Production Of Bio-based Chemicals Research Articles

Prototyping of microbial chassis for the biomanufacturing of high-value chemical targets

Metabolic engineering technologies have been employed with increasing success over the last three decades for the engineering and optimization of industrial host strains to competitively produce high-value chemical targets. To this end, continued reductions in the time taken from concept, to development, to scale-up are essential. Design-Build-Test-Learn pipelines that are able to rapidly deliver diverse chemical targets through iterative optimization of microbial production strains have been established. Biofoundries are employing in silico tools for the design of genetic parts, alongside combinatorial design of experiments approaches to optimize selection from within the potential design space of biological circuits based on multi-criteria objectives. These genetic constructs can then be built and tested through automated laboratory workflows, with performance data analysed in the learn phase to inform further design. Successful examples of rapid prototyping processes for microbially produced compounds reveal the potential role of biofoundries in leading the sustainable production of next-generation bio-based chemicals.

Strategies for the Removal of Polysaccharides from Biorefinery Lignins: Process Optimization and Techno Economic Evaluation.

The utilization of biorefinery lignins as a renewable resource for the production of bio-based chemicals and materials remain a challenge because of the high polysaccharide content of this variety of lignins. This study provides two simple methods; (i) the alkaline hydrolysis-acid precipitation method and (ii) the acid hydrolysis method for the removal of polysaccharides from polymeric biorefinery lignin samples. Both purification strategies are optimized for two different hardwood hydrolysis lignins, HL1 and HL2, containing 15.1% and 10.1% of polysaccharides, respectively. The treated lignins are characterized by polysaccharide content, molecular weight, hydroxyl content, and Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR). Preliminary techno-economic calculations are also carried out for both purification processes to assess the economic potential of these technologies. The results indicate that both protocols could be used for the purification of HL1 and HL2 hydrolysis lignins because of the minimal polysaccharide content obtained in the treated lignins. Nevertheless, from an industrial and economic perspective the acid hydrolysis technology using low acid concentrations and high temperatures is favored over the alkaline hydrolysis-acid precipitation strategy.

Advances in metabolic engineering of non-conventional yeasts

Over the past 30 years, Yarrowia lipolytica, Kluyveromyces, Pichia, Candida, Hansenula and other non-conventional yeasts have attracted wide attention because of their desirable phenotypes, such as rapid growth, capability of utilizing multiple substrates, and stress tolerance. A variety of synthetic biology tools are being developed for exploitation of their unique phenotypes, making them potential cell factories for the production of recombinant proteins and renewable bio-based chemicals. This review summarizes the gene editing tools and the metabolic engineering strategies recently developed for non-conventional yeasts. Moreover, the challenges and future perspectives for developing non-conventional yeasts into efficient cell factories for the production of useful products through metabolic engineering are discussed.

Enhanced conversion of biomass to furfurylamine with high productivity by tandem catalysis with sulfonated perlite and ω-transaminase whole-cell biocatalyst

Production of bio-based chemicals from renewable bioresource is a key driver for moving towards sustainable industry. Furfurylamine is known as an important furfural-upgrading product in organic synthesis, as well as monolithic synthetic pharmaceuticals, fibers, additives and polymers. In one-pot manner, biomass was tandemly catalyzed to furfurylamine with sulfonated Sn-PL catalyst and recombinant ω-transaminase biocatalyst. Sn-PL (2.4 wt%) catalyzed bamboo shoot shell, corncob and rice straw (75.0 g/L) to 76.5–113.0 mM furfural at 44.7–58.5 % yield in γ-valerolactone–water (2:8, v:v) at 170 ℃. The obtained biomass slurries containing furfural were biotransformed to furfurylamine at high yield (0.39−0.42 g furfurylamine/g xylan in biomass) with ω-transaminase biocatalyst using isopropylamine (3.0 mol isopropylamine/mol furfural) as amine donor at 35 ℃. Such a chemoenzymatic one-pot process combined the advantages of both solid acids and whole-cells catalysts, which provided an efficient and sustainable approach for preparing an important bio-based furan chemical furfurylamine.

Valorisation of grape stalks and pomace for the production of bio-based succinic acid by Actinobacillus succinogenes

Valorisation of food supply chain waste for the production of bio-based platform chemicals will facilitate the transition to the sustainable bio-economy era. To this end, a novel process has been developed for the conversion of grape pomace and stalks into bio-based succinic acid. Free sugars (2.55 g/100 g), a phenolic-rich extract (185.75 mg gallic acid equivalent per g extract with Antioxidant Activity Index of 1.03) and lipids (8.2 g/ 100 g) were initially extracted from grape pomace. Residual grape pomace solids were subsequently mixed with grape stalks (1:1 ratio) and treated with sodium hydroxide (1%, w/v) for lignin removal. The residual solids were treated with varying H2SO4 concentrations (3–10 %, v/v) followed by enzymatic hydrolysis using different dosages of cellulases and β-glucosidase leading to the release of 22.7 g glucose per 100 g remaining solids (48 % cellulose hydrolysis). Actinobacillus succinogenes fed-batch cultures carried out with the crude hydrolysate as fermentation medium and free sugars from grape pomace as feeding solution resulted in 40.2 g/L succinic acid concentration with a yield of 0.67 g/g and productivity of 0.79 g/L/h. This study proposes for the first time a sustainable process for valorising grape pomace and stalks for efficient bio-based succinic acid production integrated with the extraction of value-added fractions.

A New Approach to Produce Succinic Acid Through a Co-Culture System.

Microorganisms can produce a wide range of bio-based chemicals that can be used in various industrial applications as molecules of interest. In the present work, an analysis of the power production by pure culture, co-culture, and sequential culture was performed. In this study, both the mono-culture and the co-culture strategies of Actinobacillus succinogenes with Saccharomyces cerevisiae as carbon sources to produce succinic acid using glucose and fructose were examined. The cultures were performed in batch mode and a great attention was paid to the co-culture system to improve the biosynthetic pathway between A. succinogenes and S. cerevisiae by combining these two strains in a single fermentation process. Under microaerobic and anaerobic conditions, the process was characterized in terms of sugars concentration, cell density, metabolites, yield (mol-C products/ mol-C sugars), the temperature conditions for productivity, and pH. The results showed that the process could consume glucose and fructose and could adapt to different concentrations of the two sugars more quickly than by a single organism and the best results were obtained in a sequential co-culture recording 0.27 mol L-1 of succinic acid concentration and a volumetric productivity of 0.3 g L-1 h-1. Under the investigated operating conditions, the combination of these two strains in a single reactor produced a significant amount of succinic acid (0.70 mol-C SA/mol-C substrates). A simultaneous and sequential co-culture strategy can be a powerful new approach in the field of bio-based chemical production.

Catalytic upcycling paper sludge for the recovery of minerals and production of renewable high-grade biofuels and bio-based chemicals

Paper sludge is a solid waste by-product abundantly produced in the paper industry and contains fine minerals and lignocellulosic biomass. In this contribution, the ex-situ catalytic fast pyrolysis of paper sludge with a high mineral content of ca. 71 wt% is reported for the first time and demonstrated in a pilot-scale unit (feeding rate of 11.3 kg h−1) using a granular Na2CO3/γ-Al2O3 catalyst (loading of 650 g) to produce a high-grade bio-oil at a fast pyrolysis temperature of 475 ± 5 °C (pre-screened using a pyroprobe) and a catalytic upgrading temperature of 500 °C. Besides, > 99 wt% minerals in the paper sludge could be recovered, including paper fillers such as CaCO3 (major) and talc. The bio-oil obtained at a carbon yield of 21 C.% has a low oxygen content of 3.2 wt%, a low total acidity number of 5.2 mg KOH g−1, low H2O content of 0.7 wt%, and a high higher heating value of 40.9 MJ kg−1. It consists of value-added bio-based chemicals such as paraffins, olefins, and low molecular weight aromatics. The results demonstrate that the use of paper sludge to recover minerals and to obtain fuels and chemicals using ex-situ catalytic pyrolysis is technically feasible at a pilot plant scale, which will be of importance for the development of future bio-based economy.

Getting your hands dirty: A data digging exercise to unearth the EU's bio-based chemical sector

Under the auspices of the EU's new Circular Economy Action Plan and Bioeconomy Strategy, the usage of sustainably renewable biomass for bio-based chemicals is a part-solution for addressing the multidimensional challenges of (inter alia) growth and employment, food and energy security, climate change and biodiversity. Unfortunately, the lack of a formal system of European data classification and collection presents a major obstacle to measuring, monitoring and ex-ante modelling of the bio-based chemicals sector, which clouds the ability to make science-based policy and legislative judgements. Employing a combination of different data sources and plausible assumptions, this paper seeks to overcome some of these data gaps through the compilation of a meaningful set of economic and sustainability indicators for specific bio-based chemical activities and products. Due to the variety of data sources employed for each indicator, a data quality index is constructed, whilst rigorous comparisons with other studies and further critical discussion reaffirms the general observation of poor data quality. Subject to these data and methodological limitations, this paper analyses the performance of bio-based chemical industries. As long as official data sources lack adequate information systems, the current paper serves as a springboard for lowering the data ‘entry costs' behind this intricate sector, encouraging further knowledge-sharing and serving as a replication template for other regions.

Production of 5-Hydroxymethylfurfural Derived Cassava Peels Using Deep Eutectic Solvent Based Choline Chloride

The conversion of lignocellulosic biomass to biofuel as potential sources of transportation fuels, and for being both non-toxic and biodegradable. 5- Hydroxymethylfurfural (5-HMF) has been discovered to be a precursor for biofuel production and can be produced from biomass, which is readily available, renewable, and sustainable. Cellulose content in cassava peels is an opportunity to produce bio-based chemical products called 5-hydroxymethylfurfural. This study aims to determine the proper condition of glucose dehydration reaction of cassava peels hydrolysis. The optimum condition of dehydration reaction in this study was a glucose mass ratio: deep eutectic solvent of 1:6 and a reaction temperature of 80 ᵒC and the highest yield of 5-hydroxymethylfurfural using deep eutectic solvents (DES) based on choline chloride/oxalic acid was 70.22% and using DES based on choline chloride/oxalic acid was 64.50% at 5.70% glucose initial concentration using 1.5% H2SO4 catalyst on hydrolysis reaction cellulose of cassava peels. Physicochemical properties of deep eutectic solvents (DES) were pH of 5.8, density of 1.1574 gr/cm3 and viscosity of 119.33 cP. The results in this study indicate that the addition of DES choline chloride/oxalic acid can increase the yield of 5-hydroxymethylfurfural obtained.

Reprogramming the metabolism of Klebsiella pneumoniae for efficient 1,3-propanediol production

The production of 1,3-propanediol (1,3-PD) from glycerol by Klebsiella pneumoniae is limited by synthesis of numerous byproducts. Among them, the accumulation of acetate has the largest negative impact on the fermentation performance. To address the acetate overflow caused by knocking out lactate dehydrogenase, alcohol dehydrogenase and succinate dehydrogenase, several metabolic engineering manipulations were conducted. First, acetate was reduced through enhancing the acetate assimilation pathway by overexpressing heterologous acetyl-CoA synthetase. Then, the polyhydroxybutyrate (PHB) synthesis pathway was introduced to further reprogram the intracellular carbon metabolism. As a result, the best performed strain Kpr-6 produced up to 91.2 g/L extracellular 1,3-PD and 2.56 g/L intracellular PHB which can be easily separated from each other, while the acetate was dramatically reduced. The metabolic engineering strategies developed in this study would be helpful for constructing the microbial cell factory for other similar bio-based chemical production.

Deep Eutectic Solvents as Catalysts for Upgrading Biomass

Deep eutectic solvents (DESs) have emerged as promising green solvents, due to their versatility and properties such as high biodegradability, inexpensiveness, ease of preparation and negligible vapor pressure. Thus, DESs have been used as sustainable media and green catalysts in many chemical processes. On the other hand, lignocellulosic biomass as an abundant source of renewable carbon has received ample interest for the production of biobased chemicals. In this review, the state of the art of the catalytic use of DESs in upgrading the biomass-related substances towards biofuels and value-added chemicals is presented, and the gap in the knowledge is indicated to direct the future research.

Bio-based production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) with modulated monomeric fraction in Escherichia coli.

In this study, we applied metabolic engineering and bioprocessing strategies to enhance heterologous production of an important biodegradable copolymer, i.e., poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), with a modulated 3-hydroxyvalerate (3-HV) monomeric fraction from structurally unrelated carbon of glycerol in engineered Escherichia coli under different oxygenic conditions. We used our previously derived propanologenic (i.e., 1-propanol-producing) E. coli strain with an activated genomic Sleeping beauty mutase (Sbm) operon as a host for heterologous expression of the phaCAB operon. The 3-HV monomeric fraction was modulated by regulating dissimilated carbon flux channeling from the tricarboxylic acid (TCA) cycle into the Sbm pathway for biosynthesis of propionyl-CoA, which is a key precursor to (R)-3-hydroxyvaleryl-CoA (3-HV-CoA) monomer. The carbon flux channeling was regulated either by manipulating a selection of genes involved in the TCA cycle or varying oxygenic condition of the bacterial culture. With these consolidated strategies being implemented, we successfully achieved high-level PHBV biosynthesis with a wide range of 3-HV monomeric fraction from ~ 4 to 50 mol%, potentially enabling the fine-tuning of PHBV mechanical properties at the biosynthesis stage. We envision that similar strategies can be applied to enhance bio-based production of chemicals derived from succinyl-CoA. KEY POINTS: • TCA cycle engineering was applied to enhance 3-HV monomeric fraction in E. coli. • Effects of oxygenic conditions on 3-HV incorporation into PHBV in E. coli were investigated. • Bacterial cultivation for high-level PHBV production in engineered E. coli was performed.

Process design and scale-up study for the production of polyol-based biopolymers from sawdust

Sawdust is a by-product of the forestry industry which can be used as raw material for the production of added-value bio-based chemicals, leading to the development of a circular economy based on this organic resource. One of the most promising approaches for the reuse of sawdust is the production of polyols, which in turn can be used as raw material for the production of biopolymers. In this work, this process is analyzed to develop a sustainable alternative for the processing of forestry waste, obtaining biodegradable polymers than can be re-incorporated into the production cycle. A conceptual design and scale up of the process for the production of polyols out of sawdust and glycerol is developed based on experimental data. The process has been designed following the premise of being as environmentally sustainable as possible, but using simple equipment feasible to be deployed and operated by the timber facilities. Therefore, sawdust is used as raw material and energy source. The process is modeled using an equation based approach to perform a techno-economic analysis of it. The proportions of sawdust devoted to polyol production and thermal energy generation and power consumption are determined, as well as capital and operating expenses. In addition, a scale-up study to determine the effect of the economies of scale on the economic performance of the process proposed is carried out, as well as a sensitivity analysis in order to assess the influence of the raw materials price on the production cost. The results show that, for usual market prices of glycerol and starch, the production cost ranges from 0.10 to 0.66 USD per kilogram of biopolymer produced.

Microbial production of multiple short-chain primary amines via retrobiosynthesis

Bio-based production of many chemicals is not yet possible due to the unknown biosynthetic pathways. Here, we report a strategy combining retrobiosynthesis and precursor selection step to design biosynthetic pathways for multiple short-chain primary amines (SCPAs) that have a wide range of applications in chemical industries. Using direct precursors of 15 target SCPAs determined by the above strategy, Streptomyces viridifaciens vlmD encoding valine decarboxylase is examined as a proof-of-concept promiscuous enzyme both in vitro and in vivo for generating SCPAs from their precursors. Escherichia coli expressing the heterologous vlmD produces 10 SCPAs by feeding their direct precursors. Furthermore, metabolically engineered E. coli strains are developed to produce representative SCPAs from glucose, including the one producing 10.67 g L−1 of iso-butylamine by fed-batch culture. This study presents the strategy of systematically designing biosynthetic pathways for the production of a group of related chemicals as demonstrated by multiple SCPAs as examples.

Efficient reduction of 5-hydroxymethylfurfural to 2, 5-bis (hydroxymethyl) furan by a fungal whole-cell biocatalyst

Biocatalytic reduction of biomass-derived 5-hydroxymethylfurfural (HMF) to 2,5-bis(hydroxymethyl)furan (BHMF) is considered as a sustainable method and has attracted great attention. In this work, a yeast-like fungus, Aureobasidium subglaciale F134 was discovered as a whole-cell biocatalyst for BHMF synthesis from selective reduction of HMF. Its resting cells exhibited high tolerance and excellent selectivity to HMF in a broad pH range. The catalytic capacity of the cells can be substantially improved by Zn2+ ion. Under the optimized reaction conditions, 180 mM HMF substrate was converted into BHMF with a yield of 82 % and 97 % selectivity in 9 h. In a fed-batch conversion, 430 mM of BHMF product was obtained within 15 h, giving a productivity of 3.7 g/l per hour. In addition, it was found that the cells could also selectively convert furfural to form furfuryl alcohol. Discovery of A. subglaciale F134 cells for BHMF synthesis broadens the biocatalyitc toolbox for sustainable production of bio-based chemicals and biofuels from biomass-derived HMF. In addition, the results provide more information regarding the identification of related enzymes in future work.

Is Steam Explosion a Promising Pretreatment for Acid Hydrolysis of Lignocellulosic Biomass?

For the production of sugars and biobased platform chemicals from lignocellulosic biomass, the hydrolysis of cellulose and hemicelluloses to water-soluble sugars is a crucial step. As the complex structure of lignocellulosic biomass hinders an efficient hydrolysis via acid hydrolysis, a suitable pretreatment strategy is of special importance. The pretreatment steam explosion was intended to increase the accessibility of the cellulose fibers so that the subsequent acid hydrolysis of the cellulose to glucose would take place in a shorter time. Steam explosion pretreatment was performed with beech wood chips at varying severities with different reaction times (25–34 min) and maximum temperatures (186–223 °C). However, the subsequent acid hydrolysis step of steam-exploded residue was performed at constant settings at 180 °C with diluted sulfuric acid. The concentration profiles of the main water-soluble hydrolysis products were recorded. We showed in this study that the defibration of the macrofibrils in the lignocellulose structure during steam explosion does not lead to an increased rate of cellulose hydrolysis. So, steam explosion is not a suitable pretreatment for acid hydrolysis of hardwood lignocellulosic biomass.

Glutaric acid production by systems metabolic engineering of an l-lysine–overproducing Corynebacterium glutamicum

There is increasing industrial demand for five-carbon platform chemicals, particularly glutaric acid, a widely used building block chemical for the synthesis of polyesters and polyamides. Here we report the development of an efficient glutaric acid microbial producer by systems metabolic engineering of an l-lysine-overproducing Corynebacterium glutamicum BE strain. Based on our previous study, an optimal synthetic metabolic pathway comprising Pseudomonas putida l-lysine monooxygenase (davB) and 5-aminovaleramide amidohydrolase (davA) genes and C. glutamicum 4-aminobutyrate aminotransferase (gabT) and succinate-semialdehyde dehydrogenase (gabD) genes, was introduced into the C. glutamicum BE strain. Through system-wide analyses including genome-scale metabolic simulation, comparative transcriptome analysis, and flux response analysis, 11 target genes to be manipulated were identified and expressed at desired levels to increase the supply of direct precursor l-lysine and reduce precursor loss. A glutaric acid exporter encoded by ynfM was discovered and overexpressed to further enhance glutaric acid production. Fermentation conditions, including oxygen transfer rate, batch-phase glucose level, and nutrient feeding strategy, were optimized for the efficient production of glutaric acid. Fed-batch culture of the final engineered strain produced 105.3 g/L of glutaric acid in 69 h without any byproduct. The strategies of metabolic engineering and fermentation optimization described here will be useful for developing engineered microorganisms for the high-level bio-based production of other chemicals of interest to industry.

Succinic acid production from pulp and paper industry waste: A transcriptomic approach

The fermentative production of biobased chemicals and polymers using crude lignocellulose hydrolysates is challenging due to the presence of various inhibitory compounds and multiple sugars. This study evaluates the metabolic response of Actinobacillus succinogenes for the production of succinic acid using spent sulphite liquor (SSL) as feedstock derived from industrial acidic sulphite pulping of Eucalyptus globulus hardwood. A transcriptomic approach led to significant insights on gene regulation of the major metabolic pathways (glycolysis, pentose phosphate pathway, TCA cycle, pyruvate metabolism and oxidative phosphorylation) in batch cultures carried out on SSL and compared with glucose and xylose. Significantly overexpressed genes in SSL compared to glucose and xylose were fructose biphosphate aldolase (> 1.18-fold change) in the catabolism, phosphoenolpyruvate carboxykinase (> 1.59-fold change) and malate dehydrogenase (> 1.49-fold change) in the TCA cycle, citrate lyase (> 1.7-fold change), dihydrolipoamide dehydrogenase (> 0.88-fold change), pyruvate dehydrogenase E2 (> 1.63-fold change) and pyruvate formate lyase (> 0.61-fold change), involved in acetyl-CoA pathways. Finally, C4 tricarboxylic transporters were overexpressed (DCU (> 1.61-fold change) and 0079 (> 4.19-fold change). SSL was responsible for the upregulation of genes involved in the TCA cycle and oxidative phosphorylation, while xylose showed similar results with SSL in the oxidative phosphorylation.

Genome Sequence Analysis of Clostridium tyrobutyricum, a Promising Microbial Host for Human Health and Industrial Applications.

Clostridium tyrobutyricum is a promising microbial host for the anaerobic production of bio-based chemicals, especially butyric acid. At the same time, it also has great potential as a probiotic for the production of short-chain fatty acids in the intestines. However, due to the insufficient knowledge of the genetic characteristics of this organism, there has been little progress in its genetic engineering to date. Here, we present the complete genome sequence of C. tyrobutyricum L319, which consists of a circular chromosome and a plasmid with a G + C content of 31.69%, encompassing approximately 3.09Mb with 3052 protein-coding genes. Functional gene annotation revealed better results than previous studies based on KEGG pathway classification. Furthermore, we obtained detailed functional characterization of 93 genes previously annotated as putative proteins. Genomic analysis revealed that this organism contains multiple genes encoding enzymes involved in the CRISPR-Cas systems, substrate utilization, isopeptide and ester bonds, transcriptional regulation, and oxidative stress. The safety evaluation at genetic level indicated that this organism does not possess transferable resistance genes, invasive defensive pathogenicity factors, or harmful enzymes. The genome sequence data analyzed in this study will be available for further research and will facilitate the further understanding and development of potential applications of C. tyrobutyricum.

Selective production of ethyl levulinate from levulinic acid by lipase-immobilized mesoporous silica nanoflowers composite

Mesoporous silica nanoflowers bearing -NH2 groups were synthesized by the hydrolysis of tetraethyl orthosilicate (TEOS) with reverse microemulsion method, following with the grafting of -NH2 groups by the post modification with (3-Aminopropyl) trimethoxysilane (APTMS). The lipase from C. antarctica was immobilized on the as-synthesized amino-grafted mesoporous silica nanoflowers to fabricate the lipase-immobilized mesoporous silica nanoflowers composite, which was applied for the catalytic transformation of biomass-derived levulinic acid to biofuel ethyl levulinate (EL), and exhibited excellent catalytic activity. An ethyl levulinate yield as high as 99.5% could be achieved at 40 °C in 8 h reaction time, which was much higher than that catalyzed by the free lipase (67.9%) under the identical conditions. The immobilized lipase showed good stability and recyclability that ethyl levulinate yields above 68% could be remained after seven recycle times. This work represents a novel strategy to construct the immobilized biocatalyst for the production of bio-based chemicals.