Bioethanol fermentation of concentrated rice straw hydrolysate using co-culture of Saccharomyces cerevisiae and Pichia stipitis

Rice straw is one of the abundant lignocellulosic feed stocks in the world and has been selected for producing ethanol at an economically feasible manner. It contains a mixture of sugars (hexoses and pentoses).Biphasic acid hydrolysis was carried out with sulphuric acid using rice straw. After acid hydrolysis, the sugars, furans and phenolics were estimated. The initial concentration of sugar was found to be 16.8 g L⁻¹. However to increase the ethanol yield, the initial sugar concentration of the hydrolysate was concentrated to 31 g L⁻¹ by vacuum distillation. The concentration of sugars, phenols and furans was checked and later detoxified by over liming to use for ethanol fermentation. Ethanol concentration was found to be 12 g L⁻¹, with a yield, volumetric ethanol productivity and fermentation efficiency of 0.33 g L⁻¹ h⁻¹, 0.4 g g⁻¹ and 95%, respectively by co-culture of OVB 11 (Saccharomyces cerevisiae) and Pichia stipitis NCIM 3498.     

Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanol by Pichia stipitis

Fermentation of acid hydrolysate of water-hyacinth (Eichhornia crassipes), a free floating aquatic plant has been investigated for ethanol production. The dilute acid treatment has been applied to utilize the maximum hemicellulosic content of the water-hyacinth. The goal of this work was to investigate, both experimentally and theoretically using mathematical tools, a fermentative system utilizing water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate as a substrate for ethanol production using Pichia stipitis. It was found that 72.83% of xylose was converted to ethanol with a yield of 0.425 g_p/g_s and productivity of 0.176 g_p/L/h. An appropriate mathematical model was developed to explain theoretically the bioconversion of this hemicellulose acid hydrolysate to ethanol and the model was tested statistically to check the validity of the model.     

Biological treatment of eucalypt spent sulphite liquors: a way to boost the production of second generation bioethanol

The fermentation of reducing sugars from hardwood (eucalypt) spent sulphite liquor (HSSL) into ethanol by Pichia (Scheffersomyces) stipitis is hindered by concomitant inhibitors of microbial metabolism. The conditions for the HSSL biological treatment step by Paecilomyces variotii were evaluated and optimised. Two different strategies of reactor operation were compared using single batch (B) and sequential batch reactor (SBR). Biological treatment of HSSL in the SBR revealed the best results with respect to the removal of microbial inhibitors. Also, most of inhibitory compounds, acetic acid, gallic acid, pyrogallol, amongst others, were removed from HSSL by P. variotii before the ethanol fermentation. The bio-detoxified HSSL was subjected to a successful fermentation by P. stipitis, attaining a maximum ethanol concentration of 2.4 g L⁻¹ with a yield of 0.24 g ethanol g sugars⁻¹.     

Effect of nutrient limitation and two-stage continuous fermentor design on productivities during "Clostridium ragsdalei" syngas fermentation

The effect of three limiting nutrients, calcium pantothenate, vitamin B₁₂ and cobalt chloride (CoCl₂), on syngas fermentation using “Clostridium ragsdalei” was determined using serum bottle fermentation studies. Significant results from the bottle studies were translated into single- and two-stage continuous fermentor designs. Studies indicated that three-way interactions between the three limiting nutrients, and two-way interactions between vitamin B₁₂ and CoCl₂ had a significant positive effect on ethanol and acetic acid formation. In general, ethanol and acetic acid production ceased at the end of 9 days corresponding to the production of 2.01 and 1.95 g L⁻¹ for the above interactions. Reactor studies indicated the three-way nutrient limitation in two-stage fermentor showed improved acetic acid (17.51 g g⁻¹ cells) and ethanol (14.74 g g⁻¹ cells) yield compared to treatments in single-stage fermentors. These results further support the hypothesis that it is possible to modulate the product formation by limiting key nutrients during C. ragsdalei syngas fermentation.     

Ethanol production from syngas by Clostridium strain P11 using corn steep liquor as a nutrient replacement to yeast extract

The feasibility of replacing yeast extract (YE) by corn steep liquor (CSL), a low cost nutrient source, for syngas fermentation to produce ethanol using Clostridium strain P11 was investigated. About 32% more ethanol (1.7 g L⁻¹) was produced with 20 g L⁻¹ CSL media in 250-mL bottle fermentations compared to media with 1 g L⁻¹ YE after 360 h. Maximum ethanol concentrations after 360 h of fermentation in a 7.5-L fermentor with 10 and 20 g L⁻¹ CSL media were 8.6 and 9.6 g L⁻¹, respectively, which represent 57% and 60% of the theoretical ethanol yields from CO. Only about 6.1 g L⁻¹ of ethanol was obtained in the medium with 1 g L⁻¹ YE after 360 h, which represents 53% of the theoretical ethanol yield from CO. The use of CSL also enhanced butanol production by sevenfold compared to YE in bottle fermentations. These results demonstrate that CSL can replace YE as the primary medium component and significantly enhance ethanol production by Clostridium strain P11.     

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.     

Development of sequential-co-culture system (Pichia stipitis and Zymomonas mobilis) for bioethanol production from Kans grass biomass

Sequential-co-culture technique was investigated in this study for the production of bioethanol from relatively cheaper lignocellulosic biomass of Kans grass (Saccharum spontaneum). The consortium of Pichia stipitis and Zymomonas mobilis was used to develop a suitable sequential-co-culture system. The Kans grass biomass was hydrolyzed in such a manner that the two sugar fractions, xylose rich and glucose rich were generated (a separate study). The P. stipitis cells and respective fermentation media (xylose rich) were fed to the fermentation vessel, after the set fermentation time Z. mobilis cells and respective media were fed to the same vessel. Different strategies have been followed and experiments were conducted initially at flask level. The selected strategy was then applied at bioreactor level using both synthetic fermentation media and Kans grass hydrolysate media to compare the kinetic parameters. The sequential addition of cultures with their respective media and imposed process conditions, showed better utilization of total sugars added (>95%). Microaerobic condition for P. stipitis and strictly anaerobic condition for Z. mobilis fermentation were found significant. The average ethanol yield (Y_p/s) and overall volumetric productivity (r_po) were found as 0.453 g_p/g_s and 1.580 g/l/h respectively for Kans grass hydrolysate media and 0.474 g_p/g_s and 2.901 g/l/h respectively for synthetic fermentation media.     

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.     

Ethanol production from sweet sorghum juice using very high gravity technology: Effects of carbon and nitrogen supplementations

Ethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP01 was investigated under very high gravity (VHG) fermentation and various carbon adjuncts and nitrogen sources. When sucrose was used as an adjunct, the sweet sorghum juice containing total sugar of 280 g l⁻¹, 3 g yeast extract l⁻¹ and 5 g peptone l⁻¹ gave the maximum ethanol production efficiency with concentration, productivity and yield of 120.68 ± 0.54 g l⁻¹, 2.01 ± 0.01 g l⁻¹ h⁻¹ and 0.51 ± 0.00 g g⁻¹, respectively. When sugarcane molasses was used as an adjunct, the juice under the same conditions gave the maximum ethanol concentration, productivity and yield with the values of 109.34 ± 0.78 g l⁻¹, 1.52 ± 0.01 g l⁻¹ h⁻¹ and 0.45 ± 0.01 g g⁻¹, respectively. In addition, ammonium sulphate was not suitable for use as a nitrogen supplement in the sweet sorghum juice for ethanol production since it caused the reduction in ethanol concentration and yield for approximately 14% when compared to those of the unsupplemented juices.     

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.     

Scale-up study of oxalic acid pretreatment of agricultural lignocellulosic biomass for the production of bioethanol

Building on our laboratory-scale optimization, oxalic acid was used to pretreat corncobs on the pilot-scale. The hydrolysate obtained after washing the pretreated biomass contained 32.55 g/l of xylose, 2.74 g/l of glucose and low concentrations of inhibitors. Ethanol production, using Scheffersomyces stipitis, from this hydrolysate was 10.3 g/l, which approached the predicted value of 11.9 g/l. Diafiltration using a membrane system effectively reduced acetic acid in the hydrolysate, which increased the fermentation rate. The hemicellulose content of the recovered solids decreased from 27.86% before pretreatment to only 6.76% after pretreatment. Most of the cellulose remained in the pretreated biomass. The highest ethanol production after simultaneous saccharification and fermentation (SSF) of washed biomass with S. stipitis was 21.1 g/l.     

Enhanced resistance of Saccharomyces cerevisiae to vanillin by expression of lacA from Trametes sp. AH28-2

Saccharomyces cerevisiae is affected by the presence of certain phenolic compounds such as vanillin during fermentation of pretreated lignocellulosic hydrolysates. Since vanillin can be polymerized in the presence of laccase into compounds with lower toxicity, the laccase gene, lacA, from Trametes sp. AH28-2 was fused to the α-factor signal sequence and transferred into S. cerevisiae CEN.PK strains for secretory expression. Furthermore, the chaperone gene, KAR2, was overexpressed to promote the translocation of laccase. In the presence of 8 mmol/L vanillin, a shorter lag phase was observed in the lacA gene expressing strains. The vanillin-specific conversion rate of the lacA-expressing strain BSJX0A2 was 0.069 g g⁻¹ biomass h⁻¹, while it was 0.065 g g⁻¹ biomass h⁻¹ in the reference strain.     

pH-control modes in a 5-L stirred-tank bioreactor for cell biomass and exopolysaccharide production by Tremella fuciformis spore

The effect of pH-control modes on cell growth and exopolysaccharide production by Tremella fuciformis was evaluated in a 5-L bioreactor. The results show that the maximal dry cell weight (DCW) and exopolysaccharide production were 23.57 and 4.48 g L⁻¹ in pH-stat fermentation, where the maximal specific growth rate (μ_max) and specific production rate of exopolysaccharide (P_P/X) were 1.03 and 0.24 d⁻¹, respectively; under pH-shift cultivation, the maximal DCW and exopolysaccharide production were 30.57 and 3.90 g L⁻¹, where the μ_max and P_P/X were 1.21 and 0.06 d⁻¹. Unlike batch fermentation, maximal DCW and exopolysaccharide production merely reached 15.04 and 2.0 g L⁻¹, where the μ_max and P_P/X were 0.86 and 0.05 d⁻¹, respectively. These results suggest that a pH-stat strategy is a more efficient way of performing the fermentation process to increase exopolysaccharide production. Furthermore, this research has also proved that the three-stage pH-control mode is effective for cell growth.     

Purification and characterization of two extracellular endochitinases from Massilia timonae

Two extracellular chitinases (designated as Chi-56 and Chi-64) produced by Massilia timonae were purified by ion-exchange chromatography, ammonium sulfate precipitation, and gel-filtration chromatography. The molecular mass of Chi-56 was 56 kDa as determined by both SDS-PAGE and gel-filtration chromatography. On the other hand, Chi-64 showed a molecular mass of 64 kDa by SDS-PAGE and 28 kDa by gel-filtration chromatography suggesting that its properties may be different from those of Chi-56. The optimum temperature, optimum pH, pI, K_m, and V_max of Chi-56 were 55 °C, pH 5.0, pH 8.5, 1.1 mg mL⁻¹, and 0.59 μmol μg⁻¹ h⁻¹, respectively. For Chi-64, these values were 60 °C, pH 5.0, pH 8.5, 1.3 mg mL⁻¹, and 1.36 μmol μg⁻¹ h⁻¹, respectively. Both enzymes were stimulated by Mn²⁺ and inhibited by Hg²⁺, and neither showed exochitinase activity. The N-terminal sequences of Chi-56 and Chi-64 were determined to be Q-T-P-T-Y-T-A-T-L and Q-A-D-F-P-A-P-A-E, respectively.     

Butyric acid fermentation in a fibrous bed bioreactor with immobilized Clostridium tyrobutyricum from cane molasses

Butyrate fermentation by immobilized Clostridium tyrobutyricum was successfully carried out in a fibrous bed bioreactor using cane molasses. Batch fermentations were conducted to investigate the influence of pH on the metabolism of the strain, and the results showed that the fermentation gave a highest butyrate production of 26.2 g l⁻¹ with yield of 0.47 g g⁻¹ and reactor productivity up to 4.13 g l⁻¹ h⁻¹ at pH 6.0. When repeated-batch fermentation was carried out, long-term operation with high butyrate yield, volumetric productivity was achieved. Several cane molasses pretreatment techniques were investigated, and it was found that sulfuric acid treatment gave better results regarding butyrate concentration (34.6 ± 0.8 g l⁻¹), yield (0.58 ± 0.01 g g⁻¹), and sugar utilization (90.8 ± 0.9%). Also, fed-batch fermentation from cane molasses pretreated with sulfuric acid was performed to further increase the concentration of butyrate up to 55.2 g l⁻¹.     

Bicarbonate use in three aquatic plants

Our study aimed to test the ability of aquatic plants to use bicarbonate when acclimated to three different bicarbonate concentrations. To this end, we performed experiments with the three species Ceratophyllum demersum, Egeria densa, Lagarosiphon major to determine photosynthetic rates under varying bicarbonate concentrations. We measured bicarbonate use efficiency, photosynthetic performance and respiration. For all species, our results revealed that photosynthetic rates were highest in replicates grown at low alkalinity. Thus, E. densa had approx. five times higher rates at low (264 ± 15 μmol O₂ g⁻¹ DW h⁻¹) than at high alkalinity (50 ± 27 μmol O₂ g⁻¹ DW h⁻¹), C. demersum had three times higher rates (336 ± 95 and 120 ± 31 μmol O₂ g⁻¹ DW h⁻¹), and L. major doubled its rates at low alkalinity (634 ± 114 and 322 ± 119 μmol O₂ g⁻¹ DW h⁻¹). Similar results were obtained for bicarbonate use efficiency by E. densa (136 ± 44 and 43 ± 10 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹) and L. major (244 ± 29 and 82 ± 24 μmol O₂ mequiv. L⁻¹ g⁻¹ DW h⁻¹). As to C. demersum, efficiency was high but unaffected by alkalinity, indicating high adaptation ability to varied alkalinities. A pH drift experiment supported these results. Overall, our results suggest that the three globally widespread worldwide species of our study adapt to low inorganic carbon availability by increasing their efficiency of bicarbonate use.     

3-Phenyllactic acid production by substrate feeding and pH-control in fed-batch fermentation of Lactobacillus sp. SK007

3-Phenyllactic acid (PLA), which is produced by some strains of lactic acid bacteria (LAB), is a known antimicrobial agent with a broad spectrum. Batch and fed-batch fermentation by the strain Lactobacillus sp. SK007 for PLA production have been reported. With batch fermentation without pH-control, PLA production yield was 2.42 g L⁻¹. When fed-batch fermentation by Lactobacillus sp. SK007 was conducted in 3 L initial volume with pH-control at 6.0 and intermittent feeding, which was developed after fermentation for 12 h and every 2 h with 120 mL 100 g L⁻¹ PPA phenylpyruvic acid (PPA) and 50 mL 500 g L⁻¹ glucose each time, PLA production yield reached 17.38 g L⁻¹. The final conversion ratio of PPA to PLA was 51.1%, and the PLA production rate was 0.241 g L⁻¹ h⁻¹. This indicated that PPA was the ideal substrate for PLA fermentation production, and fed-batch fermentation with intermittent PPA feeding and pH-control was an effective approach to improve PLA production yield.     

Production of xylooligosaccharides by xylanase from Pichia stipitis based on xylan preparation from triploid Populas tomentosa

Xylooligosaccharides (XOS) with DP 2-4 are important synbiotics used as food ingredients based on its prebiotic characteristics. In this work, the production of XOS from lignocellulosic material was performed by combined chemical-enzymatic methods. Xylan was prepared from triploid Populas tomentosa, and bioconverted into XOS by crude xylanase solution obtained from Pichia stipitis. The effects of reaction time, temperature, enzyme dosage, and pH value on the production of XOS were fully evaluated. Under the optimal condition (25 U g⁻¹ substrate, pH 5.4 and 50 °C), 36.8% of the xylan preparation was converted to XOS, equivalent to 3.95 mg/mL of the hydrolyzate. Xylobiose, xylotriose and xylotetrose were analyzed to be the main products of the enzymatic hydrolyzate, which together accounted for over 95% of the released oligosaccharides. Meanwhile, the effect of sonication pretreatment on the conversion efficiency of the xylan preparation was also investigated.     

A novel co-culture process with Zymomonas mobilis and Pichia stipitis for efficient ethanol production on glucose/xylose mixtures

An efficient conversion of glucose and xylose is a requisite for a profitable process of bioethanol production from lignocellulose. Considering the approaches available for this conversion, co-culture is a simple process, employing two different organisms for the fermentation of the two sugars. An innovative fermentation scheme was designed, co-culturing immobilized Zymomonas mobilis and free cells of Pichia stipitis in a modified fermentor for the glucose and xylose fermentation, respectively. A sugar mixture of 30 g/l glucose and 20 g/l of xylose was completely converted to ethanol within 19 h. This gave a volumetric ethanol productivity of 1.277 g/l/h and an ethanol yield of 0.49–0.50 g/g, which is more than 96% of the theoretical value. Extension of this fermentation scheme to sugarcane bagasse hydrolysate resulted in a complete sugar utilisation within 26 h; ethanol production peaked at 40 h with a yield of 0.49 g/g. These values are comparable to the best results reported. Cell interaction was observed between Z. mobilis and P. stipitis. Viable cells of Z. mobilis inhibited the cell activity of P. stipitis and the xylose fermentation. Z. mobilis showed evidence of utilising a source other than glucose for growth when co-cultured with P. stipitis.     

Primary production,respiration and calcification of the temperate free-living coralline alga Lithothamnion corallioides

Calcification and primary production responses to irradiance in the temperate coralline alga Lithothamnion corallioides were measured in summer 2004 and winter 2005 in the Bay of Brest. Coralline algae were incubated in dark and clear bottles exposed to different irradiances. Net primary production reached 1.5 μmol C g⁻¹ dry wt h⁻¹ in August and was twice as high as in January–February. Dark respiration showed significant seasonal variations, being three-fold higher in summer. Maximum calcification varied from 0.6 μmol g⁻¹ dry wt h⁻¹ in summer 2004 to 0.4 μmol g⁻¹ dry wt h⁻¹ in winter 2005. According to P–E curves and the daily course of irradiance, estimated daily net production and calcification reached 131 μg C g⁻¹ dry wt and 970 μg CaCO₃ g⁻¹ dry wt in summer 2004, and 36 μg C g⁻¹ dry wt and 336 μg CaCO₃ g⁻¹ dry wt in winter 2005. The net primary production of natural L. corallioides populations in shallow waters was estimated at 10–600 g C m⁻² y⁻¹, depending on depth and algal biomass. The mean annual calcification of L. corallioides populations varied from 300 to 3000 g CaCO₃ m⁻². These results are similar to those reported for tropical coralline algae in terms of carbon and carbonate productivity. Therefore, L. corallioides can be considered as a key element of carbon and carbonate cycles in the shallow coastal waters where they live.