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.     

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.     

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.     

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.     

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.     

Alkyl-methylimidazolium ionic liquids affect the growth and fermentative metabolism of Clostridium sp

In this study, the effect of ionic liquids, 1-ethyl-3-methylimidazolium acetate [EMIM][Ac], 1-ethyl-3-methylimidazolium diethylphosphate [EMIM][DEP], and 1-methyl-3-methylimidazolium dimethylphosphate [MMIM][DMP] on the growth and glucose fermentation of Clostridium sp. was investigated. Among the three ionic liquids tested, [MMIM][DMP] was found to be least toxic. Growth of Clostridium sp. was not inhibited up to 2.5, 4 and 4 g L⁻¹ of [EMIM][Ac], [EMIM][DEP] and [MMIM][DMP], respectively. [EMIM][Ac] at <2.5 g L⁻¹, showed hormetic effect and stimulated the growth and fermentation by modulating medium pH. Total organic acid production increased in the presence of 2.5 and 2 g L⁻¹ of [EMIM][Ac] and [MMIM][DMP]. Ionic liquids had no significant influence on alcohol production at <2.5 g L⁻¹. Total gas production was affected by ILs at ?2.5 g L⁻¹ and varied with type of methylimidazolium IL. Overall, the results show that the growth and fermentative metabolism of Clostridium sp. is not impacted by ILs at concentrations below 2.5 g L⁻¹.     

Development of an industrial ethanol-producing yeast strain for efficient utilization of cellobiose

The BGL1 gene, encoding β-glucosidase in Saccharomycopsis fibuligera, was intracellular, secreted or cell-wall associated expressed in an industrial strain of Saccharomyces cerevisiae. The obtained recombinant strains were studied under aerobic and anaerobic conditions. The results indicated that both the wild type and recombinant strain expressing intracellular β-glucosidase cannot grow in medium using cellobiose as sole carbon source. As for the recombinant EB1 expressing secreted enzyme and WB1 expressing cell-wall associated enzyme, the maximum specific growth rates (μ_max) could reach 0.03 and 0.05 h⁻¹ under anaerobic conditions, respectively. Meanwhile, the surface-engineered S. cerevisiae utilized 5.2 g cellobiose L⁻¹ and produced 2.3 g ethanol L⁻¹ in 48 h, while S. cerevisiae secreting β-glucosidase into culture broth used 3.6 g cellobiose L⁻¹ and produced 1.5 g ethanol L⁻¹ over the same period, but no-full depletion of cellobiose were observed for both the used recombinant strains. The results suggest that S. cerevisiae used in industrial ethanol production is deficient in cellobiose transporter. However, when β-glucoside permease and β-glucosidase were co-expressed in this strain, it could uptake cellobiose and showed higher growth rate (0.11 h⁻¹) on cellobiose.     

In silico optimization and low structured kinetic model of poly[(R)-3-hydroxybutyrate] synthesis by Cupriavidus necator DSM 545 by fed-batch cultivation on glycerol

Glycerol was utilized by Cupriavidus necator DSM 545 for production of poly-3-hydroxybutyrate (PHB) in fed-batch fermentation. Maximal specific growth rates (0.12 and 0.3 h⁻¹) and maximal specific non-growth PHB production rate (0.16 g g⁻¹ h⁻¹) were determined from two experiments (inocula from exponential and stationary phase). Saturation constants for nitrogen (0.107 and 0.016 g L⁻¹), glycerol (0.05 g L⁻¹), non-growth related PHB synthesis (0.011 g L⁻¹) and nitrogen/PHB related inhibition constant (0.405 g L⁻¹), were estimated. Five relations for specific growth rate were tested using mathematical models. In silico performed optimization procedures (varied glycerol/nitrogen ratio and feeding) has resulted in a PHB content of 70.9%, shorter cultivation time (23 h) and better PHB yield (0.347 g g⁻¹). Initial concentration of biomass 16.8 g L⁻¹ and glycerol concentration in broth between 3 and 5 g L⁻¹ were decisive factors for increasing of productivity.     

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.     

Lactic acid production by Lactobacillus sp. RKY2 in a cell-recycle continuous fermentation using lignocellulosic hydrolyzates as inexpensive raw materials

Continuous lactic acid fermentations were conducted using lignocellulosic hydrolyzates and corn steep liquor as inexpensive raw materials. Lactic acid concentrations decreased with increases in the dilution rate, whereas the residual substrate concentrations increased. However, lactic acid yields were maintained at more than 0.90 g g⁻¹ over all cases experimented. The cell-recycle cultivation system exerted positive effects on fermentation efficiency, including volumetric productivity, which is attributable to the retention of cells in the bioreactor. The cell-recycle continuous fermentation of lignocellulosic hydrolyzates yielded a lactic acid productivity of 6.7 g l⁻¹ h⁻¹ for a dilution rate of 0.16 h⁻¹ using 30 g l⁻¹ of corn steep liquor and 1.5 g l⁻¹ of yeast extract as nutrients. The productivity (6.7 g l⁻¹ h⁻¹) acquired by the cell-recycle continuous fermentation of lignocellulosic hydrolyzates was 1.6 times higher than the lactic acid productivity yielded in the continuous fermentation without cell-recycle system.     

Enhanced ethanol production by fermentation of rice straw hydrolysate without detoxification using a newly adapted strain of Pichia stipitis

An enhanced inhibitor-tolerant strain of Pichia stipitis was successfully developed through adaptation to acid-treated rice straw hydrolysate. The ethanol production obtained by fermentation of NaOH-neutralized hydrolysate without detoxification using the adapted P. stipitis was comparable to fermentation of overliming-detoxified hydrolysate. The ethanol yield using the adapted P. stipitis with both types of hydrolysate at pH 5.0 achieved 0.45 g_p g_s⁻¹, which is equivalent to 87% of the maximum possible ethanol conversion. Furthermore, the newly adapted P. stipitis demonstrated significantly enhanced tolerance to sulfate and furfural despite the fact that both inhibitors had not been removed from the hydrolysate by NaOH neutralization. Finally, the ethanol conversion could be maintained at 60% and above when the neutralized hydrolysate contained 3.0% sulfate and 1.3 g L⁻¹ furfural.     

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.     

Cultivation of microalgae for oil production with a cultivation strategy of urea limitation

The microalgae, Chlorella sp., were cultivated in various culture modes to assess biomass and lipid productivity in this study. In the batch mode, the biomass concentrations and lipid content of Chlorella sp. cultivated in a medium containing 0.025–0.200 g L⁻¹ urea were 0.464–2.027 g L⁻¹ and 0.661–0.326 g g⁻¹, respectively. The maximum lipid productivity of 0.124 g d⁻¹ L⁻¹ occurred in a medium containing 0.100 g L⁻¹ urea. In the fed-batch cultivation, the highest lipid content was obtained by feeding 0.025 g L⁻¹ of urea during the stationary phase, but the lipid productivity was not significantly increased. However, a semi-continuous process was carried out by harvesting the culture and renewing urea at 0.025 g L⁻¹ each time when the cultivation achieved the early stationary phase. The maximum lipid productivity of 0.139 g d⁻¹ L⁻¹ in the semi-continuous culture was highest in comparison with those in the batch and fed-batch cultivations.     

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.     

Improved production of monoclonal antibodies through oxygen-limited cultivation of glycoengineered yeast

Glycoengineering technology can elucidate and exploit glycan related structure-function relationships for therapeutic proteins. Glycoengineered yeast has been established as a safe, robust, scalable, and economically viable expression platform. It has been found that specific productivity of antibodies in glycoengineered Pichia pastoris is a non-linear function of specific growth rate that is dictated by a limited methanol feed rate. The optimal carbon-limited cultivation requires an exponential methanol feed rate with an increasing biomass concentration and more significantly an increase in heat and mass transfer requirements that often become the limiting factor in scale-up. Both heat and mass transfer are stoichiometrically linked to the oxygen uptake rate. Consequently an oxygen-limited cultivation approach was evaluated to limit the oxygen uptake rate and ensure robust and reliable scale-up. The oxygen-limited process not only limited the maximum oxygen uptake rate (and consequently the required heat removal rate) in mut+ P. pastoris strains but also enabled extension of the induction phase leading to an increased antibody concentration (1.9 g L⁻¹ vs. 1.2 g L⁻¹), improved N-glycan composition and galactosylation, and reduced antibody fragmentation. Furthermore, the oxygen-limited process was successfully scaled to manufacturing pilot scale and thus presents a promising process option for the glycoengineered yeast protein expression platform.     

Vertical tubular photobioreactor for semicontinuous culture of Cyanobium sp

We evaluated the kinetic culture characteristics of the microalgae Cyanobium sp. grown in vertical tubular photobioreactor in semicontinuous mode. Cultivation was carried out in vertical tubular photobioreactor for 2 L, in 57 d, at 30 °C, 3200 Lux, and 12 h light/dark photoperiod. The maximum specific growth rate was found as 0.127 d⁻¹, when the culture had blend concentration of 1.0 g L⁻¹, renewal rate of 50%, and sodium bicarbonate concentration of 1.0 g L⁻¹. The maximum values of productivity (0.071 g L⁻¹ d⁻¹) and number of cycles (10) were observed in blend concentration of 1.0 g L⁻¹, renewal rate of 30%, and bicarbonate concentration of 1.0 g L⁻¹. The results showed the potential of semicontinuous cultivation of Cyanobium sp. in closed tubular bioreactor, combining factors such as blend concentration, renewal rate, and sodium bicarbonate concentration.     

Development of chemically defined media supporting high cell density growth of Ketogulonicigenium vulgare and Bacillus megaterium

The immediate precursor of L-ascorbic acid, or vitamin C, is 2-keto-l-gulonic acid (2-KLG). This is commonly produced commercially by Ketogulonicigenium vulgare and Bacillus megaterium, using corn steep liquor powder (CSLP) as an organic nitrogen source. In this study, the effects of the individual CSLP components (amino acids, vitamins, and metal elements) on 2-KLG production were evaluated, with the aim of developing a complete, chemically defined medium for 2-KLG production. Forty components of CSLP were analyzed, and key components were correlated to biomass, 2-KLG productivity, and consumption rate of L-sorbose. Glycine had the greatest effect, followed by serine, biotin, proline, nicotinic acid, and threonine. The combination of 0.28 g L⁻¹ serine, 0.36 g L⁻¹glycine, 0.18 g L⁻¹ threonine, 0.28 g L⁻¹proline, 0.19 g L⁻¹nicotinic acid, and 0.62 mg L⁻¹biotin in a chemically defined medium produced the highest maximum biomass concentration (4.2 × 10⁹ cfu mL⁻¹), 2-KLG concentration (58 g L⁻¹), and yield (0.76 g g⁻¹) after culturing for 28 h.     

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.     

Biological treatment of olive mill wastewater by non-conventional yeasts

The ability of lipolytic yeasts to grow on olive mill wastewater (OMW)-based medium and to produce high-value compounds while degrading this waste, was tested. OMW collected from three-phase olive mills from the North region of Portugal were characterized and used. OMW with COD ranging from 100 g L⁻¹ to 200 g L⁻¹ were supplemented with yeast extract and ammonium chloride. Studies of OMW consumption were carried out in batch cultures of Candida rugosa, Candida cylindracea and Yarrowia lipolytica. All strains were able to grow in the OMW-based media, without dilution, to consume reducing sugars and to reduce COD. C. cylindracea was the best strain concerning the lipase production and the reduction of phenolic compounds and COD. For all strains, the phenols degradation was quite difficult, mostly when more easily degradable carbon source is still present in the medium. Among the phenolic compounds tested catechol is the most inhibitory to the cells.