Optimal design and experimental validation of a three-zone simulated moving bed process based on the Amberchrom-CG161C adsorbent for continuous removal of acetic acid from biomass hydrolyzate

In the production process of bio-ethanol from biomass, acetic acid is recognized as the key impurity to be removed from the sugar components that are generated by hydrolyzing biomass. In regard to this issue, it has recently been confirmed that the Amberchrom-CG161C resin was highly qualified as the adsorbent of a simulated moving bed (SMB) process for continuous separation of acetic acid from the biomass hydrolyzate, i.e., sugars. However, the previous study on the Amberchrom-CG161C SMB with the aforementioned separation goal has been limited to only a theoretical work, including some batch-chromatography tests. The experimental validation of such an Amberchrom-CG161C SMB process, including its optimal design, was attempted in this article. This task began by assembling the experimental unit of the SMB process with three zones. Its operating conditions were then optimized by using genetic algorithm. Under the optimized operating conditions, the relevant three-zone SMB experiment was conducted. The assay of all the resultant product samples verified that the SMB separation of interest was performed successfully as designed. The experimental data were also found to agree closely with the model predictions. Finally, a partial-discard strategy was applied to maintain the sugar product concentration as high as possible.     

Development of a three-zone simulated moving bed process based on partial-discard strategy for continuous separation of valine from isoleucine with high purity,high yield,and high product concentration

The issue of separating valine from isoleucine has been a major concern in the biotechnological process for production of valine. To address this issue, an optimal three-zone simulated moving bed (SMB) process for continuous separation of valine was developed in this study. It was first found that an Amberchrom-CG161C resin was highly suitable for the adsorbent of such SMB process. The adsorption isotherm and mass-transfer parameters of valine and isoleucine on the Amberchrom-CG161C adsorbent were then determined through multiple frontal experiments. The determined parameters were used in the next stage of optimizing the SMB for valine separation, which was performed on the basis of genetic algorithm. For the optimized SMB process, a partial-discard strategy was applied to the raffinate port in order to make a further improvement in the valine product concentration. Finally, the optimized SMB based on the partial-discard strategy was tested experimentally using the self-assembled SMB equipment. The experimental results showed that the developed process in this study was highly effective in continuous separation of valine from isoleucine while ensuring the attainment of high product concentration. The experimental data for the SMB effluent histories and the SMB column profiles were also in close agreement with the model predictions.     

Effects of culture redox potential on succinic acid production by Corynebacterium crenatum under anaerobic conditions

During mixed-acid fermentation by Corynebacterium crenatum under anaerobic conditions, two moles of NADH are required to synthesize 1 mol of succinic acid. In this work, four controlled culture redox potentials and different carbon sources with different oxidation states were used to investigate the possibility of enhancing the succinic acid production by increasing the availability of NADH. When the culture redox potential was ?300 mV, the yield of succinic acid was 0.31 g/g, representing a 72% increase compared with the yield when the culture redox potential was ?40 mV. Meanwhile, the molar ratio of succinic acid/lactic acid increased from 0.27 to 0.48. When 0.1% neutral red was added to the acid production medium, the yield of succinic acid was 0.25 g/g, and the molar ratio of succinic acid/lactic acid was 0.38. Both values were higher than those obtained from glucose only (0.19 g/g, 0.26) or gluconate (0.05 g/g, 0.18). A higher NADH/NAD⁺ ratio and increased enzymatic activity could be achieved to enhance the succinic acid production by manipulating the culture to a more reductive environment.     

Improving the lactic acid production of Actinobacillus succinogenes by using a novel fermentation and separation integration system

This work describes the development of a novel integrated system for lactic acid production by Actinobacillus succinogenes. Fermentation and separation were integrated with the use of a microfiltration (MF) membrane, and lactic acid was recovered by resin adsorption following MF. The fermentation broth containing residual sugar and nutrients was then recycled back into the fermenter after lactic acid adsorption. This novel approach overcame the problem of product inhibition and extended the cell growth period from 41 h to 120 h. Production of lactic acid was improved by 23% to 183.4 g L⁻¹. The overall yield and productivity for glucose were 0.97 g g⁻¹ and 1.53 g L⁻¹ h⁻¹, respectively. These experimental results indicate that the integrated system could benefit continuous production of lactic acid at high levels.     

Purification of l-(+)-lactic acid from pre-treated fermentation broth using vapor permeation-assisted esterification

The vapor permeation-assisted esterification of l-(+)-lactic acid and ethanol was investigated using a zeolite membrane. Pervaporation and vapor permeation were initially investigated for dehydration performances, and the latter showed much better results. The molecular sieve property of the membrane resulted in a high separation factor of over 1000 for all conditions. The maximum flux was 10.24 kg/(m² h) at a feed temperature of 145 °C, a water feed concentration of 10 wt%, and a feed pressure of 4.0 bars, respectively. For vapor permeation-assisted esterification using synthetic solutions, the productivity and ethyl lactate yield strongly depended on the dehydration rate. Realistic purifications were performed with fermentation broths of Pediococcus pentosaceus as the lactic acid producer. Experimental results revealed that most of the lactic acid was converted into ethyl lactate at the final stage of the reaction. After distillation and hydrolysis, high purity l-(+)-lactic acid was obtained with more than 95% recovery yields.     

Two-step acid and alkaline ethanolysis/alkaline peroxide fractionation of sugarcane bagasse and rice straw for production of polylactic acid precursor

Sugarcane bagasse and rice straw were subjected to acid and alkaline ethanolysis and sequential enzymatic hydrolysis to produce glucose for lactic acid production. Influence of physico-chemical treatments using ultrasonic bath and ultrasonic probe was studied compared with mechanical stirring. The results showed that the highest glucose yield with least contamination of xylose was obtained from acid ethanolysis fractionation (5 N H₂SO₄ + 50%, v/v ethanol) when stirred at 90 °C for 4 h. Alkaline ethanolysis accomplished high amount of both glucose and xylose released, however it was not favorable substrate for homofermentative lactic acid bacteria. In order to enhance enzymatic hydrolysis of acid ethanolysis fractionated samples, lignin was subsequently removed by the second step alkaline/peroxide delignification. The maximum lactic acid was obtained at 23.6 ± 0.2 g/L from Lactobacillus casei fermentation after 72 h when hydrolysate from two-step acid hydrolysis and alkaline/peroxide fractionated sugarcane bagasse containing 24.6 g/L initial glucose concentration was used as substrate.     

Fermentative production of succinic acid from straw hydrolysate by Actinobacillus succinogenes

In this work, straw hydrolysates were used to produce succinic acid by Actinobacillus succinogenes CGMCC1593 for the first time. Results indicated that both glucose and xylose in the straw hydrolysates were utilized in succinic acid production, and the hydrolysates of corn straw was better than that of rice or wheat straw in anaerobic fermentation of succinic acid. However, cell growth and succinic acid production were inhibited when the initial concentration of sugar, which was from corn straw hydrolysate (CSH), was higher than 60 g l^?1. In batch fermentation, 45.5 g l^?1 succinic acid concentration and 80.7% yield were attained after 48 h incubation with 58 g l^?1 of initial sugar from corn straw hydrolysate in a 5-l stirred bioreactor. While in fed-batch fermentation, concentration of succinic acid achieved 53.2 g l^?1 at a rate of 1.21 g l^?1 h^?1 after 44 h of fermentation. Our work suggested that corn straw could be utilized for the economical production of succinic acid by A. succinogenes.     

Effects of redox potential control on succinic acid production by engineered Escherichia coli under anaerobic conditions

The effects of oxido-reduction potential (ORP) control on succinic acid production have been investigated in Escherichia coli LL016. In LL016, two CO₂ fixation pathways were achieved and NAD⁺ supply was enhanced by co-expression of heterologous pyruvate carboxylase (PYC) and nicotinic acid phosphoribosyltransferase (NAPRTase). During anaerobic fermentation, cell growth and metabolite distribution were changed with redox potential levels in the range of −200 to −400 mV. From the results, the ORP level of −400 mV was preferable, which resulted in the high succinic acid concentration (28.6 g/L) and high succinic acid productivity (0.33 g/L/h). Meanwhile, the yield of succinic acid at the ORP level of −400 mV was 39% higher than that at the ORP level of −200 mV. In addition, a higher NADH/NAD⁺ ratio and increased enzyme activities were also achieved by regulating the culture to a more reductive environment, which further enhanced the succinic acid production.     

Glycerol utilisation for the production of chemicals: Conversion to succinic acid,a combined experimental and computational study

An effective method for the valorisation of the main by-product of biodiesel production, i.e. glycerol is investigated in this work. It involves the biological conversion of glycerol to succinic acid, a top added-value material, which can be used as a building block for the production of various commodity and specialty chemicals. Our aim is to give new insights into this bioprocess, which has so far received little attention and is open for further investigation, through a combination of experimental and computational studies. The microorganism employed here was Actinobacillus succinogenes in batch bioreactors where glycerol was used as the sole carbon source.The highest obtained product yield, final succinate concentration and productivity were found to be equal to 1.23 g-succinate/g-glycerol, 29.3 g-succinate/L and 0.27 g-succinate/L/h, respectively. Furthermore, an unstructured model of the batch experiments was developed by considering both substrate and product inhibition. Kinetic parameters of the model were estimated by minimising the difference between experimental and predicted values. The corresponding optimisation problem was solved by using a combination of stochastic and deterministic methodologies, with the goal to probabilistically compute global minima and the resulting parameter values. The model developed can be utilised to successfully predict the concentration profiles of the five most important state variables (biomass, glycerol, succinic acid, formic acid, and acetic acid) with different initial glycerol concentrations. Scaled-up experiments in larger-scale bioreactors were used for further validation purposes. Our model can be further used to compute optimal operating/parametric conditions, which maximise yield, productivity and/or the final succinic acid concentration.     

Modelling and simulation of continuous L (+) lactic acid production from sugarcane juice in membrane integrated hybrid-reactor system

Modelling and simulation was done for a two-stage membrane-integrated hybrid reactor system for continuous production of L (+) lactic acid under non-neutralizing conditions. The model captures microbial conversion of sugar cane juice to lactic acid under substrate–product inhibitions with downstream purification by nanofiltration. All the major phenomena and the governing parameters like fluid flow, feed dilution, substrate–product inhibitions, Donnan and steric effects during micro and nanofiltration for cell recycle, product separation and purification have been reflected in the modelling. The model describes a green, integrated continuous process of direct lactic acid production starting with a cheap, renewable carbon source. The highest lactic acid concentration achieved after the final stage of nanofiltration was 66.97 g/L at 13 kg/cm² operating pressure when the overall productivity reached 12.40 g/(L h). The developed model could successfully predict production, purification and transport of lactic acid through two stage membrane modules. Performance of the model was very good as indicated in the high overall correlation coefficient (R² > 0.980) and the low relative error (RE < 0.1).     

CO2 fixation for succinic acid production by engineered Escherichia coli co-expressing pyruvate carboxylase and nicotinic acid phosphoribosyltransferase

In wild-type Escherichia coli, 1 mol of CO₂ was fixated in 1 mol of succinic acid generation anaerobically. The key reaction in this sequence, catalyzed by phosphoenolpyruvate carboxylase (PPC), is carboxylation of phosphoenolpyruvate to oxaloacetate. Although inactivation of pyruvate formate-lyase and lactate dehydrogenase is found to enhance the PPC pathway for succinic acid production, it results in excessive pyruvic acid accumulation and limits regeneration of NAD⁺ from NADH formed in glycolysis. In other organisms, oxaloacetate is synthesized by carboxylation of pyruvic acid by pyruvate carboxylase (PYC) during glucose metabolism, and in E. coli, nicotinic acid phosphoribosyltransferase (NAPRTase) is a rate-limiting enzyme of the NAD(H) synthesis system. To achieve the NADH/NAD⁺ ratio decrease as well as carbon flux redistribution, co-expression of NAPRTase and PYC in a pflB, ldhA, and ppc deletion strain resulted in a significant increase in cell mass and succinic acid production under anaerobic conditions. After 72 h, 14.5 g L⁻¹ of glucose was consumed to generate 12.08 g L⁻¹ of succinic acid. Furthermore, under optimized condition of CO₂ supply, the succinic acid productivity and the CO₂ fixation rate reached 223.88 mg L⁻¹ h⁻¹ and 83.48 mg L⁻¹ h⁻¹, respectively.     

Comparison of Amberchrom-CG161C and Dowex99 as the adsorbent of a four-zone simulated moving bed process for removal of acetic acid from biomass hydrolyzate

One of the important steps in the application of biomass to producing sugars, which can be converted into bio-ethanol and other valuable chemicals by fermentation, is to hydrolyze the biomass components by sulfuric acid. It was reported that such a hydrolysis entailed the generation of acetic acid, which has been recognized as a key impurity to be surely removed from the biomass hydrolyzate for ensuring high fermentability of the hydrolyzed sugars. Regarding such a removal task, there has been a previous application of a simulated moving bed (SMB) process based on the Dowex99 adsorbent, whose performance, however, was limited by low selectivity between acetic acid and sugars. To overcome such a limitation, another adsorbent alternative to Dowex99 was searched in this study. It was found that Amberchrom-CG161C allowed higher selectivity between acetic acid and sugars than Dowex99. To investigate the relative superiority of Amberchrom-CG161C over Dowex99 as the adsorbent of an SMB process for removing acetic acid from the biomass hydrolyzate, the two SMB processes based on Amberchrom-CG161C and Dowex99 were optimized using the SMB optimization tool based on standing wave design (SWD) method. The optimization results revealed that the Amberchrom-CG161C SMB outperformed the Dowex99 SMB by a wide margin.     

Aerobiosis–anaerobiosis transition has a significant impact on organic acid production by Corynebacterium glutamicum

Corynebacterium glutamicum is known to produce organic acids under anaerobic culture conditions, in particular, lactic, succinic, and acetic acids. Our study is focused on acetic and succinic acid production using a lactate dehydrogenase-deficient strain of C. glutamicum. Usually, with this bacterium, the organic acid production process is based on an initial aerobic growth phase, followed by a rapid deoxygenation and an anaerobic production phase. In our study, we demonstrated that this strategy was unfavorable for the production of organic acids. Conversely, we showed that applying the best transition strategy based on progressive deoxygenation significantly increased the concentration of organic acids up to 640%. This was observed either by applying controlled dissolved oxygen concentrations or by decreasing the steps of gas flow rates. Our results also showed that applying constant oxygen transfer flux throughout the culture, and thus in the absence of the anaerobic phase, promoted constant production yields (approximately 0.5 mol of succinate or acetate per mole of glucose). In this case, acetate production (120 mM) was favored over succinate production (132 mM), resulting in a decrease in the molar ratio of products (succinate/acetate) from 4.8 to 1.1 between progressive deoxygenation and constant OTR cultures.     

Enantioselective oxidation of racemic lactic acid to d-lactic acid and pyruvic acid by Pseudomonas stutzeri SDM

d-Lactic acid and pyruvic acid are two important building block intermediates. Production of d-lactic acid and pyruvic acid from racemic lactic acid by biotransformation is economically interesting. Biocatalyst prepared from 9 g dry cell wt l^?1 of Pseudomonas stutzeri SDM could catalyze 45.00 g l^?1 dl-lactic acid into 25.23 g l^?1 d-lactic acid and 19.70 g l^?1 pyruvic acid in 10 h. Using a simple ion exchange process, d-lactic acid and pyruvic acid were effectively separated from the biotransformation system. Co-production of d-lactic acid and pyruvic acid by enantioselective oxidation of racemic lactic acid is technically feasible.     

Metabolic engineering of Saccharomyces cerevisiae for the biotechnological production of succinic acid

The production of bio-based succinic acid is receiving great attention, and several predominantly prokaryotic organisms have been evaluated for this purpose. In this study we report on the suitability of the highly acid- and osmotolerant yeast Saccharomyces cerevisiae as a succinic acid production host. We implemented a metabolic engineering strategy for the oxidative production of succinic acid in yeast by deletion of the genes SDH1, SDH2, IDH1 and IDP1. The engineered strains harbor a TCA cycle that is completely interrupted after the intermediates isocitrate and succinate. The strains show no serious growth constraints on glucose. In glucose-grown shake flask cultures, the quadruple deletion strain Δsdh1Δsdh2Δidh1Δidp1 produces succinic acid at a titer of 3.62 g L^?1 (factor 4.8 compared to wild-type) at a yield of 0.11 mol (mol glucose)^?1. Succinic acid is not accumulated intracellularly. This makes the yeast S. cerevisiae a suitable and promising candidate for the biotechnological production of succinic acid on an industrial scale.     

Characterization of d-amino acid aminotransferase from Lactobacillus salivarius

We searched a UniProt database of lactic acid bacteria in an effort to identify d-amino acid metabolizing enzymes other than alanine racemase. We found a d-amino acid aminotransferase (d-AAT) homologous gene (UniProt ID: Q1WRM6) in the genome of Lactobacillus salivarius. The gene was then expressed in Escherichia coli, and its product exhibited transaminase activity between d-alanine and α-ketoglutarate. This is the first characterization of a d-AAT from a lactic acid bacterium. L. salivarius d-AAT is a homodimer that uses pyridoxal-5′-phosphate (PLP) as a cofactor; it contains 0.91 molecules of PLP per subunit. Maximum activity was seen at a temperature of 60 °C and a pH of 6.0. However, the enzyme lost no activity when incubated for 30 min at 30 °C and pH 5.5 to 9.5, and retained half its activity when incubated at pH 4.5 or 11.0 under the same conditions. Double reciprocal plots of the initial velocity and d-alanine concentrations in the presence of several fixed concentrations of α-ketoglutarate gave a series of parallel lines, which is consistent with a Ping-Pong mechanism. The K_m values for d-alanine and α-ketoglutarate were 1.05 and 3.78 mM, respectively. With this enzyme, d-allo-isoleucine exhibited greater relative activity than d-alanine as the amino donor, while α-ketobutylate, glyoxylate and indole-3-pyruvate were all more preferable amino acceptors than α-ketoglutarate. The substrate specificity of L. salivarius d-AAT thus differs greatly from those of the other d-AATs so far reported.     

Continuous succinic acid fermentation by Actinobacillus succinogenes

Fermentations were performed in an external recycle bioreactor using CO₂ and d-glucose at feed concentrations of 20 and 40 g L⁻¹. Severe biofilm formation prevented kinetic analysis of suspended cell (‘chemostat’) fermentation, while perlite packing enhanced the volumetric productivity by increasing the amount of immobilised cells. The highest productivity of 6.35 g L⁻¹ h⁻¹ was achieved at a dilution rate of 0.56 h⁻¹. A constant succinic acid yield of 0.69 ± 0.02 g/(g of glucose consumed) was obtained and found to be independent of the dilution rate, transient state and extent of biofilm build-up – approximately 56% of the carbon that formed phosphoenolpyruvate ended up as succinate. Byproduct analysis indicated that pyruvate oxidation proceeded solely via the formate-lyase pathway. Cell growth and corresponding biofilm formation were rapid at dilution rates higher than 0.35 h⁻¹ when the product concentrations were low (succinic acid < 10 g L⁻¹), while minimal growth was observed at succinic acid concentrations above this threshold.     

l-Lactic acid production from liquefied cassava starch by thermotolerant Rhizopus microsporus: Characterization and optimization

The thermotolerant Rhizopus microsporus DMKU 33 capable of producing l-lactic acid from liquefied cassava starch was isolated and characterized for its phylogenetic relationship and growth temperature and pH ranges. The concentrations of (NH₄)₂SO_4, KH₂PO₄, MgSO₄ and ZnSO₄·7H₂O in the fermentation medium was optimized for lactic acid production from liquefied cassava starch by Rhizopus microsporus DMKU 33 in shake-flasks at 40 °C. The fermentation was then studied in a stirred-tank bioreactor with aeration at 0.75 vvm and agitation at 200 rpm, achieving the highest lactic acid production of 84 g/L with a yield of 0.84 g/g at pH 5.5 in 3 days. Lactic acid production was further increased to 105–118 g/L with a yield of 0.93 g/g and productivity of 1.25 g/L/h in fed-batch fermentation. R. microsporus DMKU 33 is thus advantageous to use in simultaneous saccharification and fermentation for l-lactic acid production from low-cost starchy substrates.     

Enhanced anaerobic succinic acid production by Escherichia coli NZN111 aerobically grown on gluconeogenic carbon sources

Escherichia coli strain NZN111, a pflB and ldhA double mutant of E. coli W1485, is considered a candidate of succinic acid producer. However, it is reported that this strain fails to ferment glucose anaerobically. In this study, it was demonstrated that when a gluconeogenic carbon source was used to replace glucose in aerobic culture, the NZN111 cells restored the ability to ferment glucose in the subsequent anaerobic culture with succinic acid as the major product even though no further genetic manipulation had been carried out. Activities of enzymes including phosphoenolpyruvate (PEP) carboxykinase, PEP carboxylase, isocitrate lyase, malate dehydrogenase, malic enzyme, and pyruvate kinase in the NZN111 cells aerobically grown on different carbon sources were measured, and enhanced anaplerotic and oxaloacetate-reducing activities were revealed. Furthermore, supply of MgCO₃ or NaHCO₃ greatly improved succinate production by the malate-grown NZN111 cells. At the same time, pyruvic acid production was significantly reduced. When the malate-grown cells were anaerobically cultured in a salt medium with high pH buffering capacity, succinic acid was produced at a specific productivity of 308 mg/(g DCW h) with a molar yield of 1.31 mol succinic acid/mol glucose.     

Lactic acid and biosurfactants production from hydrolyzed distilled grape marc

Acid hydrolysis of distilled grape marc, an useless agricultural residue from wineries, was carried out using dilute sulfuric acid (1–5%) at several reaction times and 130 °C, in order to obtain monomeric sugars which after supplementation with corn steep liquor (10 g/L) and yeast extract (10 g/L) were used to carry out the fermentation into lactic acid by Lactobacillus pentosus without detoxification stage. Xylose was the main sugar generated followed by glucose and arabinose. Possible inhibitor compounds such as acetic acid liberated from acetyl groups, and furfural and hydroxymethylfurfural generated by sugars dehydration, were produced as degradation byproducts. The hydrolysis stage was optimized by using an incomplete factorial design where the independent variables were the percentage of catalyzer, the reaction time and the temperature. The optima conditions in terms of xylose concentration were 3.3% H₂SO₄, 125 min and 130 °C, but due to the high furfural concentration, two other conditions using lower reaction times (30 and 77.5 min) were also selected to assay the fermentation. Although any condition was feasible to fully utilize the relatively broad spectra of sugars released by the acid hydrolysis, under the shorter reaction time the best results were achieved (Q_P = 0.476 g/L h; Y_P/S = 0.71 g/g) which represents a theoretical yield of 97%. Furthermore, L. pentosus produced 4.8 mg/L of intracellular biosurfactants, measured as biosurfactin, representing a yield of 0.60 mg of intracellular biosurfactant per g of sugars consumed.