻¹ DCW). The highest yield of isobutyryl-pyrrothine (7.6 mg g⁻¹ DCW) was observed in the presence of crotonic acid. Sorbic acid yielded the best production of butanoyl-pyrrothine (26 mg g⁻¹ DCW). Methacrylic, butyric, pyruvic and 4-bromobenzoic acids also exhibited the best production of butanoyl-pyrrothine (27–11-fold).Study of organic acid concentration showed that among the selected organic acids, pimelic acid yielded the highest specific production of thiolutin (91 mg g⁻¹ DCW) at 7.5 mM; and senecioyl-pyrrothine (11 mg g⁻¹ DCW), tigloyl-pyrrothine (9 mg g⁻¹ DCW) and butanoyl-pyrrothine (3.5 mg g⁻¹ DCW) at 5 mM. Pyruvic acid at 1.25 mM enhanced the production of senecioyl-pyrrothine (4.3 mg g⁻¹ DCW). The maximum production of tigloyl-pyrrothine (18.6 mg g⁻¹ DCW) was observed in the presence of tiglic acid at 2.5 mM. Maximum production of isobutyryl-pyrrothine was observed in the presence of 7.5 mM tiglic acid. In addition, methacrylic acid (at 5 mM) and butyric acid (at 2.5 mM) enhanced the production of butanoyl-pyrrothine (26 and 20 times, respectively).The above results can be employed in the optimisation of the culture medium for the production of dithiolopyrrolone in higher quantities.     

The simultaneous utilization of kinetic analysis and flow cytometry in the assessment of Lactobacillus rhamnosus ATCC 7469 physiological states produced by increasing oxygen limitation levels and lactic acid accumulation

Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1 h⁻¹ and 0.6 h⁻¹. At 0.45 h⁻¹, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD⁺ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids.The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D = 0.20 h⁻¹, 8% at D = 0.30 h⁻¹, 14% at D = 0.45 h⁻¹ and 26% for D = 0.62 h⁻¹, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment.     

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus for acetic acid production

Mixed culture of Saccharomyces cerevisiae and Acetobacter pasteurianus was carried out for high yield of acetic acid. Acetic acid production process was divided into three stages. The first stage was the growth of S. cerevisiae and ethanol production, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. The second stage was the co-culture of S. cerevisiae and A. pasteurianus, fermentation temperature and aeration rate were maintained at 34 °C and 0.4 vvm, respectively. The third stage was the growth of A. pasteurianus and production of acetic acid, fermentation temperature and aeration rate were controlled at 32 °C and 0.2 vvm, respectively. Inoculation volume of A. pasteurianus and S. cerevisiae was 16% and 0.06%, respectively. The average acetic acid concentration was 52.51 g/L under these optimum conditions. To enhance acetic acid production, a glucose feeding strategy was subsequently employed. When initial glucose concentration was 90 g/L and 120 g/L glucose was fed twice during fermentation, acetic acid concentration reached 66.0 g/L.