Raw Glycerol Research Articles

Expression and Characterization of a Class III Alcohol Dehydrogenase Gene from Gluconobacter frateurii in the Presence of Methanol during Glyceric Acid Production from Glycerol

Some acetic acid bacteria have been shown to produce large amounts of glyceric acid (GA) from glycerol, which is a by-product of biodiesel fuel (BDF) production. Previously, a Gluconobacter strain was found that produced decreased amounts of GA from glycerol in the presence of methanol, a major ingredient of raw glycerol derived from the BDF industry. Thus, a comparative transcriptome analysis of Gluconobacter frateurii NBRC103465 was performed to investigate changes in gene expression during GA production from glycerol in the presence of methanol. Cells grown with methanol showed upregulated expression of a class III alcohol dehydrogenase homolog (adhC(Gf)) and decreased GA production. adhC(Gf) was cloned and expressed heterologously in Escherichia coli, and the presence of an additional protein with an approximate molecular mass of 39 kDa in the cytosol of the recombinant E. coli cells was identified by SDS-PAGE. Activity measurements of the cytosol revealed that the translational product of adhC(Gf) exhibited formaldehyde dehydrogenase activity in the presence of nicotinamide adenine dinucleotide and glutathione. Gluconobacter frateurii cells grown in 1% methanol-containing glycerol were found to have fivefold higher formaldehyde dehydrogenase activity than cells grown without methanol, suggesting that adhC(Gf) in G. frateurii cells functions in the dissimilation of methanol-derived formaldehyde.

Enhanced 1,3-propanediol production by a newly isolated Citrobacter freundii strain cultivated on biodiesel-derived waste glycerol through sterile and non-sterile bioprocesses

The production of 1,3-propanediol (PD) by a newly isolated Citrobacter freundii strain [FMCC-B 294 (VK-19)] was investigated. Different grades of biodiesel-derived glycerol were employed. Slightly lower PD biosynthesis was observed in batch experiments only when crude glycerol from waste-cooking oil trans-esterification was utilized and only at elevated initial substrate concentrations employed. Batch bioreactor cultures revealed the capability of the strain to tolerate elevated amounts of substrate (glycerol up to 170g/L) and produce quantities of PD in such high substrate concentrations. Nevertheless, maximum PD quantities (45.9g/L) were achieved at lower initial glycerol concentrations (∼100g/L) employed, suggesting some inhibition exerted due to the increased initial substrate concentrations. In order to improve PD production, a fed-batch fermentation was carried out and 68.1g/L of PD were produced (the highest PD quantity achieved by C. freundii strains so far) with yield per glycerol consumed ∼0.40g/g and volumetric productivity 0.79g/L/h. Aiming to perform a more economical and eco-friendlier procedure, batch and fed-batch fermentations under completely non-sterile conditions were carried out. During non-sterilized fed-batch process, 176g/L of raw glycerol were converted to 66.3g/L of PD, suggesting the potentiality of the non-sterile fermentation by C. freundii FMCC-B 294.

Lactic acid production by Lactobacillus sp. from biodiesel derived raw glycerol

Lactic Acid (LA) is a versatile compound with extensive industrial usage. Lactic Acid Bacteria (LAB) are renowned for their capacity to ferment carbohydrates to LA. In the biodiesel industry, bioconversion of residual glycerol to monomers of biopolymers, such as LA, is an alternative for usage of this waste as raw material to produce a high value added biotechnological compound. This research evaluates LA production through fermentation processes using a Lactobacillus sp. native strain and raw glycerol from the biodiesel industry. Native strain was isolated from the processing of dairy, fruit, and vegetable products and through cultures in Man, Rogosa & Sharpe agar (MRS). After an adaptation phase, the fermentative capability of the strain was evaluated through chemical quantification of metabolites using Nuclear Magnetic Resonance (1H-NMR); fermentative potential was compared with a Lactobacillus sp. reference strain (ATCC 7469). The native strain Lactobacillus sp. CYP4 showed a higher bioconversion potential (LA concentration: 39.41 mM, conversion percentage: 39.27%, at 24 h, volumetric productivity: 1.28 g.L-1.h-1, yield: 0.71 g·g-1) from raw glycerol, when compared to the reference strain.

High yield production of extracellular recombinant levansucrase by Bacillus megaterium

In this study, a high yield production bioprocess with recombinant Bacillus megaterium for the production of the extracellular enzyme levansucrase (SacB) was developed. For basic optimization of culture parameters and nutrients, a recombinant B. megaterium reporter strain that produced green fluorescent protein under control of a vector-based xylose-inducible promoter was used. It enabled efficient microtiter plate-based screening via fluorescence analysis. A pH value of pH 6, 20 % of dissolved oxygen, 37 °C, and elevated levels of biotin (100 μg L(-1)) were found optimal with regard to high protein yield and reduced overflow metabolism. Among the different compounds tested, fructose and glycerol were identified as the preferred source of carbon. Subsequently, the settings were transferred to a B. megaterium strain recombinantly producing levansucrase SacB based on the plasmid-located xylose-inducible expression system. In shake flask culture under the optimized conditions, the novel strain already secreted the target enzyme in high amounts (14 U mL(-1) on fructose and 17.2 U mL(-1) on glycerol). This was further increased in high cell density fed-batch processes up to 55 U mL(-1), reflecting a levansucrase concentration of 0.52 g L(-1). This is 100-fold more than previous efforts for this enzyme in B. megaterium and more than 10-fold higher than reported values of other extracellular protein produced in this microorganism so far. The recombinant strain could also handle raw glycerol from biodiesel industry which provided the same amount and quality of the recombinant protein and suggests future implementation into existing biorefinery concepts.

Carotenoid Production by Phaffia rhodozyma Using Raw Glycerol as an Additional Carbon Source

Abstract This paper presents pure glycerol and raw glycerol as additional carbon sources in the production of carotenoids by Phaffia rhodozyma NRRL Y-17268. Batch cultures in shaken flasks were performed in order to verify the effects of temperature cultivation (20 and 25ºC) and pure glycerol concentration (0, 10 and 40 g.L-1). The increase from 20 to 25ºC led to a significant increase (p<0.05) in cell concentration for all the pure glycerol concentrations, reaching the maximum levels of 8.9 g.L-1 of biomass (168 h), specific production of carotenoids (SPC) of 91.0 μg.g-1 (72 h) and volumetric production of carotenoids (VPC) of 2.0 μg.mL-1 (168 h) with 40 g.L-1 of pure glycerol at 25ºC. The use of 40 g.L-1 of raw glycerol at the same temperature resulted in a carotenogenic production showing no significant difference (p>0.05) in pure glycerol, reaching the maximum values of 8.3 g.L-1 of biomass (168 h) and within 120 h SPC 275.5 μg.g-1 and VPC 1.7 μg.mL-1. Therefore, the pure glycerol and raw glycerol can be used as additional carbon sources in the culture medium of P. rhodozyma.

Optimization of lipid production in the oleaginous bacterium Rhodococcus erythropolis growing on glycerol as the sole carbon source

The growth of an oleaginous bacteria strain Rhodococcus erythropolis on glycerol was studied. Lipid accumulation was influenced by the glycerol concentration, nitrogen source, nitrogen concentration, pH medium, incubation time and aeration rate. Under the best optimized flask culture medium; 30 g/L glycerol plus 0.75 g/L urea, incubation temperature at 30°C, agitation rate of 150 rpm and 96 h of cultivation time, 3.93 g/L of dry biomass, 1.84 g/L cellular lipid accumulation and 45.8% accumulated lipid of total dry biomass were produced. The scaling up of R. erythropolis in bioreactors yielded the biomass and lipid content of 11.74 g/L and 14.1% of total dry biomass, respectively. The lipid composition of oleaginous microorganisms contained a high proportion of C16 and C18 fatty acids. The extracted lipids were mainly 16.48% C14:1, 16.69% C16:1, 20.16% C16:0, 18.90% C18:1. R. erythropolis could be directed to using raw glycerol obtained from biodiesel by-product as substrate, in order to accumulate lipids for biodiesel production. Key words : Rhodococcus eryhropolis , glycerol, lipid accumulation, biodiesel, fatty acid composition.

Application of a two‐stage temperature control strategy to enhance 1,3‐propanediol productivity by Clostridium butyricum

AbstractBACKGROUND: The purpose of the present work was to enhance 1,3‐propanediol productivity during the batch cultivation on a type of raw glycerol by application of a two‐stage temperature control strategy.RESULTS: First, the effect of the raw glycerol on microbial growth and 1,3‐propanediol production was investigated. The highest 1,3‐propanediol productivity, 1.93 g L−1 h−1, was achieved when the initial raw glycerol concentration was 6% (v/v). Second, the effect of temperature on microbial growth and 1,3‐propanediol production was investigated and kinetic analysis was carried out. The results indicated that 37 °C favored microbial growth while 35 °C was best for 1,3‐propanediol production. Finally, a two‐stage temperature control strategy was applied in 1,3‐propanediol production. The incubation temperature was kept at 37 °C from inoculation to 2 h and then switched to 35 °C. Compared with batch cultivations at 35 and 37 °C, the fermentation time was shortened from 10 to 9.2 h, resulting in an increase in 1,3‐propanediol productivity of around 11%.CONCLUSION: 1,3‐propanediol productivity was enhanced effectively by application of a two‐stage temperature control strategy. © 2012 Society of Chemical Industry

Bioconversion of crude glycerol from biodiesel production to hydrogen

This study evaluates the potential of bioconversion of crude glycerol, discharged from biodiesel production plant, to hydrogen (H2) by an enriched microbial community. Microbial community was enriched from activated sludge in a medium amended with 2.5 g/L of crude glycerol. Optimal cultivation parameters for H2 production such as initial pH, cultivation temperature and substrate concentration were investigated. H2 yields from raw glycerol at optimal conditions (pH 6.5; 40 °C and 1 g/L raw glycerol) were 1.1 ± 0.1 mol-H2/mol-glycerolconsumed. H2 production was associated with acetate-butyrate type fermentation, along with ethanol as one of the end products. Kinetic experiments on H2 production from pure and crude glycerol indicated the absence of any inhibitory effects from the impurities present in crude glycerol. The community analysis revealed that the enriched microbial consortium was dominated mainly by Clostridium species.

Variation of the by-product spectrum during α-ketoglutaric acid production from raw glycerol by overexpression of fumarase and pyruvate carboxylase genes in Yarrowia lipolytica

The yeast Yarrowia lipolytica secretes high amounts of various organic acids, like citric, isocitric, pyruvic (PA), and α-ketoglutaric (KGA) acids, triggered by growth limitation and excess of carbon source. This is leading to an increased interest in this non-conventional yeast for biotechnological applications. To improve the KGA production by Y. lipolytica for an industrial application, it is necessary to reduce the amounts of by-products, e.g., fumarate (FU) and PA, because production of by-products is a main disadvantage of the KGA production by this yeast. We have examined whether the concentration of secreted organic acids (main product KGA and PA as major by-product and FU, malate (MA), and succinate (SU) as minor by-products) can be influenced by a gene-dose-dependent overexpression of fumarase (FUM) or pyruvate carboxylase (PYC) genes under KGA production conditions. Recombinant Y. lipolytica strains were constructed, which harbor multiple copies of the respective FUM1, PYC1 or FUM1, and PYC1 genes. Overexpression of the genes FUM1 and PYC1 resulted in strongly increased specific enzyme activities during cultivation of these strains on raw glycerol as carbon source in bioreactors. The recombinant Y. lipolytica strains showed different product selectivity of the secreted organic acids KGA, PA, FU, MA, and SU. Concentrations of the by-products FU, MA, SU, and PA decreased significantly at overproduction of FUM and increased at overproduction of PYC and also of FUM and PYC simultaneously. In contrast, the production of KGA with the multicopy strains H355A(FUM1) and H355A(FUM1-PYC1) was comparable with the wild-type strain H355 or slightly lower in case of H355(PYC1). KGA productivity was not changed significantly compared with strain H355 whereas product selectivity of the main product KGA was increased in H355A(FUM1).

Determination of kinetic parameters of 1,3-propanediol fermentation by Clostridium diolis using statistically optimized medium

1,3-propanediol (1,3-PD) is a chemical compound of immense importance primarily used as a raw material for fiber and textile industry. It can be produced by the fermentation of glycerol available abundantly as a by-product from the biodiesel plant. The present study was aimed at determination of key kinetic parameters of 1,3-PD fermentation by Clostridium diolis. Initial experiments on microbial growth inhibition were followed by optimization of nutrient medium recipe by statistical means. Batch kinetic data from studies in bioreactor using optimum concentration of variables obtained from statistical medium design was used for estimation of kinetic parameters of 1,3-PD production. Direct use of raw glycerol from biodiesel plant without any pre-treatment for 1,3-PD production using this strain investigated for the first time in this work gave results comparable to commercial glycerol. The parameter values obtained in this study would be used to develop a mathematical model for 1,3-PD to be used as a guide for designing various reactor operating strategies for further improving 1,3-PD production. An outline of protocol for model development has been discussed in the present work.

Design and analysis of biorefineries based on raw glycerol: Addressing the glycerol problem

Glycerol as a low-cost by-product of the biodiesel industry can be considered a renewable building block for biorefineries. In this work, the conversion of raw glycerol to nine added-value products obtained by chemical (syn-gas, acrolein, and 1,2-propanediol) or bio-chemical (ethanol, 1,3-propanediol, d-lactic acid, succinic acid, propionic acid, and poly-3-hydroxybutyrate) routes were considered. The technological schemes for these synthesis routes were designed, simulated, and economically assessed using Aspen Plus and Aspen Icarus Process Evaluator, respectively. The techno-economic potential of a glycerol-based biorefinery system for the production of fuels, chemicals, and plastics was analyzed using the commercial Commercial Sale Price/Production Cost ratio criteria, under different production scenarios. More income can be earned from 1,3-propanediol and 1,2-propanediol production, while less income would be obtained from hydrogen and succinic acid. This analysis may be useful mainly for biodiesel producers since several profitable alternatives are presented and discussed.

Propionic Acid Production from Raw Glycerol Using Commercial and Engineered Strains

Five technological schemes for propionic acid production from raw glycerol were designed, simulated, and economically assessed. Fermentative scenarios considered two different qualities of glycerol: 88 and 98 wt % with concentrations in the fermentation media from 20 to 50 g/L. Raw glycerol (60 wt %) was considered as the feedstock feeding the production process in all cases; then a purification process of raw glycerol up to the required quality was used. Simulation processes were carried out using Aspen Plus, while economic assessments were performed using Aspen Icarus Process Evaluator. Propionic acid recovery and purification processes were based on reactive–extraction with tri-n-octylamine using ethyl acetate as extractant agent. This technology results in energy-intensive processes with total energy consumption ranging from 120.4 to 138.9 MJ/kg. However, further significant improvements could be obtained using energy integration techniques. On the other hand, it was found that the production costs are directly related to the fermentation stage and decrease as follows: (i) increasing the glycerol concentration, (ii) decreasing the glycerol purity, and (iii) increasing the glycerol consumption.

English

The conversion of glycerol to 1,3-propanediol (1,3-PD) is an environmentally friendly biological process and has attracted great interests worldwide. Efficient utilization of raw glycerol could bring significant economic benefits for 1,3-PD production. In the present study, the glycerol broth from strain Zygosacharomyces rouxii JL2011 was first used as the sole carbon source for 1,3-PD production by Klebsiella pneumoniae . The result indicates that the glycerol broth affected K. pneumoniae growth and 1,3-PD yield dramatically. Therefore, the raw glycerol was further prepared by extraction and purification, and the effect of raw glycerol on 1,3-PD production was investigated. The results obtained showed that the purified glycerol could be directly utilized in shake-flask culture by K. pneumoniae with a result comparable with that attained from pure glycerol. In addition, the fed-batch culture using raw glycerol as the substrate was conducted giving 56.1 g×L -1 with a yield of 0.40 g×g -1 glycerol and productivity of 1.12 g×L -1 ×h -1 . Key words: 1,3-propanediol, Klebsiella pneumoniae, Zygosacharomyces rouxii , raw glycerol.

The effects of emulsified polydimethylsiloxane FG-10 on the oxygen transfer coefficient (kLa) and lipase production by Staphylococcus warneri EX17

BACKGROUND: In this study the effects of the addition of emulsified polydimethylsiloxane (PMDS) FG-10 on the oxygen transfer coefficient (kLa) of submerged cultures of Staphylococcus warneri EX17 and its lipase production is described. FG-10 is an emulsified silicone capable of dissolving 50 times more oxygen than water. The combined effects of FG-10 concentration and different conditions of agitation were optimized in bioreactors using statistical design tools, and the cultures were run using raw glycerol from biodiesel synthesis as the sole carbon source. RESULTS: The optimal conditions found to improve lipase production were FG-10 concentration of 11.2% (v/v) and speed agitation of 527 rpm, respectively, producing around 861 U L−1 of lipolytic activity, a maximal cell concentration of 8.4 g L−1, and a kLa of 99 h−1, values that are approximately 3 times higher than cultures without FG-10. CONCLUSIONS: This is the first report in the literature on the use of this class of chemicals as oxygen carriers in microbial cultures and its effect on kLa and lipase production, demonstrating the potential use of FG-10 in microbial cultures. Copyright © 2012 Society of Chemical Industry

Evaluation of the composition of culture medium for yeast biomass production using raw glycerol from biodiesel synthesis.

The work herewith investigated the production of yeast biomass as a source of protein, using Yarrowia lipolytica NRRL YB-423 and raw glycerol from biodiesel synthesis as the main carbon source. A significant influence of glycerol concentration, initial pH and yeast extract concentration on biomass and protein content was observed according to the 2v5-1 fractional design. These factors were further evaluated using a central composite design and response surface methodology, and an empirical model for protein content was established and validated. The biomass of Yarrowia lipolytica NRRL YB-423 reached 19.5 ± 1.0 g/L in shaken flasks cultivation, with a protein content of 20.1 ± 0.6% (w/w).

BDF副生グリセリンを原料とするグリセリン誘導体の製造および機能性化学品への応用

BDF副生グリセリンを原料とするグリセリン誘導体の製造および機能性化学品への応用

Fermentative conversion of raw glycerol into 1,3-propanediol by isolated Klebsiella pneumoniae 141B stain: Optimization of culture variables

In this study, 1,3-propanediol (1,3-PDO) production by Klebsiella pneumoniae 141B strain using raw glycerol as substrate was investigated. Taguchi L18 orthogonal array (OA) was adopted to optimize nutritional (raw glycerol, yeast extract and calcium carbonate), physiological (incubation temperature and medium pH) and microbial (inoculum level) fermentation variables. These controlling fermentation factors were selected based on one variable at a time methodology. Raw glycerol, temperature and calcium carbonate were the most significant factors among the selected factors followed by pH and inoculum level. Yeast extract, however, showed the least significance on 1,3-PDO production at individual level, while in combination, it elucidated a remarkable severity index values of 64.81 and 58.17% with pH and temperature, respectively. At optimized environment, 53% contribution towards 1,3- PDO production was observed from selected fermentation parameters. The model was experimentally validated, yielding a 1,3-PDO production of 0.62 mol mol -1 of raw glycerol, which represented a 19% increase when compared to the non-optimized medium. Key words: Raw glycerol, 1,3-propanediol, Taguchi methodology, Klebsiella pneumoniae and optimization.

Analysis of the Production Process of Optically Pure d-Lactic Acid from Raw Glycerol Using Engineered Escherichia coli Strains

Glycerol has become an ideal feedstock for producing fuels and chemicals. Here, five technological schemes for optically pure D: -lactic acid production from raw glycerol were designed, simulated, and economically assessed based on five fermentative scenarios using engineered Escherichia coli strains. Fermentative scenarios considered different qualities of glycerol (pure, 98 wt.%, and crude, 85 wt.%) with concentrations ranging from 20 to 60 g/l in the fermentation media, and two fermentation stages were also analyzed. Raw glycerol (60 wt.%) was considered as the feedstock feeding the production process in all cases; then a purification process of raw glycerol up to the required quality was required. Simulation processes were carried out using Aspen Plus, while economic assessments were performed using Aspen Icarus Process Evaluator. D: -Lactic acid recovery and purification processes were based on reactive extraction with tri-n-octylamine using dichloromethane as active extractant agent. The use of raw glycerol represents only between 2.4% and 7.8% of the total production costs. Also, the total production costs obtained of D: -lactic acid in all cases were lower than its sale price indicating that these processes are potentially profitable. Thus, the best configuration process requires the use of crude glycerol diluted at 40 g/l with total glycerol consumption and with D: -lactic acid recovering by reactive extraction. The lowest obtained total production cost was 1.015 US$/kg with a sale price/production cost ratio of 1.53.

The use of microorganisms in 1,3-Propanediol production

Recently, an increasing interest in “green” processes for the production of chemicals was observed. An attractive solution is microbial fermentation processes which use renewable feedstocks such as glycerol. However, this kind of synthesis has several limitations, among others a small range of products, low productivities, as well as difficulty in recovery and purification of products. One way to overcome these limitations is the application of metabolic engineering. This paper is a review of 1,3Propanediol (1,3-PD) characteristic and applications, of microorganisms which ferment glycerol to 1,3PD and their metabolic pathways, and finally of the metabolic engineering methods for the microbial conversion of raw glycerol to 1,3-PD.

Screening of bacterial strains capable of converting biodiesel‐derived raw glycerol into 1,3‐propanediol, 2,3‐butanediol and ethanol

AbstractThe ability of bacterial strains to assimilate glycerol derived from biodiesel facilities to produce metabolic compounds of importance for the food, textile and chemical industry, such as 1,3‐propanediol (PD), 2,3‐butanediol (BD) and ethanol (EtOH), was assessed. The screening of 84 bacterial strains was performed using glycerol as carbon source. After initial trials, 12 strains were identified capable of consuming raw glycerol under anaerobic conditions, whereas 5 strains consumed glycerol under aerobiosis. A plethora of metabolic compounds was synthesized; in anaerobic batch‐bioreactor cultures PD in quantities up to 11.3 g/L was produced by Clostridium butyricum NRRL B‐23495, while the respective value was 10.1 g/L for a newly isolated Citrobacter freundii. Adaptation of Cl. butyricum at higher initial glycerol concentration resulted in a PDmax concentration of ∼32 g/L. BD was produced by a new Enterobacter aerogenes isolate in shake‐flask experiments, under fully aerobic conditions, with a maximum concentration of ∼22 g/L which was achieved at an initial glycerol quantity of 55 g/L. A new Klebsiella oxytoca isolate converted waste glycerol into mixtures of PD, BD and EtOH at various ratios. Finally, another new C. freundii isolate converted waste glycerol into EtOH in anaerobic batch‐bioreactor cultures with constant pH, achieving a final EtOH concentration of 14.5 g/L, a conversion yield of 0.45 g/g and a volumetric productivity of ∼0.7 g/L/h. As a conclusion, the current study confirmed the utilization of biodiesel‐derived raw glycerol as an appropriate substrate for the production of PD, BD and EtOH by several newly isolated bacterial strains under different experimental conditions.