Efficient production of ethanol from crude glycerol by a Klebsiella pneumoniae mutant strain

A mutant strain of Klebsiella pneumoniae, termed GEM167, was obtained by γ irradiation, in which glycerol metabolism was dramatically affected on exposure to γ rays. Levels of metabolites of the glycerol reductive pathway, 1,3-propanediol (1,3-PD) and 3-hydroxypropionic acid (3-HP), were decreased in the GEM167 strain compared to a control strain, whereas the levels of metabolites derived from the oxidative pathway, 2,3-butanediol (2,3-BD), ethanol, lactate, and succinate, were increased. Notably, ethanol production from glycerol was greatly enhanced upon fermentation by the mutant strain, to a maximum production level of 21.5 g/l, with a productivity of 0.93 g/l/h. Ethanol production level was further improved to 25.0 g/l upon overexpression of Zymomonas mobilispdc and adhII genes encoding pyruvate decarboxylase (Pdc) and aldehyde dehydrogenase (Adh), respectively in the mutant strain GEM167.     

Use of glycerol for producing 1,3-dihydroxyacetone by Gluconobacter oxydans in an airlift bioreactor

1,3-Dihydroxyacetone can be produced by biotransformation of glycerol with glycerol dehydrogenase from Gluconobacter oxydans cells. Firstly, improvement the activity of glycerol dehydrogenase was carried out by medium optimization. The optimal medium for cell cultivation was composed of 5.6 g/l yeast extract, 4.7 g/l glycerol, 42.1 g/l mannitol, 0.5 g/l K₂HPO₄, 0.5 g/l KH₂PO₄, 0.1 g/l MgSO₄·7H₂O, and 2.0 g/l CaCO₃ with the initial pH of 4.9. Secondly, an internal loop airlift bioreactor was applied for DHA production from glycerol by resting cells of G. oxydans ZJB09113. Furthermore, the effects of pH, aeration rate and cell content on DHA production and glycerol feeding strategy were investigated. 156.3 ± 7.8 g/l of maximal DHA concentration with 89.8 ± 2.4% of conversion rate of glycerol to DHA was achieved after 72 h of biotransformation using 10 g/l resting cells at 30 °C, pH 5.0 and 1.5 vvm of aeration rate.     

Escherichia coli genome-scale metabolic gene knockout of lactate dehydrogenase (ldhA), increases succinate production from glycerol

Genome-scale metabolic model (GEM) of Escherichia coli has been published with applications in predicting metabolic engineering capabilities on different carbon sources and directing biological discovery. The use of glycerol as an alternative carbon source is economically viable in biorefinery. The use of GEM for predicting metabolic gene deletion of lactate dehydrogenase (ldhA) for increasing succinate production in Escherichia coli from glycerol carbon source remained largely unexplored. Here, I hypothesized that metabolic gene knockout of ldhA in E. coli from glycerol could increase succinate production. A proof-of-principle strain was constructed and designated as E. coli BMS5 (ΔldhA), by predicting increased succinate production in E. coli GEM and confirmed the predicted outcomes using wet cell experiments. The mutant GEM (ΔldhA) predicted 11% increase in succinate production from glycerol compared to its wild-type model (iAF1260), and the E. coli BMS5 (ΔldhA) showed 1.05 g/l and its corresponding wild-type produced .05 g/l (23-fold increase). The proof-of-principle strain constructed in this study confirmed the aforementioned hypothesis and further elucidated the fact that E. coli GEM can prospectively and effectively predict metabolic engineering interventions using glycerol as substrate and could serve as platform for new strain design strategies and biological discovery.     

Simultaneous production of 3-hydroxypropionic acid and 1,3-propanediol from glycerol by a recombinant strain of Klebsiella pneumoniae

In this study, an aldehyde dehydrogenase (ALDH) was over-expressed in Klebsiella pneumoniae for simultaneous production of 3-hydroxypropionic acid (3-HP) and 1,3-propanediol (1,3-PDO). Various genes encoding ALDH were cloned and expressed in K. pneumoniae, and expression of Escherichia colialdH resulted in the highest 3-HP titer in anaerobic cultures in shake flasks. Anaerobic fed-batch culture of this recombinant strain was further performed in a 5-L reactor. The 3-HP concentration and yield reached 24.4 g/L and 0.18 mol/mol glycerol, respectively, and at the same time 1,3-PDO achieved 49.3 g/L with a yield of 0.43 mol/mol in 24 h. The overall yield of 3-HP plus 1,3-PDO was 0.61 mol/mol. Over-expression of the E. coli AldH also reduced the yields of by-products except for lactate. This study demonstrated the possibility of simultaneous production of 3-HP and 1,3-PDO by K. pneumoniae under anaerobic conditions without supply of vitamin B₁₂.     

Using a starch-rich mutant of Arabidopsis thaliana as feedstock for fermentative hydrogen production

A mutant plant (Arabidopsis thaliana), sex1-1 (starch excess 1-1), accumulating high starch content in leaves was created to serve as better biomass feedstock for a H₂-producing strain Clostridium butyricum CGS2, which efficiently utilizes starch for H₂ production but cannot assimilate cellulosic materials. The starch content of the mutant plant increased to 10.67 mg/fresh weight, which is four times higher than that of wild type plant. Using sex1-1 mutant plant as feedstock, C. butyricum CGS2 could produce 490.4 ml/l of H₂ with a H₂ production rate of 32.9 ml/h/l. The H₂ production performance appeared to increase with the increase in the concentration of mutant plant from 2.5 to 10 g/l. The highest H₂ to plant biomass yield was nearly 49 ml/g for the mutant plant. This study successfully demonstrated the feasibility of using a starch-rich mutant plant for more effective bioH₂ production with C. butyricum CGS2.     

A novel assay for rapid in vivo determination of phenotypic stability of recombinant ethanol-producing microorganisms

A rapid empirical assay is presented for assessing the phenotypic stability of continuous cultures of recombinant bacteria containing transposed pdc and adh genes for ethanol production. The method measures spectrophotometrically the rate of colour formation when cells oxidize added ethanol to acetaldehyde in the presence of Schiff’s reagent. During chemostat cultures of the recombinant ethanologen Escherichia coli KO11 on 20 g/l glucose, assay activities were stable and high at ca 8 × 10⁻⁴ ΔOD₅₄₀/(s.OD₅₅₀), reflecting the high, stable ethanol yield (ca 95%). On 20 g/l and 50 g/l xylose, ethanol yields declined rapidly to about 60% and this was closely mirrored by the assay activities which fell to ca 1.5 ΔOD₅₄₀/(s.OD₅₅₀), only slightly higher than those measured for the parent strain. Typically taking only about an hour to perform, the assay provides a faster means of gauging the phenotypic stability of ethanol production than is possible by conventional methods.     

Production of succinate and polyhydroxyalkanoate from substrate mixture by metabolically engineered Escherichia coli

The strategic design of this study aimed at producing succinate and polyhydroxyalkanoate (PHA) from substrate mixture of glycerol/glucose and fatty acid in Escherichia coli. To accomplish this, an E. coli KNSP1 strain derived from E. coli LR1110 was constructed by deletions of ptsG, sdhA and pta genes and overexpression of phaC1 from Pseudomonas aeruginosa. Cultivation of E. coli KNSP1 showed that this strain was able to produce 21.07 g/L succinate and 0.54 g/L PHA (5.62 wt.% of cell dry weight) from glycerol and fatty acid mixture. The generated PHA composed of 58.7 mol% 3-hydroxyoctanoate (3HO) and 41.3 mol% 3-hydroxydecanoate (3HD). This strain would be useful for complete utilization of byproducts glycerol and fatty acid of biodiesel production process.     

Importance of the methyl-citrate cycle on glycerol metabolism in the yeast Yarrowia lipolytica

A novel approach to trigger lipid accumulation and/or citrate production in vivo through the inactivation of the 2-methyl-citrate dehydratase in Yarrowia lipolytica was developed. In nitrogen-limited cultures with biodiesel-derived glycerol utilized as substrate, the Δphd1 mutant (JMY1203) produced 57.7 g/L of total citrate, 1.6-fold more than the wild-type strain, with a concomitant glycerol to citrate yield of 0.91 g/g. Storage lipid in cells increased at the early growth stages, suggesting that inactivation of the 2-methyl-citrate dehydratase would mimic nitrogen limitation. Thus, a trial of JMY1203 strain was performed with glycerol under nitrogen-excess conditions. Compared with the equivalent nitrogen-limited culture, significant quantities of lipid (up to ∼31% w/w in dry weight, 1.6-fold higher than the nitrogen-limited experiment) were produced. Also, non-negligible quantities of citric acid (up to ∼26 g/L, though 0.57-fold lower than the nitrogen-limited experiment) were produced, despite remarkable nitrogen presence into the medium, indicating the construction of phenotype that constitutively accumulated lipid and secreted citrate in Y. lipolytica during growth on waste glycerol utilized as substrate.     

Enhanced ethanol production from sugarcane juice by galactose adaptation of a newly isolated thermotolerant strain of Pichia kudriavzevii

The thermotolerant yeast strain isolated from sugarcane juice through enrichment technique was identified as a strain of Pichiakudriavzevii (Issatchenkiaorientalis) through molecular characterization. The P. kudriavzevii cells adapted to galactose medium produced about 30% more ethanol from sugarcane juice than the non-adapted cells. The recycled cells could be used for four successive cycles without a significant drop in ethanol production. Fermentation in a laboratory fermenter with galactose adapted P. kudriavzevii cells at 40 °C resulted in an ethanol concentration and productivity of 71.9 g L⁻¹ and 4.0 g L⁻¹ h⁻¹, respectively from sugarcane juice composed of about 14% (w/v) sucrose, 2% (w/v) glucose and 1% (w/v) fructose. In addition to ethanol, 3.30 g L⁻¹ arabitol and 4.19 g L⁻¹ glycerol were also produced, whereas sorbitol and xylitol were not formed during fermentation. Use of galactose adapted P. kudriavzevii cells for ethanol production from sugarcane juice holds potential for scale-up studies.     

Expression of aldehyde dehydrogenase 6 reduces inhibitory effect of furan derivatives on cell growth and ethanol production in Saccharomyces cerevisiae

Yeast dehydrogenases and reductases were overexpressed in Saccharomyces cerevisiae D452-2 to detoxify 2-furaldehyde (furfural) and 5-hydroxymethyl furaldehyde (HMF), two potent toxic chemicals present in acid-hydrolyzed cellulosic biomass, and hence improve cell growth and ethanol production. Among those enzymes, aldehyde dehydrogenase 6 (ALD6) played the dual roles of direct oxidation of furan derivatives and supply of NADPH cofactor to their reduction reactions. Batch fermentation of S. cerevisiae D452-2/pH-ALD6 in the presence of 2 g/L furfural and 0.5 g/L HMF resulted in 20-30% increases in specific growth rate, ethanol concentration and ethanol productivity, compared with those of the wild type strain. It was proposed that overexpression of ALD6 could recover the yeast cell metabolism and hence increase ethanol production from lignocellulosic biomass containing furan-derived inhibitors.     

Hydrogen production by immobilized R. faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria E. harbinense B49

In order to increase the hydrogen yield from glucose, hydrogen production by immobilized Rhodopseudomonas faecalis RLD-53 using soluble metabolites from ethanol fermentation bacteria Ethanoligenens harbinense B49 was investigated. The soluble metabolites from dark-fermentation mainly were ethanol and acetate, which could be further utilized for photo-hydrogen production. Hydrogen production by B49 was noticeably affected by the glucose and phosphate buffer concentration. The maximum hydrogen yield (1.83 mol H₂/mol glucose) was obtained at 9 g/l glucose. In addition, we found that the ratio of acetate/ethanol (A/E) increased with increasing phosphate buffer concentration, which is favorable to further photo-hydrogen production. The total hydrogen yield during dark- and photo-fermentation reached its maximum value (6.32 mol H₂/mol glucose) using 9 g/l glucose, 30 mmol/l phosphate buffers and immobilized R. faecalis RLD-53. Results demonstrated that the combination of dark- and photo- fermentation was an effective and efficient process to improve hydrogen yield from a single substrate.     

Isolation of a solventogenic Clostridium sp. strain: Fermentation of glycerol to n-butanol,analysis of the bcs operon region and its potential regulatory elements

A new solventogenic bacterium, strain GT6, was isolated from standing water sediment. 16S-rRNA gene analysis revealed that GT6 belongs to the heterogeneous Clostridium tetanomorphum group of bacteria exhibiting 99% sequence identity with C. tetanomorphum 4474^T. GT6 can utilize a wide range of carbohydrate substrates including glucose, fructose, maltose, xylose and glycerol to produce mainly n-butanol without any acetone. Additional products of GT6 metabolism were ethanol, butyric acid, acetic acid, and trace amounts of 1,3-propanediol. Medium and substrate composition, and culture conditions such as pH and temperature influenced product formation. The major fermentation product from glycerol was n-butanol with a final concentration of up to 11.5 g/L. 3% (v/v) glycerol lead to a total solvent concentration of 14 g/L within 72 h. Growth was not inhibited by glycerol concentrations as high as 15% (v/v).     

Simultaneous production of citric acid and invertase by Yarrowia lipolytica SUC+ transformants

Simultaneous production of citric acid (CA) and invertase by Yarrowia lipolytica A-101-B56-5 (SUC⁺ clone) growing from sucrose, mixture of glucose and fructose, glucose or glycerol was investigated. Among the tested substrates the highest concentration of CA was reached from glycerol (57.15 g/L) with high yield (Y_CA/S = 0.6 g/g). When sucrose was used, comparable amount of CA was secreted (45 g/L) with slightly higher yield (Y_CA/S = 0.643 g/g). In all cultures amount of isocitrate (ICA) was below 2% of total citrates. Considering invertase production, the best carbon source appeared to be sucrose (72 380 U/L). The highest yield of CA and invertase biosynthesis calculated for 1 g of biomass was obtained for cells growing from glycerol (9.9 g/g and 4325 U/g, respectively). Concentrates of extra- and intracellular invertase of the highest activity were obtained from sucrose as substrate (0.5 and 1.8 × 10⁶ U/L, respectively).     

Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 mg/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3 g/l and 8.7 g/l, respectively.     

Effect of lignocellulosic inhibitory compounds on growth and ethanol fermentation of newly-isolated thermotolerant Issatchenkia orientalis

A newly isolated thermotolerant ethanologenic yeast strain, Issatchenkia orientalis IPE 100, was able to produce ethanol with a theoretical yield of 85% per g of glucose at 42 °C. Ethanol production was inhibited by furfural, hydroxymethylfurfural and vanillin concentrations above 5.56 g L⁻¹, 7.81 g L⁻¹, and 3.17 g L⁻¹, respectively, but the strain was able to produce ethanol from enzymatically hydrolyzed steam-exploded cornstalk with 93.8% of theoretical yield and 0.91 g L⁻¹ h⁻¹ of productivity at 42 °C. Therefore, I. orientalis IPE 100 is a potential candidate for commercial lignocelluloses-to-ethanol production.     

Physiological Function of Alcohol Dehydrogenases and Long-Chain (C30) Fatty Acids in Alcohol Tolerance of Thermoanaerobacter ethanolicus

A mutant strain (39E H8) of Thermoanaerobacter ethanolicus that displayed high (8% [vol/vol]) ethanol tolerance for growth was developed and characterized in comparison to the wild-type strain (39E), which lacks alcohol tolerance (<1.5% [vol/vol]). The mutant strain, unlike the wild type, lacked primary alcohol dehydrogenase and was able to increase the percentage of transmembrane fatty acids (i.e., long-chain C₃₀ fatty acids) in response to increasing levels of ethanol. The data support the hypothesis that primary alcohol dehydrogenase functions primarily in ethanol consumption, whereas secondary alcohol dehydrogenase functions in ethanol production. These results suggest that improved thermophilic ethanol fermentations at high alcohol levels can be developed by altering both cell membrane composition (e.g., increasing transmembrane fatty acids) and the metabolic machinery (e.g., altering primary alcohol dehydrogenase and lactate dehydrogenase activities).     

Techno-economic implications of improved high gravity corn mash fermentation

The performance of Saccharomyces cerevisiae MBG3964, a strain able to tolerate >18% v/v ethanol, was compared to leading industrial ethanol strain, Fermentis Ethanol Red, under high gravity corn mash fermentation conditions. Compared to the industrial ethanol strain, MBG3964 gave increased alcohol yield (140 g L⁻¹ vs. 126 g L⁻¹), lower residual sugar (4 g L⁻¹ vs. 32 g L⁻¹), and lower glycerol (11 g L⁻¹ vs. 12 g L⁻¹). After 72 h fermentation, MBG3964 showed about 40% viability, whereas the control yeast was only about 3% viable. Based on modelling, the higher ethanol tolerant yeast could increase the profitability of a corn-ethanol plant and help it remain viable through higher production, lower unit heating requirements and extra throughput. A typical 50 M gal y⁻¹ dry mill ethanol plant that sells dried distiller’s grain could potentially increase its profit by nearly $US3.4 M y⁻¹ due solely to the extra yield, and potentially another $US4.1 M y⁻¹ if extra throughput is possible.     

Continuous culture of the microalgae Schizochytrium limacinum on biodiesel-derived crude glycerol for producing docosahexaenoic acid

Crude glycerol is a major byproduct of the biodiesel industry; previous research has proved the feasibility of producing docosahexaenoic acid (DHA, 22:6 n − 3) through fermentation of the algae Schizochytrium limacinum on crude glycerol. The objective of this work is to investigate the cell growth kinetics, substrate utilization efficiency, and DHA production of the algae through a continuous culture. Steady-state biomass yield, biomass productivity, growth yield on glycerol, specific glycerol consumption rate, and fatty acid composition were investigated within the range of dilution rate (D) from 0.2 to 0.6 day⁻¹, and the range of feed crude glycerol concentration (S₀) from 15 to 120 g/L. The maximum specific growth rate was determined as 0.692 day⁻¹. The cells had a true growth yield of 0.283 g/g but with a relatively high maintenance coefficient (0.2216 day⁻¹). The highest biomass productivity of 3.88 g/L-day was obtained at D = 0.3 day⁻¹ and S₀ = 60 g/L, while the highest DHA productivity (0.52 g/L-day) was obtained at D = 0.3 day⁻¹ and S₀ = 90 g/L due to the higher DHA content at S₀ = 90 g/L. The biomass and DHA productivity of the continuous culture was comparable to those of batch culture, while lower than the fed-batch culture, mainly because of the lower DHA content obtained by the continuous culture. Overall, the results show that continuous culture is a powerful tool to investigate the cell growth kinetics and physiological behaviors of the algae growing on biodiesel-derived crude glycerol.