Alcaligenes Eutrophus and Its Role in Biodegradable Polymer Production

Introduction

By [Angel Mogie]

General

Alcaligenes eutrophus (sometimes referred as Ralstonia metallidurans) is a gram negative bacillus with an optimum-growth temperature of 30oC [1]. It is a non-spore forming, obligate aerobic, and facultatively chemolithoautotrohpic bacterium that can thrive in environments containing mM concentrations of some toxic heavy metals such as Zinc, Cadmium, Cobalt, Lead, Mercury, Nickle, and Chromium [2]. In the presence of nitrate, A. eutrhopus can grow anaerobically. It naturally synthesizes polyhydroxybutyrate (PHB), a specific type of polyhydroxyalkanoates (PHA) that is used in the manufacture of biodegradable plastic.

The Enzymes: phbC-phbA-phbB

There are three regulatory enzymes involved in the bioproduction of PHB in Alcaligenes eutrophus: 3-ketothiolase, acetoacetyl-CoA reductase, and PHA syntase, encoded by by genes phbA, phbB, and phbC respectively. The molecular genetics and mapping of these enzymes were done in E. coli using the A. eutrophus strains of H16, 11599, 1159981, PHB 2, PHB3, PHB19. In 1989, Sinskey and Peoples identified the location of phbA and phbB genes on plasmid pAeT29 of E. coli and that their expressions were under the control of Alcaligenes eutrophus promoter [1]. Later in the same year, the two researchers mapped the location phbC— phbC gene is located upstream from phbA-phbB. Additionally, they also observed a reduced activities in both thiolase and reeducate. Their results suggest that, in PHB2 and PHB3 strains, all three genes are expressed from the same promoter located upstream from phbC [1]

Moreover, Sinskey and Peoples also propose that Alcaligenes eutrophus has at least two P-ketothiolase and acetoace- tyl-CoA reductase enzymes. Thiolase and reductase enzymes, when interact with either enoyl-CoA hydratase or epimerase or both, can lead to the formation of D(-)-3-hydroxybutyryl-CoA. In E.coli the expression of phbC alone results in diminished amount of PHB synthesis and insignificant rate of PHB polymerase activity. The absence of both phbA and phB genes of A. eutrophus leads to no synthesis of D(-)-3-hydroxybutyryl-CoA. Only when the three A. eutrophus genes, phbC-phbA-phbB, are present in E. coli > 50% of PHB production was observed. They also figured that PHB production is inhibited by the presence of nitrogen. Moreover, they propose that the interaction of thiolase and/ or reducatse with the polymerase is necessary for the polymerase to function.

Heavy Metals Resistance

Among the many hydrogen-oxidizing bacteria, the Alcaligenes are characterized by their ability in forming two hydrogenases; an NAD-reducing hydrogenase found in the cytoplasm and a membrane-bound hydrogenase [1]. The hydrogen-oxidizing trait of A. eutrophus is encoded by a 450 kb plasmid, which was identified through a plasmid transfer to Hox- cells lacking the plasmid. Two gene encoding the heavy-metal resistance are located within two large megaplasmids (pMOL28=180 kbp and pMOL30=240 kbp). These two megaplasmid also carry genes that express cation-efflux pumps for both bacterial inner and outer membranes.

PHB

Discovery

Maurice Lemoigne (mid 1920s) was the first person who discovered monopolyester of identified poly(3-hydroxybutyrate) or PHB. It took a while until the discovery was known to public because it was originally written in French. Later on, PHB was chemically identified by Staudinger (1920s). However, PHB did not get the attention from public and scientists as the prototypical biodegradable thermoplastic until thirty years later. Imperial Chemical Industries Ltd. (1980s) led was the first one to do intensive studies on PHB in multiple research areas including genetic engineering, biotechnology, and the enzymes responsible for biosynthesis and biodegradation. PHB is a reverse polymer found in bacteria and it can produce both the monomer and polymer of [R]-3-hydroxybutyric acid

Synthesis in Alcaligenes eutrophus

In A. eutrophus, the synthesis of PHB involves glucose as a carbon source and three enzymes; 3-ketothiolase, acetoacetyl-CoA reductase, and PHA syntase [1]. The first enzyme, 3-ketothiolase, is responsible in carrying the condensation reaction of acetyl-CoA into acetoacetyl-CoA. Then, acetoacetyl-CoA reductase reduces acetoacetly-CoA into R(-)-3-hydroxybutynl-CoA, with NADPH serves as the reducing agent. R(-)-3-hydroxybutynl-CoA is then polymerized into PHB with the aid of PHA synthase. A (Figure X). eutrophus can produce PHB as much as 80% of their dry body weight when grown in sugar-rich but nitrogen/ phosphate-deprived media. Alcaligenes eutrophus synthesizes different kind of polymers base on the carbon source they are exposed to in the media. For example, having propionic acid, instead of glucose, as the main carbon source leads to the production of a polymer containing 3-hydroxybutyrate a (3HB) and 3-hydroxyvalerate (3HV) instead of PHB (Figure X). On the other hand, completely substituting glucose with valeric acid leads to higher production of polymer in A. eutrophus— 90% of the dry body weight.

Physical Characteristics: Benefits and disadvantages

PHB is a rigid homopolymer and is a fragile thermoplastic. It has 100% stereospecificity with the asymmetric D(-) molecule and therefore is highly crystalline[2]. Unlike many other biodegradable polymers, PHB is resistant to moisture, impermeable to oxygen, and insoluble in water [1], which makes it particularly beneficial for the food packaging industry. However, PHB’s melting point (175o C) is so close to its degrading temperature (185o C) which is why it is so fragile and hence hard to mold [2]. The fragility of PHB can be lowered by integrating co-monomer 3-hydroxyvalerate or mixing PHB with more elastic polymer. The PHB copolymer containing 3-hydroxyvalerate unit P(3HB-co-3HV) is one of the developed forms of PHB with better mechanical functions: the more the 3-hydroxyvalerate unit makes PHB tougher, more elastic (i.e. lower young’s modulus), its elongation to break increases, lowers its melting point, but does not change its degrading temperature. Hence, the physical properties of PHB can be controlled by the amount of integrated 3-hydroxyvalerate during fermentation.

How is PHB Biodegradable?

Alcaligenes eutrophus forms PHB as a way of fixing carbon and as an intercellular storage [1]. When the bacterium runs out of extracellular carbon, PHB is broken down effectively and quickly into organic compounds to be reused. In fact, A. eutrophus is not the only bacteria that degrade PHB. Lemoigne (1923-1927) discoverd that B. megaterium releases [R]-3-hydroxybutyric in an aqueous environment. Wilkinson et al. (1958) also study B. megaterium and figure out that this bacterium degrades PHB and releases both acetoacetic acid and acetic acid as byproducts. In 1962, Merrick et al., work on B. rubrum and find that the bacterium self-catabolize its “native” granules through hydrolysis with an aid of the enzyme depolymerase (sometimes referred as hydrolase). Followed by Williamson et al., in 1967, who discovered a specific dehydrogenase that convertes [R]-3-hydroxybutyric acid to acetoacetic acid. Another enzyme that carries the synthesis of acetic acid from acetoacetic acid was classified by Dawes and his team in 1973. Base on those discoveries above, the biodegradation of PHB into simple organic compounds can be done through any of these possible ways:

• PHB can be broken down through hydrolisis with the aid of depolymerase—an enzyme secreted by many bacteria and fungi. In 1965, Delafield, Doudoroff and co-workers identified some pseudomonas that treats PHB as their energy and carbon source.

• Many microorganisms have the ability to both catabolize PHB and metabolize [R]-3-hydroxybutyric acid.

• Depolymerases was found associated with long alkyl chained PHA classes.

• Polyester depolymerases are found in many different organisms and their characteristics and structure have been well studied.

Also, as Lee (1996) argues, human blood plasma also has the ability to catabolize PHB and a fairly high concentration of the degradation byproduct has been detected. Hence, it is strongly conceivable that PHB is not poisonous to either plants, mammals, or the environments.

Section 3

Include some current research, with at least one figure showing data.

Conclusion

Overall text length at least 3,000 words, with at least 3 figures.

References

[Sample reference] Takai, K., Sugai, A., Itoh, T., and Horikoshi, K. "Palaeococcus ferrophilus gen. nov., sp. nov., a barophilic, hyperthermophilic archaeon from a deep-sea hydrothermal vent chimney". International Journal of Systematic and Evolutionary Microbiology. 2000. Volume 50. p. 489-500.

Edited by student of Joan Slonczewski for BIOL 238 Microbiology, 2011, Kenyon College.