Improved Bioethanol Production Using Fusants of Saccharomyces cerevisiae and Xylose-Fermenting Yeasts

The present research deals with the development of a hybrid yeast strain with the aim of converting pentose and hexose sugar components of lignocellulosic substrate to bioethanol by fermentation. Different fusant strains were obtained by fusing protoplasts of Saccharomyces cerevisiae and xylose-fermenting yeasts such as Pachysolen tannophilus, Candida shehatae and Pichia stipitis. The fusants were sorted by fluorescent-activated cell sorter and further confirmed by molecular characterization. The fusants were evaluated by fermentation of glucose?Cxylose mixture and the highest ethanol producing fusant was used for further study to ferment hydrolysates produced by acid pretreatment and enzymatic hydrolysis of cotton gin waste. Among the various fusant and parental strains used under present study, RPR39 was found to be stable and most efficient strain giving maximum ethanol concentration (76.8?±?0.31?g L^?1), ethanol productivity (1.06?g L^?1 h^?1) and ethanol yield (0.458?g g^?1) by fermentation of glucose?Cxylose mixture under test conditions. The fusant has also shown encouraging result in fermenting hydrolysates of cotton gin waste with ethanol concentration of 7.08?±?0.142?g L^?1, ethanol yield of 0.44?g g^?1, productivity of 0.45?g L^?1?h^?1 and biomass yield of 0.40?g g^?1.     

Microbial Fed-batch Production of 1,3-Propanediol Using Raw Glycerol with Suspended and Immobilized Klebsiella pneumoniae

The production of 1,3-propanediol (1,3-PD) was investigated with Klebsiella pneumoniae DSM 4799 using raw glycerol without purification obtained from a biodiesel production process. Fed-batch cultures with suspended cells revealed that 1,3-PD production was more effective when utilizing raw glycerol than pure glycerol (productivity after 47 h of fermentation, 0.84 g?L^?1?h^?1 versus 1.51 g?L^?1?h^?1 with pure and raw glycerol, respectively). In addition, more than 80 g/L of 1,3-PD was produced using raw glycerol; this is the highest 1,3-PD concentration reported thus far for K. pneumoniae using raw glycerol. Repeated fed-batch fermentation with cell immobilization in a fixed-bed reactor was performed to enhance 1,3-PD production. Production of 1,3-PD increased with the cycle number (1.06 g?L^?1?h^?1 versus 1.61 g?L^?1?h^?1 at the first and fourth cycle, respectively) due to successful cell immobilization. During 46 cycles of fed-batch fermentation taking place over 1,460 h, a stable and reproducible 1,3-PD production performance was observed with both pure and raw glycerol. Based on our results, repeated fed batch with immobilized cells is an efficient fermentor configuration, and raw glycerol can be utilized to produce 1,3-PD without inhibitory effects caused by accumulated impurities.     

<Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> application as a prospective approach for biosynthesis of pyruvic acid from glycerol

With the problems related to chemical methods of pyruvic acid (PA) synthesis, a fast-growing interest has been observed in research aiming at reducing the production cost of PA by applying biotechnological methods. This study aimed to investigate the potential applicability of Yarrowia lipolytica Wratislavia 1.31 yeast strain for valorisation of pure and crude glycerol through the production of industrially desired PA. Conditions required for the effective PA biosynthesis, i.e., pH value, thiamine concentration, agitation, and substrate concentration, were examined in batch and fed-batch cultivation modes. The efficient production of PA occurred under the limitation of thiamine (1 µg L^?1) and was stimulated by moderate pH (4.5) and agitation (800 rev min^?1) of the culture. Under optimal conditions, Y. lipolytica Wratislavia 1.31 was able to produce 85.2 g L^?1 of PA with volumetric productivity of 0.90 g L^?1 h^?1. The yield of PA biosynthesis reached a high level of 1.03 g g^?1. Obtained results confirmed the aptitude of Y. lipolytica yeast to produce high amounts of PA from simple glycerol-containing media. Presented process was very promising and might be considered as an attractive alternative for currently used chemical methods of PA synthesis.     

Biodiesel Residual Glycerol Metabolism by Klebsiella pneumoniae: Pool of Metabolites Under Anaerobiosis and Oxygen Limitation as a Function of Feeding Rates

The metabolism of residual glycerol from biodiesel synthesis by Klebsiella pneumoniae BLh-1 was investigated in this study. Batch and fed-batch cultivations were performed in bioreactors under anaerobic and oxygen limitation conditions. Results of batch cultivations showed that the main product was 1,3-propanediol (1,3-PD) in both conditions, although the higher yields and productivities (0.46 mol mol^?1 glycerol and 1.22 g?L^?1?h^?1, respectively) were obtained under anaerobic condition. Large amounts of ethanol were also produced under batch anaerobic condition, peaking at 12.30 g?L^?1. Batch cultivations under oxygen limitation were characterized by faster growth kinetics, with higher biomass production but lower conversions of glycerol into 1,3-PD, with yields and productivities of 0.33 mol mol^?1 glycerol and 0.99 g?L^?1?h^?1, respectively. The fed-batch cultivations were carried out in order to investigate the effects of feeding of raw glycerol on cells. Fed-batch under anaerobiosis showed that 1,3-PD and ethanol concentrations increased with the feeding rate, with maximal productions of 26.12 and 19.2 g?L^?1, respectively. The oxygen limitation conditions diverted the bacterium metabolism to an elevated lactic acid formation, reaching 59 g?L^?1 in higher feeding rates of glycerol, but lowering the production of ethanol.     

Effect of Nutrients on Fermentation of Pretreated Wheat Straw at very High Dry Matter Content by Saccharomyces cerevisiae

Wheat straw hydrolysate produced by enzymatic hydrolysis of hydrothermal pretreated wheat straw at a very high solids concentration of 30% dry matter (w/w) was used for testing the effect of nutrients on their ability to improve fermentation performance of Saccharomyces cerevisiae. The nutrients tested were MgSO₄ and nitrogen sources; (NH₄)₂SO₄, urea, yeast extract, peptone and corn steep liquor. The fermentation was tested in a separate hydrolysis and fermentation process using a low amount of inoculum (0.33 g kg^?1) and a non-adapted baker’s yeast strain. A factorial screening design revealed that yeast extract, peptone, corn steep liquor and MgSO₄ were the most significant factors in obtaining a high fermentation rate, high ethanol yield and low glycerol formation. The highest volumetric ethanol productivity was 1.16 g kg^?1 h^?1 and with an ethanol yield close to maximum theoretical. The use of urea or (NH₄)₂SO₄ separately, together or in combination with MgSO₄ or vitamins did not improve fermentation rate and resulted in increased glycerol formation compared to the use of yeast extract. Yeast extract was the single best component in improving fermentation performance and a concentration of 3.5 g kg^?1 resulted in high ethanol yield and a volumetric productivity of 0.6 g kg^?1 h^?1.     

Single-cell Protein and Xylitol Production by a Novel Yeast Strain <Emphasis Type="Italic">Candida intermedia</Emphasis> FL023 from Lignocellulosic Hydrolysates and Xylose

Yeasts are good candidates to utilize the hydrolysates of lignocellulose, the most abundant bioresource, for bioproducts. This study aimed to evaluate the efficiencies of single-cell protein (SCP) and xylitol production by a novel yeast strain, Candida intermedia FL023, from lignocellulosic hydrolysates and xylose. This strain efficiently assimilated hexose, pentose, and cellubiose for cell mass production with the crude protein content of 484.2 g kg^?1 dry cell mass. SCP was produced by strain FL023 using corncob hydrolysate and urea as the carbon and nitrogen sources with the dry cell mass productivity 0.86 g L^?1 h^?1 and the yield of 0.40 g g^?1 sugar. SCP was also produced using NaOH-pretreated Miscanthus sinensis straw and corn steep liquor as the carbon and nitrogen sources through simultaneous saccharification and fermentation with the dry cell productivity of 0.23 g L^?1 h^?1 and yield of 0.17 g g^?1 straw. C. intermedia FL023 was tolerant to 0.5 g L^?1 furfural, acetic acid, and syringaldehyde in xylitol fermentation and produced 45.7 g L^?1 xylitol from xylose with the productivity of 0.38 g L^?1 h^?1 and the yield of 0.57 g g^?1 xylose. This study provides feasible methods for feed and food additive production from the abundant lignocellulosic bioresources.     

Ethanol Production from Sugarcane Bagasse Hydrolysate Using Pichia stipitis

The objective of this study was to evaluate the ethanol production from the sugars contained in the sugarcane bagasse hemicellulosic hydrolysate with the yeast Pichia stipitis DSM 3651. The fermentations were carried out in 250-mL Erlenmeyers with 100 mL of medium incubated at 200 rpm and 30 °C for 120 h. The medium was composed by raw (non-detoxified) hydrolysate or by hydrolysates detoxified by pH alteration followed by active charcoal adsorption or by adsorption into ion-exchange resins, all of them supplemented with yeast extract (3 g/L), malt extract (3 g/L), and peptone (5 g/L). The initial concentration of cells was 3 g/L. According to the results, the detoxification procedures removed inhibitory compounds from the hemicellulosic hydrolysate and, thus, improved the bioconversion of the sugars into ethanol. The fermentation using the non-detoxified hydrolysate led to 4.9 g/L ethanol in 120 h, with a yield of 0.20 g/g and a productivity of 0.04 g L^?1 h^?1. The detoxification by pH alteration and active charcoal adsorption led to 6.1 g/L ethanol in 48 h, with a yield of 0.30 g/g and a productivity of 0.13 g L^?1 h^?1. The detoxification by adsorption into ion-exchange resins, in turn, provided 7.5 g/L ethanol in 48 h, with a yield of 0.30 g/g and a productivity of 0.16 g L^?1 h^?1.     

Enzymatic Hydrolysis and Fermentation of Pretreated Cashew Apple Bagasse with Alkali and Diluted Sulfuric Acid for Bioethanol Production

The aim of this work was to optimize the enzymatic hydrolysis of the cellulose fraction of cashew apple bagasse (CAB) after diluted acid (CAB-H) and alkali pretreatment (CAB-OH), and to evaluate its fermentation to ethanol using Saccharomyces cerevisiae. Glucose conversion of 82?±?2 mg/g CAB-H and 730?±?20 mg/g CAB-OH was obtained when 2% (w/v) of solid and 30 FPU/g bagasse was used during hydrolysis at 45 °C, 2-fold higher than when using 15 FPU/g bagasse, 44?±?2 mg/g CAB-H, and 450?±?50 mg/g CAB-OH, respectively. Ethanol concentration and productivity, achieved after 6 h of fermentation, were 20.0?±?0.2 g L^?1 and 3.33 g L^?1 h^?1, respectively, when using CAB-OH hydrolyzate (initial glucose concentration of 52.4 g L^?1). For CAB-H hydrolyzate (initial glucose concentration of 17.4 g L^?1), ethanol concentration and productivity were 8.2?±?0.1 g L^?1 and 2.7 g L^?1 h^?1 in 3 h, respectively. Hydrolyzates fermentation resulted in an ethanol yield of 0.38 and 0.47 g/g glucose with pretreated CAB-OH and CAB-H, respectively. Ethanol concentration and productivity, obtained using CAB-OH hydrolyzate, were close to the values obtained in the conventional ethanol fermentation of cashew apple juice or sugar cane juice.     

Efficient utilization of inulin and glycerol as fermentation substrates in erythritol and citric acid production using <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> expressing inulinase

Inulin and glycerol were used as substrates for efficient erythritol and citric acid production by newly engineered Yarrowia lipolytica strains. Hydrolysis of inulin by the Y. lipolytica Wratislavia K1 strain was established by expressing the Kluyveromyces marxianus INU1 gene. Erythritol was produced in two stages: inulin was used for biomass formation, followed by erythritol biosynthesis initiated by glycerol addition. The highest titer of erythritol obtained, 120.9 g L^?1 with the yield of 0.6 g g^?1, was produced by the K1 INU 6 strain. Moreover, the K1 INU 6 strain in fed-batch culture produced a high amount of citric acid: 105.2 g L^?1 after 235 h from 200 g L^?1 of inulin. Maximum activity of inulinase during this culture was 14000 U g^?1 of cell dry mass. The presented study proves the potential of new Y. lipolytica transformants for efficient erythritol and citric acid production from inexpensive raw materials such as inulin and glycerol.     

A Comparison Between Shaker and Bioreactor Performance Based on the Kinetic Parameters of Xanthan Gum Production

Xanthan gum production was studied using sugarcane broth as the raw material and batch fermentation by Xanthomonas campestris pv. campestris NRRL B-1459. The purpose of this study was to optimize the variables of sucrose, yeast extract, and ammonium nitrate concentrations and to determine the kinetic parameters of this bioreaction under optimized conditions. The effects of yeast extract and ammonium nitrate concentrations for a given sucrose concentration (12.1–37.8 g L^?1) were evaluated by central composite design to maximize the conversion efficiency. In a bioreactor, the maximum conversion efficiency was achieved using 27.0 g L^?1 sucrose, 2.7 g L^?1 yeast extract, and 0.9 g L^?1 NH₄NO₃. This point was assayed in a shaker and in a bioreactor to compare bioreaction parameters. These parameters were estimated by the unstructured kinetic model of Weiss and Ollis (Biotechnol Bioeng 22:859–873, 1980) to determinate the yields (Y _P/S), the maximum growth specific rate (μ _max), and the saturation cellular concentration (X*). The parameters of the model (μ _max, X*, m, λ, α, and β) were obtained by nonlinear regression. For production of xanthan gum in a shaker, the values of μ _max and Y _P/S obtained were 0.119 h^?1 and 0.34 g g^?1, respectively, while in a bioreactor, they were 0.411 h^?1 and 0.63 g g^?1, respectively.     

Bioethanol Production from Ipomoea Carnea Biomass Using a Potential Hybrid Yeast Strain

The paper deals with the exploitation of Ipomoea carnea as a feedstock for the production of bioethanol. Dilute acid pretreatment under optimum conditions (3 %H₂SO₄, 120 °C for 45 min) produced 17.68 g L^?1 sugars along with 1.02 g L^?1 phenolics and 1.13 g L^?1 furans. A combination of overliming and activated charcoal adsorption facilitated the removal of 91.9 % furans and 94.7 % phenolics from acid hydrolysate. The pretreated biomass was further treated with a mixture of sodium sulphite and sodium chlorite and, a maximum lignin removal of 81.6 % was achieved. The enzymatic saccharification of delignified biomass resulted in 79.4 % saccharification with a corresponding sugar yield of 753.21 mg g^?1. Equal volume of enzymatic hydrolysate and acid hydrolysate were mixed and used for fermentation with a hybrid yeast strain RPRT90. Fermentation of mixed detoxified hydrolysate at 30 °C for 28 h produced ethanol with a yield of 0.461 g g^?1. A comparable ethanol yield (0.414 g g^?1) was achieved using a mixture of enzymatic hydrolysate and undetoxified acid hydrolysate. Thus, I. carnea biomass has been demonstrated to be a potential feedstock for bioethanol production, and the use of hybrid yeast may pave the way to produce bioethanol from this biomass.     

Effects of elicitors on the enhancement of asiaticoside biosynthesis in cell cultures of centella (Centella asiatica L. Urban)

In this work, the effects of elicitor concentration and elicitation day on the growth and asiaticoside production of centella cells were investigated. The results showed that 2-hydroxybenzoic acid from 50?C200 ??M and yeast extract from 2?C5 g L^?1 had different eliciting influences. The addition of 2-hydroxybenzoic acid and yeast extract to the cultures strongly enhanced asiaticoside production in centella cells. The increase in asiaticoside content induced by the addition of 100 ??M of 2-hydroxybenzoic acid and 4 g L^?1 of yeast extract at day 10 of inoculation was about 5- and 3.5-fold, respectively, as compared with that of the reference cells. In general, 2-hydroxybenzoic acid (abiotic elicitor) was more effective in enhancing asiaticoside biosynthesis than yeast extract (biotic elicitor).     

Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica

Three acetate mutants of the yeast species Yarrowia lipolytica were screened using batch cultivation. The strain Y. lipolytica 1.31 was found to be the most suitable for citric acid production from raw glycerol, a by-product of biodiesel production from rapeseed oil. At the initial concentration of glycerol of 200 g dm⁻³, the citric acid production of 124.5 g dm⁻³, yield of 0.62 g g⁻¹, and productivity of 0.88 g dm⁻³ h⁻¹ were achieved. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.     

Optimal glucose and inoculum concentrations for production of bioactive molecules by Paenibacillus polymyxa RNC-D

The production of antimicrobial metabolites by Paenibacillus polymyxa RNC-D was assessed. Two process variables, glucose and inoculum concentrations, were evaluated at different levels (5?C40 g L^?1, and at ?? _r = 2.5?C5.0 %, respectively), and their effects on biomass formation, minimal inhibitory concentration (MIC) against Escherichia coli, and surface tension reduction (STR) were studied. When the fermentation process was carried out under non-optimised conditions, the biomass, MIC, and STR achieved the following values: 0.6 g L^?1, 1 g L^?1, and 18.4 mN m^?1, respectively. The optimum glucose (16 g L^?1) and inoculum volume ratio (?? _r = 5.0 %) were defined in order to maximise the biomass formation, with a low value of MIC and high STR of extract. The experiments carried out under optimal conditions showed the following values for the dependent variables: biomass concentration 2.05 g L^?1, MIC 31.2 ??g mL^?1, and STR 10.7 mN m^?1, which represented improvement of 241.7 %, 96.9 %, and 41.9 % for the responses of biomass, MIC, and STR, respectively. This is the first recorded study on the optimisation of culture conditions for the production of antimicrobial metabolites of P. polymyxa RNC-D, and constitutes an important step in the development of strategies to modulate the production of antimicrobial molecules by this microorganism at elevated levels.     

Effect of Elicitors and Precursors on Azadirachtin Production in Hairy Root Culture of Azadirachta indica

The present study involved strategies for enhancement in in vitro azadirachtin (commercially used biopesticide) production by hairy root cultivation of Azadirachta indica. Improvement in the azadirachtin production via triggering its biosynthetic pathway in plant cells was carried out by the exogenous addition of precursors and elicitors in the growth medium. Among the different abiotic stress inducers (Ag⁺, Hg⁺², Co⁺², Cu⁺²) and signal molecules (methyl jasmonate and salicylic acid) tested, salicylic acid at 15 mg l^?1 of concentration was found to enhance the azadirachtin yield in the hairy roots to the maximum (up to 4.95 mg g^?1). Similarly, among the different biotic elicitors tested (filter-sterilized fungal culture filtrates of Phoma herbarium, Alternaria alternata, Myrothecium sp., Fusarium solani, Curvularia lunata, and Sclerotium rolfsii; yeast extract; and yeast extract carbohydrate fraction), addition of filter-sterilized fungal culture filtrate of C. lunata (1 %?v/v) resulted in maximum azadirachtin yield enhancement in hairy root biomass (up to 7.1 mg g^?1) with respect to the control (3.3 mg g^?1). Among all the biosynthetic precursors studied (sodium acetate, cholesterol, squalene, isopentynyl pyrophosphate, mavalonic acid lactone, and geranyl pyrophosphate), the overall azadirachtin production (70.42 mg l^?1 in 25 days) was found to be the highest with cholesterol (50 mg l^?1) addition as an indirect precursor in the medium.     

Improving the Performance of a Continuous Process for the Production of Ethanol from Starch

In a previous work, a continuous simultaneous saccharification and fermentation process to produce ethanol from cassava starch was studied, using a set of fixed-bed reactors. The biocatalyst consisted of glucoamylase immobilized in silica particles and co-immobilized with S. cerevisiae in pectin gel. Using 3.8 U mL^?1 _reactor and 0.05 g_{wet yeast} mL^?1 _reactor at start-up, starch hydrolysis was the rate-limiting step. Maximum ethanol productivity was 5.8 g_ethanol L^?1 h^?1, with 94.0% conversion of total reducing sugars (TRS) and 83.0% of the ethanol theoretical yield. In this work, the molar mass of the substrate and the biocatalyst particle size were reduced in an attempt to improve the bioreactor performance. The diameters of silica and pectin gel particles were reduced from 100 μm and 3–4 mm, respectively, to 60 μm and 1–1.5 mm, and the degree of substrate prehydrolysis by α-amylase was increased. The bioreactor performance was assessed for different loads of immobilized glucoamylase (2.1, 2.8, and 3.8 U mL^?1 _reactor), for the same initial cell concentration (0.05 g_{wet yeast.}mL^?1 _reactor). Feeding with 154.0 g L^?1 of TRS and using 3.8 U mL^?1 _reactor, fermentation became the rate-limiting step. Productivity reached 11.7 g L^?1 h^?1, with 97.0% of TRS conversion and 92.0% of the ethanol theoretical yield. The reactor was operated during 275 h without any indication of destabilization.     

Mineral Supplementation Increases Erythrose Reductase Activity in Erythritol Biosynthesis from Glycerol by Yarrowia lipolytica

The aim of this study was to examine the impact of divalent copper, iron, manganese, and zinc ions on the production of erythritol from glycerol by Yarrowia lipolytica and their effect on the activity of erythrose reductase. No inhibitory effect of the examined minerals on yeast growth was observed in the study. Supplementation with MnSO₄·7H₂O (25 mg l^?1) increased erythritol production by Y. lipolytica by 14.5 %. In the bioreactor culture with manganese ion addition, 47.1 g l^?1 of erythritol was produced from 100.0 g l^?1 of glycerol, which corresponded to volumetric productivity of 0.87 g l^?1 h^?1. The addition of Mn²⁺ enhanced the intracellular activity of erythrose reductase up to 24.9 U g^?1 of dry weight of biomass (DW), hence, about 1.3 times more than in the control.     

Isolation of Novel Microalgae from Acid Mine Drainage and Its Potential Application for Biodiesel Production

Microalgae were selected and isolated from acid mine drainage in order to find microalgae species which could be cultivated in low pH condition. In the present investigation, 30 microalgae were isolated from ten locations of acid mine drainage in South Korea. Four microalgae were selected based on their growth rate, morphology, and identified as strains of KGE1, KGE3, KGE4, and KGE7. The dry biomass of microalgae species ranged between 1 and 2 g L^?1 after 21 days of cultivation. The growth kinetics of microalgae was well described by logistic growth model. Among these, KGE7 has the highest biomass production (2.05?±?0.35 g L^?1), lipid productivity (0.82?±?0.14 g L^?1), and C16–C18 fatty acid contents (97.6 %). These results suggest that Scenedesmus sp. KGE 7 can be utilized for biodiesel production based on its high biomass and lipid productivity.     

Ethanol Production from Sugarcane Bagasse by Zymomonas mobilis Using Simultaneous Saccharification and Fermentation (SSF) Process

Considerable efforts have been made to utilize agricultural and forest residues as biomass feedstock for the production of second-generation bioethanol as an alternative fuel. Fermentation utilizing strains of Zymomonas mobilis and the use of simultaneous saccharification and fermentation (SSF) process has been proposed. Statistical experimental design was used to optimize the conditions of SSF, evaluating solid content, enzymatic load, and cell concentration. The optimum conditions were found to be solid content (30%), enzymatic load (25 filter paper units/g), and cell concentration (4 g/L), resulting in a maximum ethanol concentration of 60 g/L and a volumetric productivity of 1.5 g L^?1?h^?1.     

Production of Butyric Acid from Glucose and Xylose with Immobilized Cells of <Emphasis Type="Italic">Clostridium tyrobutyricum</Emphasis> in a Fibrous-bed Bioreactor

Butyric acid has many applications in chemical, food, and pharmaceutical industries. In the present study, Clostridium tyrobutyricum ATCC 25755 was immobilized in a fibrous-bed bioreactor to evaluate the performance of butyrate production from glucose and xylose. The results showed that the final concentration and yield of butyric acid were 13.70 and 0.46 g g⁻¹, respectively, in batch fermentation when 30 g L⁻¹ glucose was introduced into the bioreactor. Furthermore, high concentration 10.10 g L⁻¹ and yield 0.40 g g⁻¹ of butyric acid were obtained with 25 g L⁻¹ xylose as the carbon source. The immobilized cells of C. tyrobutyricum ensured similar productivity and yield from repeated batch fermentation. In the fed-batch fermentation, the final concentration of butyric acid was further improved to 24.88 g L⁻¹ with one suitable glucose feeding in the fibrous-bed bioreactor. C. tyrobutyricum immobilized in the fibrous-bed bioreactor would provide an economically viable fermentation process to convert the reducing sugars derived from plant biomass into the final bulk chemical (butyric acid).