Pilot-scale bioethanol production from the starch of avocado seeds using a combination of dilute acid-based hydrolysis and alcoholic fermentation by Saccharomyces cerevisiae.

BACKGROUND: A processing methodology of raw starch extraction from avocado seeds (ASs) and a sequential hydrolysis and fermentation bioprocess in just a few steps was successfully obtained for the bioethanol production by a single yeast Saccharomyces cerevisiae strain and this research was also to investigate the optimum conditions for the pretreatment of biomass and technical procedures for the production of bioethanol. It successfully resulted in high yields and productivity of all the experiments from the laboratory scale and the pilot plant. Ethanol yields from pretreated starch are comparable with those in commercial industries that use molasses and hydrolyzed starch as raw materials. RESULTS: Before the pilot-scale bioethanol production, studies of starch extraction and dilute sulfuric acid-based pretreatment was carefully conducted. The amount of starch extracted from dry and fresh avocado seed was 16.85 g ± 0.34 g and 29.79 ± 3.18 g of dry starch, representing a yield of ∼17% and 30%, respectively. After a dilute sulfuric acid pretreatment of starch, the released reducing sugars (RRS) were obtained and the hydrolysate slurries containing glucose (109.79 ± 1.14 g/L), xylose (0.99 ± 0.06 g/L), and arabinose (0.38 ± 0.01 g/L). The efficiency of total sugar conversion was 73.40%, with a productivity of 9.26 g/L/h. The ethanol fermentation in a 125 mL flask fermenter showed that Saccharomyces cerevisiae (Fali, active dry yeast) produced the maximum ethanol concentration, pmax at 49.05 g/L (6.22% v/v) with a yield coefficient, Yp/s of 0.44 gEthanol/gGlucose, a productivity or production rate, rp at 2.01 g/L/h and an efficiency, Ef of 85.37%. The pilot scale experiments of the ethanol fermentation using the 40-L fermenter were also successfully achieved with essentially good results. The values of pmax,Yp/s, rp, and Ef of the 40-L scale were at 50.94 g/L (6.46% v/v), 0.45 gEthanol/gGlucose, 2.11 g/L/h, and 88.74%, respectively. Because of using raw starch, major by-products, i.e., acetic acid in the two scales were very low, in ranges of 0.88-2.45 g/L, and lactic acid was not produced, which are less than those values in the industries. CONCLUSIONS: The sequential hydrolysis and fermentation process of two scales for ethanol production using the combination of hydrolysis by utilizing dilute sulfuric acid-based pretreatment and fermentation by a single yeast Saccharomyces cerevisiae strain is practicable and feasible for realistic and effective scale-up strategies of bioethanol production from the starch of avocado seeds.

Co-Fermentation of Glucose-Xylose Mixtures from Agroindustrial Residues by Ethanologenic Escherichia coli: A Study on the Lack of Carbon Catabolite Repression in Strain MS04.

The production of biofuels, such as bioethanol from lignocellulosic biomass, is an important task within the sustainable energy concept. Understanding the metabolism of ethanologenic microorganisms for the consumption of sugar mixtures contained in lignocellulosic hydrolysates could allow the improvement of the fermentation process. In this study, the ethanologenic strain Escherichia coli MS04 was used to ferment hydrolysates from five different lignocellulosic agroindustrial wastes, which contained different glucose and xylose concentrations. The volumetric rates of glucose and xylose consumption and ethanol production depend on the initial concentration of glucose and xylose, concentrations of inhibitors, and the positive effect of acetate in the fermentation to ethanol. Ethanol yields above 80% and productivities up to 1.85 gEtOH/Lh were obtained. Furthermore, in all evaluations, a simultaneous co-consumption of glucose and xylose was observed. The effect of deleting the xyIR regulator was studied, concluding that it plays an important role in the metabolism of monosaccharides and in xylose consumption. Moreover, the importance of acetate was confirmed for the ethanologenic strain, showing the positive effect of acetate on the co-consumption rates of glucose and xylose in cultivation media and hydrolysates containing sugar mixtures.

Ethanol production by Escherichia coli from detoxified lignocellulosic teak wood hydrolysates with high concentration of phenolic compounds.

Teak wood residues were subjected to thermochemical pretreatment, enzymatic saccharification, and detoxification to obtain syrups with a high concentration of fermentable sugars for ethanol production with the ethanologenic Escherichia coli strain MS04. Teak is a hardwood, and thus a robust deconstructive pretreatment was applied followed by enzymatic saccharification. The resulting syrup contained 60 g l-1 glucose, 18 g l-1 xylose, 6 g l-1 acetate, less than 0.1 g l-1 of total furans, and 12 g l-1 of soluble phenolic compounds (SPCs). This concentration of SPC is toxic to E. coli, and thus two detoxification strategies were assayed: (1) treatment with Coriolopsis gallica laccase followed by addition of activated carbon and (2) overliming with Ca(OH)2. These reduced the phenolic compounds by 40% and 76%, respectively. The detoxified syrups were centrifuged and fermented with E. coli MS04. Cultivation with the overlimed hydrolysate showed a 60% higher volumetric productivity (0.45 gETOH l-1 hr-1). The bioethanol/sugar yield was over 90% in both strategies.

Sequential enzymatic saccharification and fermentation of ionic liquid and organosolv pretreated agave bagasse for ethanol production.

Agave bagasse (AGB) has gained recognition as a drought-tolerant biofuel feedstock with high productivity in semiarid regions. A comparative analysis of ionic liquid (IL) and organosolv (OV) pretreatment technologies in AGB was performed using a sequential enzymatic saccharification and fermentation (SESF) strategy with cellulolytic enzymes and the ethanologenic Escherichia coli strain MS04. After pretreatment, 86% of xylan and 45% of lignin were removed from OV-AGB, whereas IL-AGB reduced lignin content by 28% and xylan by 50% when compared to the untreated biomass. High glucan (>90%) and xylan (>83%) conversion was obtained with both pretreated samples. During the fermentation stage (48h), 12.1 and 12.7kg of ethanol were produced per 100kg of untreated AGB for IL and OV, respectively. These comparative analyses showed the advantages of SESF using IL and OV in a biorefinery configuration where a better understanding of AGB recalcitrance is key for future applications.

Production of d-lactate from sugarcane bagasse and corn stover hydrolysates using metabolic engineered Escherichia coli strains.

In this study, the lactogenic Escherichia coli strain JU15 was used and modified to produce d-lactate (d-LA) from plant hydrolysates with a minimal nutrient addition in pH controlled fermenters. Results showed that strain JU15 produces d-LA with high yield and productivity in laboratory simulated hydrolysate media and actual sugar cane bagasse hemicellulosic hydrolysate. Strain JU15 showed sequential carbon source utilization and acetic acid production. The l-lactic and acetic acid production pathways were deleted in JU15, resulting strain AV03 (JU15 ΔpoxB, ΔackA-pta, ΔmgsA), which showed simultaneous consumption of glucose and xylose and no acetic acid production in the simulated hydrolysate. The d-LA yield from hydrolysate sugars was close to 0.95gD-LA/gsugars in all cases. Our results show that d-LA can be produced from plant hydrolysates in simple batch fermentation processes with a high productivity using engineered E. coli strains at fermenter scales from 0.2 up to 10L.

Non-severe thermochemical hydrolysis of stover from white corn and sequential enzymatic saccharification and fermentation to ethanol.

A parametric study, with an initial load of 15%w/w of dry stover from white corn, was conducted to evaluate the sequential thermochemical hydrolysis (TH), enzymatic saccharification (ES) and fermentation of the whole slurry with ethanologenic Escherichia coli. The TH was designed to release the maximum amount of xylose with a concomitant formation of minimal amounts of furans. It was found that 29.0% or 93.2% of the xylan was recovered as free xylose at 130°C after 8 min in the presence of 1% or 2%w/w H2SO4 and produced only 0.06 or 0.44 g/L of total furans, respectively. After 24h of ES, 76.14-77.18 g/L of monosaccharides (pentoses and hexoses) were obtained. These slurries, which contained 0.03-0.26 g/L of total furans and 5.14-5.91 g/L of acetate, were fermented with 3.7 g/L of ethanologenic E. coli to produce 24.5-23.5 g/L of ethanol.

Production of Cinnamic and p-Hydroxycinnamic Acids in Engineered Microbes.

The aromatic compounds cinnamic and p-hydroxycinnamic acids (pHCAs) are phenylpropanoids having applications as precursors for the synthesis of thermoplastics, flavoring, cosmetic, and health products. These two aromatic acids can be obtained by chemical synthesis or extraction from plant tissues. However, both manufacturing processes have shortcomings, such as the generation of toxic subproducts or a low concentration in plant material. Alternative production methods are being developed to enable the biotechnological production of cinnamic and (pHCAs) by genetically engineering various microbial hosts, including Escherichia coli, Saccharomyces cerevisiae, Pseudomonas putida, and Streptomyces lividans. The natural capacity to synthesize these aromatic acids is not existent in these microbial species. Therefore, genetic modification have been performed that include the heterologous expression of genes encoding phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities, which catalyze the conversion of l-phenylalanine (l-Phe) and l-tyrosine (l-Tyr) to cinnamic acid and (pHCA), respectively. Additional host modifications include the metabolic engineering to increase carbon flow from central metabolism to the l-Phe or l-Tyr biosynthetic pathways. These strategies include the expression of feedback insensitive mutant versions of enzymes from the aromatic pathways, as well as genetic modifications to central carbon metabolism to increase biosynthetic availability of precursors phosphoenolpyruvate and erythrose-4-phosphate. These efforts have been complemented with strain optimization for the utilization of raw material, including various simple carbon sources, as well as sugar polymers and sugar mixtures derived from plant biomass. A systems biology approach to production strains characterization has been limited so far and should yield important data for future strain improvement.

Production of cinnamic and p-hydroxycinnamic acid from sugar mixtures with engineered Escherichia coli.

BACKGROUND: The aromatic compounds cinnamic acid (CA) and p-hydroxycinnamic acid (pHCA) are used as flavoring agents as well as precursors of chemicals. These compounds are present in plants at low concentrations, therefore, complex purification processes are usually required to extract the product. An alternative production method for these aromatic acids is based on the use of microbial strains modified by metabolic engineering. These biotechnological processes are usually based on the use of simple sugars like glucose as a raw material. However, sustainable production processes should preferably be based on the use of waste material such as lignocellulosic hydrolysates. RESULTS: In this study, E. coli strains with active (W3110) and inactive phosphoenolpyruvate:sugar phosphotransferase system (PTS) (VH33) were engineered for CA and pHCA production by transforming them with plasmids expressing genes encoding phenylalanine/tyrosine ammonia lyase (PAL/TAL) enzymes from Rhodotorula glutinis or Arabidopsis thaliana as well as genes aroGfbr and tktA, encoding a feedback inhibition resistant version of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase and transketolase, respectively. The generated strains were evaluated in cultures with glucose, xylose or arabinose, as well as a simulated lignocellulosic hydrolysate containing a mixture of these three sugars plus acetate. Production of CA was detected in strains expressing PAL/TAL from A. thaliana, whereas both CA and pHCA accumulated in strains expressing the enzyme from R. glutinis. These experiments identified arabinose and W3110 expressing PAL/TAL from A. thaliana, aroGfbr and tktA as the carbon source/strain combination resulting in the best CA specific productivity and titer. To improve pHCA production, a mutant with inactive pheA gene was generated, causing an 8-fold increase in the yield of this aromatic acid from the sugars in a simulated hydrolysate. CONCLUSIONS: In this study the quantitative contribution of active or inactive PTS as well as expression of PAL/TAL from R. glutinis or A. thaliana were determined for production performance of CA and pHCA when growing on carbon sources derived from lignocellulosic hydrolysates. These data will be a useful resource in efforts towards the development of sustainable technologies for the production of aromatic acids.