Bio-based Succinate Research Articles

Effect of enzymatic versus titanium dioxide/silicon dioxide catalyst on crystal structure of ‘green’ poly[(butylene succinate)‐co‐(dilinoleic succinate)] copolymers

AbstractFocusing on an eco‐friendly approach, biodegradable poly[(butylene succinate)‐co‐(dilinoleic succinate)] (PBS‐DLS) copolymers with 70:30 (wt%) ratio of hard to soft segments were successfully synthesized via various processes and catalytic systems. In this approach, biobased succinate was polymerized with renewable 1,4‐butanediol and dimer linoleic diol to obtain ‘green’ copolyesters as sustainable alternatives to petroleum‐based materials. In the first procedure, a two‐step synthesis in diphenyl ether was performed using Candida antarctica lipase B (CAL‐B) as a biocatalyst. A second material was produced via two‐step melt polycondensation in the presence of heterogeneous titanium dioxide/silicone dioxide (C‐94) catalyst. The obtained PBS‐DLS copolyesters were further characterized in regard to their number‐average molecular weight (Mn), chemical structure, thermal transition temperatures and crystallization behavior. Here, digital holographic microscopy was used to study the crystallization behavior of synthesized segmented copolyesters for the first time. Using this technique, it was possible to reveal the twisting of crystalline regions in formed spherulites and observe the differences in crystallization behavior of copolyesters depending on the type of catalyst used in their synthesis. Structural characterization indicated random and blocky structure of copolymers depending on the type of catalyst. Mn was noticeably higher in the case of PBS‐DLS 70:30 copolymer catalyzed using C‐94 than PBS‐DLS 70:30 synthesized with the use of CAL‐B. However, the degree of crystallinity was lower for polymer catalyzed with the heterogeneous catalyst. Furthermore, differential scanning calorimetric thermal analysis revealed that synthesized copolyesters exhibit low glass transition temperature as well as high melting point which are typical for thermoplastic elastomers. © 2020 Society of Chemical Industry

A Genomic Perspective on the Potential of Wild-Type Rumen Bacterium Enterobacter sp. LU1 as an Industrial Platform for Bio-Based Succinate Production.

Enterobacter sp. LU1, a wild-type bacterium originating from goat rumen, proved to be a potential succinic acid producer in previous studies. Here, the first complete genome of this strain was obtained and analyzed from a biotechnological perspective. A hybrid sequencing approach combining short (Illumina MiSeq) and long (ONT MinION) reads allowed us to obtain a single continuous chromosome 4,636,526 bp in size, with an average 55.6% GC content that lacked plasmids. A total of 4425 genes, including 4283 protein-coding genes, 25 ribosomal RNA (rRNA)-, 84 transfer RNA (tRNA)-, and 5 non-coding RNA (ncRNA)-encoding genes and 49 pseudogenes, were predicted. It has been shown that genes involved in transport and metabolism of carbohydrates and amino acids and the transcription process constitute the major group of genes, according to the Clusters of Orthologous Groups of proteins (COGs) database. The genetic ability of the LU1 strain to metabolize a wide range of industrially relevant carbon sources has been confirmed. The genome exploration indicated that Enterobacter sp. LU1 possesses all genes that encode the enzymes involved in the glycerol metabolism pathway. It has also been shown that succinate can be produced as an end product of fermentation via the reductive branch of the tricarboxylic acid cycle (TCA) and the glyoxylate pathway. The transport system involved in succinate excretion into the growth medium and the genes involved in the response to osmotic and oxidative stress have also been recognized. Furthermore, three intact prophage regions ~70.3 kb, ~20.9 kb, and ~49.8 kb in length, 45 genomic islands (GIs), and two clustered regularly interspaced short palindromic repeats (CRISPR) were recognized in the genome. Sequencing and genome analysis of Enterobacter sp. LU1 confirms many earlier results based on physiological experiments and provides insight into their genetic background. All of these findings illustrate that the LU1 strain has great potential to be an efficient platform for bio-based succinate production.

Valorising fermentation effluent rich in short-chain fatty acids and sugars for biohydrogen via photofermentation by Rhodobacter sphaeroides KKU-PS1

Growing fermentative chemical production will increase effluents from industrial fermentations containing short-chain fatty acids and residual sugars, which are exploitable for biohydrogen through photofermentation. Previous studies have concentrated on single substrates and photofermentation study using fermentation effluent from bio-succinate production containing residual sugars and short-chain organic acids has yet to be reported to the best of authors’ knowledge. Rhodobacter sphaeroides KKU-PS1 grown on succinate was used for hydrogen production from medium containing mixture of substrates mimicking final effluent from bio-based succinate production. Prior to that, hydrogen producibility test with succinate-only medium was carried out. Photofermentation from succinate by this strain yielded 1217 ml H2/l of maximum cumulative hydrogen with maximum hydrogen rate of 6.7 ml H2/l/h, comparable to malate which was previously reported as best single substrate for the strain. Hydrogen production profiles using mixed substrates was well-fitted by modified Gompertz model with maximum cumulative hydrogen and maximum hydrogen production rate of 1005 ml H2/l and 4.1 ml H2/l/h, respectively. Only glucose, xylose and succinate followed modified Gompertz model for substrate consumption. Instantaneous succinate consumption compared to extended lag time of 100h for consumption of both sugars indicated higher affinity towards short-chain fatty acid utilization during initial growth phase. Xylose showed highest overall substrate consumption signifying its importance for hydrogen generation, which continued after stationary growth phase started reaching a total of 91.9% consumption. Significant remaining substrate levels other than xylose suggested that the process was not inhibited by limited substrates. The study highlighted potential of fermentation effluents containing mixed substrates for biohydrogen, with further optimization needed.

Prediction of reaction knockouts to maximize succinate production by Actinobacillus succinogenes.

Succinate is a precursor of multiple commodity chemicals and bio-based succinate production is an active area of industrial bioengineering research. One of the most important microbial strains for bio-based production of succinate is the capnophilic gram-negative bacterium Actinobacillus succinogenes, which naturally produces succinate by a mixed-acid fermentative pathway. To engineer A. succinogenes to improve succinate yields during mixed acid fermentation, it is important to have a detailed understanding of the metabolic flux distribution in A. succinogenes when grown in suitable media. To this end, we have developed a detailed stoichiometric model of the A. succinogenes central metabolism that includes the biosynthetic pathways for the main components of biomass—namely glycogen, amino acids, DNA, RNA, lipids and UDP-N-Acetyl-α-D-glucosamine. We have validated our model by comparing model predictions generated via flux balance analysis with experimental results on mixed acid fermentation. Moreover, we have used the model to predict single and double reaction knockouts to maximize succinate production while maintaining growth viability. According to our model, succinate production can be maximized by knocking out either of the reactions catalyzed by the PTA (phosphate acetyltransferase) and ACK (acetyl kinase) enzymes, whereas the double knockouts of PEPCK (phosphoenolpyruvate carboxykinase) and PTA or PEPCK and ACK enzymes are the most effective in increasing succinate production.

Re-engineering Escherichia coli KJ122 to enhance the utilization of xylose and xylose/glucose mixture for efficient succinate production in mineral salt medium.

Escherichia coli KJ122 was previously engineered to produce high concentration and yield of succinate in mineral salt medium containing glucose and sucrose under anaerobic conditions. However, this strain does not efficiently utilize xylose. To improve the xylose uptake and utilization in the strain KJ122, xylFGH and xylE genes were individually and simultaneously deleted. E. coli KJ12201 (KJ122::ΔxylFGH) exhibited superior abilities in growth, xylose consumption, and succinate production compared to those of the parental strain KJ122. However, E. coli KJ12202 (KJ122::ΔxylE) lessened xylose consumption due to an ATP deficit for metabolizing xylose thus making succinate production from xylose not preferable. Moreover, E. coli KJ12203 (KJ122::ΔxylFGHΔxylE) exhibited an impaired growth on xylose due to lacking of xylose transporters. After performing metabolic evolution, the evolved KJ12201-14T strain exhibited a great improvement in succinate production from pure xylose with higher concentration and productivity about 18 and 21%, respectively, compared to KJ12201 strain. During fed-batch fermentation, KJ12201-14T also produced succinate from xylose at a concentration, yield, and overall productivity of 84.6±0.7g/L, 0.86±0.01g/g and 1.01±0.01g/L/h, respectively. KJ12201 and KJ12201-14T strains co-utilized glucose/xylose mixture without catabolite repression. Both strains produced succinate from glucose/xylose mixture at concentration, yield, and overall and specific productivities of about 85g/L, 0.85g/g, 0.70g/L/h, and 0.44g/gCDW/h, respectively. Based on our results, KJ12201 and KJ12201-14T strains exhibited a greater performance in succinate production from xylose containing medium than those of other published works. They would be potential strains for the economic bio-based succinate production from xylose.

Bio-based succinate from sucrose: High-resolution 13C metabolic flux analysis and metabolic engineering of the rumen bacterium Basfia succiniciproducens

Succinic acid is a platform chemical of recognized industrial value and accordingly faces a continuous challenge to enable manufacturing from most attractive raw materials. It is mainly produced from glucose, using microbial fermentation. Here, we explore and optimize succinate production from sucrose, a globally applied substrate in biotechnology, using the rumen bacterium Basfia succiniciproducens DD1. As basis of the strain optimization, the yet unknown sucrose metabolism of the microbe was studied, using 13C metabolic flux analyses. When grown in batch culture on sucrose, the bacterium exhibited a high succinate yield of 1molmol−1 and a by-product spectrum, which did not match the expected PTS-mediated sucrose catabolism. This led to the discovery of a fructokinase, involved in sucrose catabolism. The flux approach unraveled that the fructokinase and the fructose PTS both contribute to phosphorylation of the fructose part of sucrose. The contribution of the fructokinase reduces the undesired loss of the succinate precursor PEP into pyruvate and into pyruvate-derived by-products and enables increased succinate production, exclusively via the reductive TCA cycle branch. These findings were used to design superior producers. Mutants, which (i) overexpress the beneficial fructokinase, (II) lack the competing fructose PTS, and (iii) combine both traits, produce significantly more succinate. In a fed-batch process, B. succiniciproducens ΔfruA achieved a titer of 71gL−1 succinate and a yield of 2.5molmol−1 from sucrose.

Performance and mechanism analysis of succinate production under different transporters in Escherichia coli

Succinic acid is a platform chemical with potential for bio-based synthesis. However, the production of bio-based succinate is limited because of insufficient succinate efflux capacity in the late stage of fermentation. In the present study, three different transporters, which have been reported to be responsible for C4-dicarboxylates transport, were employed for investigation of the transport capacity of succinate in Escherichia coli. After engineered strains were constructed, the fermentative production of succinic acid was studied in serum bottles and 3 L of fermentor. The results demonstrated that engineered strain showed better efflux capacity than control strain under high concentration of succinate. The highest production of succinate was 68.66 g/L, while the NCgl2130 transporter may be the best candidate for succinate export in E. coli. Further research showed that the expression levels and relative enzyme activities involved in the metabolic pathway all increased markedly, and the maximum activities of PPC, PCK, PYK, and MDH increased by 1.50, 1.38, 1.28, and 1.27-fold in recombinant E. coli AFP111/pTrc99a-NCgl2130, respectively. Moreover, the maximum level of intracellular ATP increased by 23.79% in E. coli AFP111/pTrc99a-NCgl2130. Taken together, these findings indicated that engineered transporters can improve succinate production by increasing key enzyme activities and intracellular ATP levels. To the best of thew authors’ knowledge, this is the first report on a mechanism to improve succinate production by engineered transporters. This strategy set up a foundation for improving the biosynthesis of other C4-dicarboxylates, such as fumaric acid and malic acid.

Inducing hyperosmotic stress resistance in succinate-producing Escherichia coli by using the response regulator DR1558 from Deinococcus radiodurans

Abstract Bio-based succinate synthesis process suffers from low production efficiency, especially in the late stage of fermentation because of high osmotic stress. In this study, DR1558 (a response regulator in tow-component signal transduction systems) from Deinococcus radiodurans was overexpressed in Escherichia coli Suc260. High concentration glucose and NaCl impact experiment results showed that the recombinant strain (E. coli Suc270) displayed higher tolerance to osmotic stress. Fermentation in a 3-L fermenter showed that the dry cell weight, glucose consumption, and succinic acid titer of Suc270 were 2.06, 90.2, and 55.4 g/L, which is an increase of 1.50-, 1.38-, and 1.26-fold, respectively, compared to those of Suc260. Moreover, the lag phase was shortened by 10 h compared to that of the control strain Suc260. Real-time PCR results showed that DR1558 could enhance the expression level of the genes involved in glucose utilization, ATP synthesis, and osmoprotectant (glycine betaine, trehalose) synthesis pathways. Intracellular ATP and osmoprotectant content analysis further showed that Suc270 accumulated much more intracellular ATP, glycine betaine, and trehalose than Suc260. In conclusion, DR1558 could affect ATP metabolism and the synthesis of osmoprotectants and thus enhance the cell’s tolerance against osmotic stress.

Biotechnological route for sustainable succinate production utilizing oil palm frond and kenaf as potential carbon sources.

Due to the world's dwindling energy supplies, greater thrust has been placed on the utilization of renewable resources for global succinate production. Exploration of such biotechnological route could be seen as an act of counterbalance to the continued fossil fuel dominance. Malaysia being a tropical country stands out among many other nations for its plenty of resources in the form of lignocellulosic biomass. To date, oil palm frond (OPF) contributes to the largest fraction of agricultural residues in Malaysia, while kenaf, a newly introduced fiber crop with relatively high growth rate, holds great potential for developing sustainable succinate production, apart from OPF. Utilization of non-food, inexhaustible, and low-cost derived biomass in the form of OPF and kenaf for bio-based succinate production remains largely untapped. Owing to the richness of carbohydrates in OPF and kenaf, bio-succinate commercialization using these sources appears as an attractive proposition for future sustainable developments. The aim of this paper was to review some research efforts in developing a biorefinery system based on OPF and kenaf as processing inputs. It presents the importance of the current progress in bio-succinate commercialization, in addition to describing the potential use of different succinate production hosts and various pretreatments-saccharifications under development for OPF and kenaf. Evaluations on the feasibility of OPF and kenaf as fermentation substrates are also discussed.

Development of structure–property relationships that allow independent control of glass transition temperature, melting temperature, and rheology in a library of bio-based succinate polyester polyols

A library of 20 bio-based succinate (SA) diol, co-diol, and co-diacid polyester polyols (PESPs) was generated and characterized, giving detailed understanding for tuning of their thermal transitions and rheological parameters. SA and 1,3-propanediol, 1,4-butanediol (BDO), 1,6-hexanediol, 2,2-dimethyl-1,3-propanediol, 3-methyl-1,5-pentanediol, diethylene glycol (DEG), adipic acid, or sebacic acid, and the blended diols were oligomerized to 1000 and 2000 Da Mn. The SA PESP melting point and its ability to crystallize can be set by controlling the structures and ratios of co-diols or co-diacids. We also show that the PESPs follow the expected Arrhenius temperature–viscosity relationship, but with a clear break in activation energy between PESPs with and without pendent methyl substituents and with DEG. PESP glass transition temperature, melting points, and rheology can be controlled independently by use of co-monomers with and without pendent methyl groups and with DEG.

Coupled CO2 fixation from ethylene oxide off-gas with bio-based succinic acid production by engineered recombinant Escherichia coli

Industrial exhaust gases contributed heavily to anthropogenic CO2 emissions. In this study, a novel approach of capturing CO2 from ethylene oxide off-gas and converting it into a platform C4 chemical was established by using succinate-producing E. coli. Fermentation results showed that the acetaldehyde in the off-gas was the main factor inhibiting cell metabolic capability, thus IrrE which is a global regulator from Deinococcus radiodurans was used to improve cell resistance to acetaldehyde. A final succinic acid titer of 68.12gL−1 with a CO2 fixation rate of 4.7mmolL−1h−1 was achieved. Notably, this coupled process not only realized the CO2 fixation from ethylene oxide off-gas but also provided a potential cost-effective gas source which holds potential application for bio-based succinate production.

High production yield and specific productivity of succinate from cassava starch by metabolically-engineeredEscherichia coliKJ122

BACKGROUND Succinate has been widely used in chemical industries and its microbial production is a desirable option. The feasibility of bio-based succinate production on an industrial scale strongly depends on the utilization of cheaper renewable resources. The use of cassava starch may make the fermentation process of succinate more economically competitive. RESULTS Simultaneous saccharification and fermentation (SSF) of succinate production was performed in minimal salt medium containing a cheap cassava starch. The parameters affecting succinate production from cassava starch by E. coli KJ122 were optimized. A succinate concentration of 70.1 ± 0.1 g Ll−1 was produced with a yield of 1.00 ± 0.01 g g−1 substrate and productivity of 0.97 ± 0.01 g L−1h−1 during batch SSF in a 2 L fermentor under optimized conditions. Further improvements in succinate concentration (82.5 ± 0.7 g L−1), yield (1.03 ± 0.01 g g−1 glucose), and average and specific productivities of 1.15 ± 0.01 g L−1h−1, and 456 mg g CDW−1 h−1, respectively, were observed in fed-batch SSF. CONCLUSION Escherichia coli KJ122 produced a succinate yield of 92% of theoretical maximum and the highest specific productivity ever reported. Succinate production yield and specific productivity from cassava starch in this study are superior to other previously published works. Escherichia coli KJ122 may be used as a biocatalyst for cost-effective succinate production. © 2016 Society of Chemical Industry

Physical Properties of Bio-Based Polyurethane Foams from Bio-Based Succinate Polyols

The goal of this work is to study the physical properties of bio-based polyurethane (PU) foams prepared from synthesized bio-based succinate polyols. Bio-based succinate polyols were synthesized by condensation reaction using succinic acid (SA) and four types of glycols that are diethylene glycol (DEG), triethylene glycol (TEG), tetraethylene glycol (TTEG) and glycerol (Gly). The effects of glycol types (single or mixed glycols) used in the prepared polyols toward physical properties of PU foams were studied. Physical and mechanical properties of the prepared PU foams were taken relatively to PU foam obtained from a commercial polyol. The results revealed that hydroxyl number (OH number) and viscosity of prepared polyols decreased as the length of the glycol chains increased (DEG < TEG < TTEG). However, the polyols prepared from mixed glycols showed higher viscosity than those prepared from single glycol. Optical micrographs images showed that the PU foams prepared from mixed-glycol polyols had smaller cell dimension and more closed cell contents compared to ones prepared from single-glycol polyols. Foam formation time and density of foams prepared from DEG-glycerol mixed glycol polyol were similar to one prepared from commercial polyol (POLIMAXX A8360). Moreover, compressive strength of foam was higher than one prepared from commercial polyol.

Metabolic engineering of Escherichia coli for the production of succinic acid from glucose

Bio-based succinate production from renewable resources has prospective economic and environmental benefits that caused heightened interest towards the study of succinate-producing microorganisms. The pathways of succinate formation have been well studied, and microorganisms that are capable of biomass convertion into the target substance (bacteria of the genera Actinobacillus, Anaerobiospirillum, and Mannheimia) have been isolated and characterized; however, the realization of economically feasible industrial processes using native producers still remains a challenge. Traditionally, the Escherichia coli bacterium has been used as a workhouse to develop new processes for the biosynthesis of many valuable chemicals due to the extensive knowledge of its metabolism, available genetic tools, and good growth characteristics, combined with low nutrient requirements. This review is focused on modern rational approaches to the construction of recombinant E. coli strains that efficiently produce succinic acid from glucose.

Green upgrading of succinate using dimethyl carbonate for a better integration with fermentative production

In this work we present a new process alternative comprising bio-based succinate production, its recovery by ion exchange and further upgrading to dimethyl succinate via O-alkylation by direct downstream catalysis. The use of dimethyl carbonate as alkylating agent permits the in-process generation of a mobile bicarbonate anion, playing the role of counter-ion during succinate capture and then transported upstream where is used as inorganic carbon source and neutralizing agent required for most bacterial succinate fermentations. Succinate recovery using a strong anion exchange resin in the bicarbonate form presented good equilibrium characteristics and column performance. Subsequently, dimethyl succinate was produced in high yield (96%) using dimethyl carbonate as a solvent and reagent and the kinetics of the system were described by a pseudo-first order model. Throughout the reaction and in the presence of water, it was verified that the resin is effectively regenerated to its bicarbonate form maintaining its ion exchange capacity. As a main advantage, the validated concept leads to an improved bio-based process for succinate to ester derivatives where no stoichiometric waste is produced, contrary to conventional acidification–esterification operations.

Fermentative Succinate Production: An Emerging Technology to Replace the Traditional Petrochemical Processes

Succinate is a valuable platform chemical for multiple applications. Confronted with the exhaustion of fossil energy resources, fermentative succinate production from renewable biomass to replace the traditional petrochemical process is receiving an increasing amount of attention. During the past few years, the succinate-producing process using microbial fermentation has been made commercially available by the joint efforts of researchers in different fields. In this review, recent attempts and experiences devoted to reduce the production cost of biobased succinate are summarized, including strain improvement, fermentation engineering, and downstream processing. The key limitations and challenges faced in current microbial production systems are also proposed.

Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli

Abstract Sucrose-utilizing genes (cscKB and cscA) from Escherichia coli KO11 were cloned and expressed in a metabolically engineered E. coli KJ122 to enhance succinate production from sucrose. KJ122 harboring a recombinant plasmid, pKJSUC, was screened for the efficient sucrose utilization by growth-based selection and adaptation. KJ122-pKJSUC-24T efficiently utilized sucrose in a low-cost medium to produce high succinate concentration with less accumulation of by-products. Succinate concentrations of 51 g/L (productivity equal to 1.05 g/L/h) were produced from sucrose in anaerobic bottles, and concentrations of 47 g/L were produced in 10 L bioreactor within 48 h. Antibiotics had no effect on the succinate production by KJ122-pKJSUC-24T. In addition, succinate concentrations of 62 g/L were produced from sugarcane molasses in anaerobic bottles, and concentrations of 56 g/L in 10 L bioreactor within 72 h. These results demonstrated that KJ122-pKJSUC-24T would be a potential strain for bio-based succinate production from sucrose and sugarcane molasses.

Production of succinic acid from sucrose and sugarcane molasses by metabolically engineered Escherichia coli

Sucrose-utilizing genes (cscKB and cscA) from Escherichia coli KO11 were cloned and expressed in a metabolically engineered E. coli KJ122 to enhance succinate production from sucrose. KJ122 harboring a recombinant plasmid, pKJSUC, was screened for the efficient sucrose utilization by growth-based selection and adaptation. KJ122-pKJSUC-24T efficiently utilized sucrose in a low-cost medium to produce high succinate concentration with less accumulation of by-products. Succinate concentrations of 51g/L (productivity equal to 1.05g/L/h) were produced from sucrose in anaerobic bottles, and concentrations of 47g/L were produced in 10L bioreactor within 48h. Antibiotics had no effect on the succinate production by KJ122-pKJSUC-24T. In addition, succinate concentrations of 62g/L were produced from sugarcane molasses in anaerobic bottles, and concentrations of 56g/L in 10L bioreactor within 72h. These results demonstrated that KJ122-pKJSUC-24T would be a potential strain for bio-based succinate production from sucrose and sugarcane molasses.

Is it possible to produce succinic acid at a low pH?

Bio-based succinate is still a matter of special emphasis in biotechnology and adjacent research areas. The vast majority of natural and engineered producers are bacterial strains that accumulate succinate under anaerobic conditions. Recently, we succeeded in obtaining an aerobic yeast strain capable of producing succinic acid at low pH. Herein, we discuss some difficulties and advantages of microbial pathways producing “succinic acid” rather than “succinate”. It was concluded that the peculiar properties of the constructed yeast strain could be clarified in view of a distorted energy balance. There is evidence that in an acidic environment, the majority of the cellular energy available as ATP will be spent for proton and anion efflux. The decreased ATP:ADP ratio could essentially reduce the growth rate or even completely inhibit growth. In the same way, the preference of this elaborated strain for certain carbon sources could be explained in terms of energy balance. Nevertheless, the opportunity to exclude alkali and mineral acid waste from microbial succinate production seems environmentally friendly and cost-effective.

Prospects for a bio-based succinate industry

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.