Ethanol production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> and <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> in the presence of orange-peel oil

Summary Two ethanologenic yeasts, Saccharomyces cerevisiae and Kluyveromyces marxianus, were used to ferment sugar solutions modeling hydrolyzed Valencia orange peel waste at 37°C. Orange stripper oil produced from orange peel was added in various amounts to determine its effect on ethanol production. The minimum peel oil concentration that inhibited ethanol production was determined after 24, 48 and 72 h and the two yeasts were compared to one another in terms of ethanol yield. Minimum inhibitory peel oil concentrations for ethanol production were 0.05% at 24 h, 0.10% at 48 h, and 0.15% at 72 h for both yeasts. S. cerevisiae produced more ethanol than K. marxianus at each time point.     

Single cell protein production from mandarin orange peel

Summary As the hydrolysis of mandarin orange peel with macerating enzyme (40°C, 24 h) produced 0.59 g g^–1 reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120°C with 0.8 N H₂SO₄), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel.When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (g g^–1) were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30°C using 100 g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts.     

Production of pyruvic acid from 1,2-propanediol byPseudomonas sp. strain TB-135 which does not require thiamine

Summary Pseudomonas sp. strain TB-135 produces D-lactic acid from 1,2-propanediol (1,2-PD) and requires Ca²⁺, Mg²⁺ and Fe²⁺ for growth but does not require thiamine. The strain produced pyruvic acid only under Fe²⁺-deficient conditions and the addition of Cu²⁺ increased pyruvic acid production. Under optimal conditions (0.03 ppm of FeSO₄ and 0.5ppm of CuSO₄), the strain accumulated 14 mg pyruvic acid par ml after 3 days of cultivation. The thiamine concentration in the cells grown on Fe²⁺-deficient medium was about 6% of that in the cells grown on Fe²⁺-sufficient medium, though pyruvate dehydrogenase (EC 1.2.4.1) activities of both types of cells were the same. We conclude that the low thiamine content of the cells is responsible for the acid production.     

Identification of Flavone Compounds in Eighteen Gramineae Species

Conditions for tryptophan synthesis from pyruvic acid, indole and NH₄Cl by Enterobacter aerogenes AHU 1540 having a high tryptophanase activity, were investigated using a reaction mixture containing 1.7% of pyruvic acid. Under optimum conditions, 16.4g/liter of tryptophan was accumulated after 24 hr of incubation.

Agaricus campestris AHU 9382 produced pyruvic acid in amounts of 22 ~ 26.5 g/liter from 5% of glucose after 3-days shaking culture. When E. aerogenes was added to this fermentation broth together with indole and NH₄Cl, pyruvic acid produced was rapidly converted to tryptophan and yields of tryptophan as high as 15 g/liter were obtained after 12 hr of incubation. Furthermore, pyruvic acid fermentation by Saccharomyces exiguus AHU 3110 or Corynebacterium sp. 37-3A could also be used as a pyruvic acid source for subsequent tryptophan production.     

Optimization of enzymatic hydrolysis and fermentation conditions for improved bioethanol production from potato peel residues

The aim of this work was the optimization of the enzyme hydrolysis of potato peel residues (PPR) for bioethanol production. The process included a pretreatment step followed by an enzyme hydrolysis using crude enzyme system composed of cellulase, amylase and hemicellulase, produced by a mixed culture of Aspergillus niger and Trichoderma reesei. Hydrothermal, alkali and acid pretreatments were considered with regards to the enhancement of enzyme hydrolysis of potato peel residues. The obtained results showed that hydrothermal pretreatment lead to a higher enzyme hydrolysis yield compared to both acid and alkali pretreatments. Enzyme hydrolysis was also optimized for parameters such as temperature, pH, substrate loading and surfactant loading using a response surface methodology. Under optimized conditions, 77 g L^?1 of reducing sugars were obtained. Yeast fermentation of the released reducing sugars led to an ethanol titer of 30 g L^?1 after supplementation of the culture medium with ammonium sulfate. Moreover, a comparative study between acid and enzyme hydrolysis of potato peel residues was investigated. Results showed that enzyme hydrolysis offers higher yield of bioethanol production than acid hydrolysis. These results highlight the potential of second generation bioethanol production from potato peel residues treated with onsite produced hydrolytic enzymes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:397–406, 2017     

Impact of acetaldehyde- and pyruvic acid-bound sulphur dioxide on wine lactic acid bacteria

Aims: To investigate the impact of acetaldehyde‐ and pyruvic acid‐bound sulphur dioxide on wine lactic acid bacteria (LAB). Methods and Results: Growth studies were performed where Oenococcus oeni, Pediococcus parvulus, Ped. damnosus and Lactobacillus hilgardii were inoculated into media containing various concentrations of acetaldehyde or pyruvic acid and an equimolar concentration of SO₂ at pH 3·50 and 3·70. Low concentrations of acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to the growth of all bacteria although acetaldehyde‐bound SO₂ was generally more inhibitory than pyruvic acid‐bound SO₂. Inhibition was greater at pH 3·50 than 3·70, and Lact. hilgardii was the most sensitive to acetaldehyde‐bound SO₂, while O. oeni was the most sensitive to pyruvic acid‐bound SO₂. Degradation of SO₂‐bound acetaldehyde was observed for all LAB, and aside from O. oeni, there was also complete degradation of SO₂‐bound pyruvic acid at both pH values. O. oeni only degraded pyruvic acid at pH 3·70. Degradation of SO₂‐bound acetaldehyde or pyruvic acid did not correlate with bacterial growth as inhibition was always observed in media containing bound SO₂. Conclusions: Acetaldehyde‐ and pyruvic acid‐bound SO₂ were inhibitory to wine LAB growth at concentrations as low as 5 mg l^?1. Despite this inhibition, all wine LAB degraded SO₂‐bound acetaldehyde and pyruvic acid suggesting that bound SO₂ may have a bacteriostatic rather than bacteriocidal action. Significance and Impact of the Study: Sulphur dioxide bound to acetaldehyde or pyruvic acid is inhibitory to growth of wine LAB and must be considered when conducting the malolactic fermentation or controlling the growth of spoilage bacteria such as Pediococcus and Lactobacillus.     

Reverse Reaction of Malic Enzyme for HCO3 − Fixation into Pyruvic Acid to Synthesize L-Malic Acid with Enzymatic Coenzyme Regeneration

Malic enzyme [L-malate: NAD(P)⁺ oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)⁺). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO₃ ^? fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD⁺, and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO₃ ^? and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 °C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD⁺.     

Pyruvic Acid Production by an F1-ATPase-defective Mutant of Escherichia coli W1485lip2

An F₁-ATPase-defective mutant, TBLA-1, was constructed by the transduction of a defective gene for the a subunit of F₁-ATPase, atpA401, into Escherichia coli W1485lip2, a lipoic acid-requiring pyruvic acid producer. The pyruvic acid production of the strain TBLA-1 was found to be improved markedly compared with that of strain W1485lip2. In cultures using a jar fermentor, the strain W1485lip2 consumed 50 g/liter of glucose and produced 25 g/liter of pyruvic acid after culture for 32 h, while strain TBLA-1 consumed the same amount of glucose, and produced more than 30 g/liter of pyruvic acid in a 24-h culture. A revertant, No. 63–1, derived from the strain TBLA-1, had a normal level of F₁-ATPase activity, and showed a similar pattern of pyruvic acid production to that of strain W1485lip2.     

Transformation of Citric Acid to Acetic Acid,Acetoin and Diacetyl by Wine Making Lactic Acid Bacteria

A decrease in citric acid and increases in acetic acid, acetoin and diacetyl were found in the test red wine after inoculation of intact cells of Leuconostoc mesenteroides subsp. lactosum ATCC 27307. a malo-lactic bacterium, grown on the malate plus citrate-medium. Citric acid in the buffer solution was transformed to acetic acid, acetoin and diacetyl in the pH range of 2 to 6 after inoculation with intact cells of this bacterial species. It was concluded that citric acid in wine making involving malolactic fermentation, at first, was converted by citrate lyase to acetic and oxaloacetic acids, and the latter was successively transformed by decarboxylation to pyruvic acid which was subsequently converted to acetoin, diacetyl and acetic acid.

Both the activities of citrate lyase and acetoin formation from pyruvic acid in the dialyzed cell-free extract were optimal at pH 6.0. Divalent cations such as Mn²⁺, Mg²⁺, Co²⁺ and Zn²⁺ activated the citrate lyase. The citrate lyase was completely inhibited by EDTA, Hg²⁺ and Ag²⁺ . The acetoin formation from pyruvic acid was significantly stimulated by thiamine pyrophosphate and CoCl₂, and inhibited by oxaloacetic acid. Specific activities of the citrate lyase and acetoin formation were considerably variable among the six strains of malo-lactic bacteria examined. Some activities of irreversible reduction of diacetyl to acetoin were found in the cell-free extracts of four of the malo-lactic bacteria strains and the optimal pH was 6.0 for this activity of Leu. mesenteroides.     

Tryptophan Production by a Lipoic Acid Auxotroph of Enterobacter aerogenes Having Both Pyruvic Acid Productivity and High Tryptophanase Activity

A new process for tryptophan production was established using a lipoic acid auxotrophic mutant, Enterobacter aerogenes l-12, which has both pyruvic acid productivity and tryptophanase activity. The process consists of the production of pyruvic acid from glucose by the washed cells and the subsequent conversion of the acid to tryptophan by the tryptophanase itself in the presence of indole and NH₄C1.

To prepare washed cells of which the tryptophanase activity and the pyruvic acid productivity were both high, it was best to culture the strain in a medium containing 1 % Polypepton, 0.2 % glucose, 3 μg/1 dl-lipoic acid, 0.05 % l-tryptophan, and mineral salts. The optimum composition of the reaction mixture for the pyruvic acid production by the washed cells was established. Under these conditions, 17 g/1 of pyruvic acid was accumulated from 5 % glucose after 36 hr of incubation. Thus, the conversion of the pyruvic acid to tryptophan was done by adding indole, NH₄C1, pyridoxal-5′-phosphate, Triton X-100, and KOH to adjust the pH to 9.0 to the above reaction mixture. As a result, the pyruvic acid was rapidly converted to tryptophan, and the concentration of 14 g/1 was obtained after 36 hr (total 72 hr).     

Microbiology and physiology of Cachaça (Aguardente) fermentations

Cachaça (aguardente) is a rum-style spirit made from sugar cane juice by artisanal methods in Brazil. A study was made of the production, biochemistry and microbiology of the process in fifteen distilleries in Sul de Minas. Identification of 443 yeasts showed Saccharomyces cerevisiae to be the predominant yeast but Rhodotorula glutinis and Candida maltosa were predominant in three cases. Bacterial infection is a potential problem, particularly in older wooden vats, when the ratio of yeasts:bacteria can be 10:1 or less. A study of daily batch fermentations in one distillery over one season in which 739 yeasts were identified revealed that S. cerevisiae was the predominant yeast. Six other yeast species showed a daily succession: Kluyveromyces marxianus, Pichia heimii and Hanseniaspora uvarum were present only at the beginning, Pichia subpelliculosa and Debaryomyces hansenii were detected from mid to the end of fermentation, and Pichia methanolica appeared briefly after the cessation of fermentation. Despite a steady influx of yeasts from nature, the species population in the fermenter was stable for at least four months suggesting strong physiological and ecological pressure for its maintenance. Cell densities during the fermentation were: yeasts – 4 × 10⁸/ml; lactic acid bacteria – 4 × 10⁵/ml; and bacilli – 5 × 10⁴/ml. Some acetic acid bacteria and enterobacteriaceae appeared at the end. Sucrose was immediately hydrolysed to fructose and glucose. The main fermentation was complete after 12 hours but not all fructose was utilised when harvesting after 24 hours.     

Cellular content of the Krebs cycle keto acids in yeasts grown on different nitrogen sources,including hydroxylamine

The cellular pool of Krebs cycle keto acids was followed as a function of growth in three yeasts. The keto acids were analyzed as silylated methoximes by quantitative gas chromatography with capillary glass columns. The 2-oxoglutaric acid content was strikingly high in the hydroxylamine(HA)-tolerant, HA-utilizing Endomycopsis lipolytica when compared to that in the nitrate-utilizing yeast Cryptococcus albidus and Saccharomyces cerevisiae, requiring fully reduced nitrogen for growth. The content in E. lipolytica increased throughout the log phase to maxima of about 200–250 g per g dry weight in HA and ammonia media. These amounts are 20–25 times greater than those attained in the two other yeasts. The cellular content of pyruvic acid was at a maximum early in the log phase, amounting to 50–70 g per g dry weight for all yeasts. The oxalacetic acid content never exceeded 9 g per g dry weight in any of the yeasts. Oximeformation, for which keto acid production is a prerequisite, is discussed as part of the HA-tolerance.Abbreviations HA hydroxylamine - GLC gas-liquid chromatography - BSTFA N,O-bis-(trimethylsilyl)-trifluoroacetamid - BTPPC benzyltriphenylphosphonium chloride     

Pyruvic acid production by a lipoic acid auxotroph of Escherichia coliW1485

A lipoic acid auxotroph of Escherichia coli K-12, strain W1485lip2 (ATCC25645), produced pyruvic acid aerobically from glucose under the lipoic acid-deficient conditions, while the prototrophic parent strain, W1485 (ATCC12435), produced 2-oxoglutaric acid aas the main product. The mechanism of the pyruvic acid production by strain W1485lip2 was found to be the impaired oxidative decarboxylation of pyruvic acid caused by the decrease in the activity of pyruvate dehydrogenase complex under the conditions of lipoic acid deficiency. Under the optimum culture conditions using the pH-controlled jar fermentor, 25.5 g/l pyruvic acid was obtained from 50 g/l glucose after the culture for 32–40 h at pH6.0. The relationship between the pyruvic acid productivity and the pyruvate dehydrogenase complex activity in jar-fermentor culture was discussed.     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

Fermentation of galacturonic acid and other sugars in orange peel hydrolysates by the ethanologenic strain of Escherichia coli

Summary Enzymatic hydrolysates of orange peel contain relatively high levels of galacturonic acid and arabinose which are not fermentable to ethanol by yeasts. We observed complete utilization of both sugars during fermentation of peel hydrolysates by the ethanologenic construct of E. coli KO11. The bacterium exhibits a novel pattern of galacturonic acid fermentation producing equimolar amounts of acetate and ethanol accompanied by carbon dioxide.     

The importance of aeration strategy in fuel alcohol fermentations contaminated with Dekkera/Brettanomyces yeasts

Whole corn mash fermentations infected with industrially-isolated Brettanomyces yeasts were not affected even when viable Brettanomyces yeasts out-numbered Saccharomyces yeasts tenfold at the onset of fermentation. Therefore, aeration, a parameter that is pivotal to the physiology of Dekkera/Brettanomyces yeasts, was investigated in mixed culture fermentations. Results suggest that aeration strategy plays a significant role in Dekkera/Brettanomyces-mediated inhibition of fuel alcohol fermentations. Although growth of Saccharomyces cerevisiae was not impeded, mixed culture fermentations aerated at rates of ≥20 ml air l⁻¹ mash min⁻¹ showed decreased ethanol yields and an accumulation of acetic acid. The importance of aeration was examined further in combination with organic acid(s). Growth of Saccharomyces occurred more rapidly than growth of Brettanomyces yeasts in all conditions. The combination of 0.075% (w/v) acetic acid and contamination with Brettanomyces TK 1404W did not negatively impact the final ethanol yield under fermentative conditions. Aeration, however, did prove to be detrimental to final ethanol yields. With the inclusion of aeration in the control condition (no organic acid stress) and in each fermentation containing organic acid(s), the final ethanol yields were decreased. It was therefore concluded that aeration strategy is the key parameter in regards to the negative effects observed in fuel alcohol fermentations infected with Dekkera/Brettanomyces yeasts.     

Pyruvic Acid Production from 1,2-Propanediol by Thiamin-requiring Acinetobacter sp. 80-M

For the purpose of producing pyruvic acid from 1,2-propanediol (PD), three PD-utilizing and thiamin-requiring microorganisms were isolated from soil. All the isolated strains were found to be pyruvic acid producers. Among them, strain 80-M was the best producer and was identified as an Acinetobacter sp. With this bacterium, the conditions were optimized for pyruvic acid production from PD by two methods: growing cell and resting cell methods. The amount of thiamin added to the medium remarkably affected the pyruvic acid production using either method. Under optimal conditions, 14.6 and 10.0 mg/ml of pyruvate as a sodium salt were produced from 20 mg/ml of PD by the growing and resting cell methods, respectively. In the resting cell system, only PD-grown cells showed a significant pyruvate productivity from PD.     

The cytosolic pathway of l-malic acid synthesis in Saccharomyces cerevisiae: the role of fumarase

Saccharomyces cerevisiae accumulates l-malic acid but only minute amounts of fumaric acid. A ¹³C-nuclear magnetic resonance study following the label from glucose to l-malic acid indicates that the l-malic acid is synthesized from pyruvic acid via oxaloacetic acid. From this, and from previously published studies, we conclude that a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to l-malic acid is responsible for the l-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to l-malic acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards l-malic acid; the K _m for l-malic acid is very high indicating that l-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more l-malic acid). (c) According to ¹³C NMR studies there is no interconversion of cytosolic l-malic and fumaric acids.     

Synthetic media for production of quality xanthan gum in 20 liter fermentors

Xanthan gum is a heteropolysaccharide synthesized by Xanthomonas campestris NRRL B-1459 and is composed of D -glucose, D -mannose, and D -glucuronic acid, in addition to acetic and pyruvic acids. Different amounts of pyruvic acid ketal are found in various preparations which can influence the viscosities of dilute xanthan solutions. Polysaccharide production on synthetic media was studied in small-scale fermentors. Fermentation conditions were established for production of both high and low pyruvic acid gums (about 4 and 2% pyruvic acid, respectively). Low nitrogen [0.1% (NH₄)₂HPO₄] and air (0.25 vol/liter/min) levels favor production of low pyruvate gum; increasing (NH₄)₂HPO₄ to 0.15%, adding K₂HPO₄, and increasing the air flow to 1.5 vol/liter/min favored production of normal gum. Both processes gave xanthan yields of 50 to 60%, based on 2.5% initial D -glucose substrate, in two to three days. Differences in pyruvic acid content and in the quantity of gum produced under a given set of conditions were attributed to strain variability. Substrains were isolated that have desirable characteristics for production of xanthan gum; i.e., the ability to give good yields of high-pyruvate gum when grown on both complex and synthetic media.     

Improved ATP supply enhances acid tolerance of Candida glabrata during pyruvic acid production

Aims: A major problem in industrial fermentation of organic acids with micro‐organisms is to ensure a suitable pH in the culture broth. To circumvent this problem, we investigated the effect of citrate, which is a widely used auxiliary energy co‐substrate, on cell growth, organic acid production and pH homeostasis among extracellular environment, cytoplasm and vacuole, in the pyruvic acid production by Candida glabrata CCTCC M202019 under different pH conditions. Methods and Results: Analysis of intracellular ATP regeneration, cytoplasmic and vacuolar pH values under different culture conditions points towards a relief of stress when C. glabrata is exposed to lower pH, if citrate is added. When 50 mmol l^?1 citrate was added to the culture medium, the intracellular ATP concentrations increased by 20·5% (pH 5·5), 20·4% (pH 5·0) and 39·3% (pH 4·5), and higher pH gradients among the culture broth, cell cytoplasm and vacuoles resulted. As a consequence, the cell growth and pyruvic acid production of C. glabrata CCTCC M202019 were significantly improved under pH 5·0 and 4·5. Conclusions: The acid tolerance of yeast can be improved by enhancing the ATP supply, which helps to maintain higher pH gradients in the system. Significance and Impact of the Study: The results presented here expand our understanding of the physiological characteristics in eukaryotic micro‐organisms under low pH conditions and provide a potential route for the further improvement of organic acids production process by process optimization or metabolic engineering.