Strains for Production of PHA

Many bacteria have been found to produce PHA on different levels, for example, over 30% of soil bacteria were found to produce PHA. Most of these are SCL PHA, while most of the bacteria living in oil-contaminated locations, namely, oily aqueous environments or oily soils accumulate MCL PHA. Among these, few of them have been found to make copolymers of SCL and MCL monomers. 

Large cell sizes are important for easy separation and more PHA accumulation although those are not always easily found. Moreover, if the strain is able to produce more than one PFLA, that would be even better. Finally, PHA produced should have 

Obviously, all the production strains currently employed do not have all the good properties combined. 

Besides the hydrolytic cleavage of y-butyrolactone by NaOH,4-hydroxybutyric add (4-HBA) and different isomers can be produced biotechnologically. Apart from the fact that 4-HBA is a drug (intravenous narcotic, liquid ecstasy, knockout drops), P4HB could also be manufactured by polycondensation starting from 4-HB. 

Growth to high density PM A curttcni Rapid gio Mh Control la hie structures Cantmllable PI IA Mw Simple stibsimte 

---

Fig. 1. Construction of plasmid pl04613C2ReABstb/ which is used to transform/ad mutant E. coli strains for production of SCL-MCL PHA copolymers.

The uses of low specificity PHA synthases for production of PHA with functional groups for chemical modifications, and PHA with controllable compositions, including block PHA copolymers The use of special strain and mutated PHA synthases The making of PHA diols and the block copolymerization with other polymers... 

Though there are several reports on production of PHAs from cheap earbon substrates by wild type producers, the polymer concentration and the eontent obtained were relatively low than those obtained using purified carbon somces. Therefore there is a need for the development of more efficient fermentation strategies for production of PHA from cheap carbon sources using different microbial strains. 

Paracoccus denitrificans, a facultatively methylotrophic bacterium, is able to grow and accumulate PHAs on methanol. Recombinant P. denitrificans strains with increased expression levels of all PHA synthetic enzymes were investigated for the enhanced production of PHA. The PHA content and PHA accumulation rate of recombinant P. denitrificans with homologous overexpression of PHA synthase were 2 and 2.7 times higher, respectively, than those of the wild strain, suggesting that the step of PHA synthase was limited in PHA biosynthesis [111]. 

Recombinant Escherichia coli strains harboring the heterologous PHA biosynthesis genes have been shown to be suitable for the high-level production of PHAs. In addition, several engineered metabolic pathways for the synthesis of various RHAs have recently been established in recombinant E. coli (16-18). 

PH As discovered by Lemoigne [23] more than 70 years ago. The P(3HB) is produced and stored inside the bacterial cell walls in granules. Alcaligenes eutrophus, which was the first strain used for the semi-industrial production of PHAs, can accumulate large quantities of P(3HB) as discrete intercellular granules by careful control of the fermentation process, i.e., up to 80% of the weight of the dried cell can be in the form of P(3HB) granules [7]. 

Successful large-scale production of PHA is largely determined by the availability and constant supply of cheap fermentative substrates. At the same time, the overall cost involved in the production of this biodegradable polymeric material needs to be controlled and reduced in order to penetrate and compete in the world s commodity market. Palm oil has been identified as suitable carbon feedstock and potential strains capable of utilizing this raw material have been discussed in the above sections. However, waste disposals from palm oil mill and the amount of energy needed for PHA production are other issues that require equivalent attention when PHA is produced in large scale. 

The application of whey, a main by-product in the manufacturing of dairy products and cheese, has been widely explored in the production of PHA. Whey serves as an excellent carbon and energy source as lactose makes up 70% of the total dry matter. Whey retentate is mainly composed of a-lactalbumin and P-lactoglobulin proteins which can be employed as a nitrogen source for the enhanced cultivation of microbial PHA-producing strains [2, 34, 35]. However, the inability of some PHA-producing strains to utilise raw whey has... 

Fig. 5 Systems biotechnology for the development of an efficient PHA production system. Optimization of aU steps, from the upstream process (strain development) and the midstream process (fermentation) to the downstream process (recovery), is required for the economic production of PHAs having desired characteristics...

The production of PHA using residual oil from biotechnological rhamnose production as a carbon source for growth of C. necator H16 (the nomenclature in the article was "Ralstonia eutropha") andP. oleovorans-was described by Fiichtenbusch et al. (2000). The strains accumulated PHA at 41.3 and 38.9%, respectively, of the cell dry mass when they were cultivated in defined media with oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from C. necator was identified as PHB homopolyester, whereas the PHA isolated from P. oleovorans consisted, typically for this type of PHA-accumulating organism, of (P)-3-hydroxyhexanoicacid, (P)-3-hydroxyoctanoicacid, (/ )-3-hydroxydecanoic acid and (P)-3-hydroxydodecanoic acid. Approximately 20-25% of the carbon components of the residual oil were converted into PHA. Up to 80% of cell dry mass of PHB homopolyester from different plant oils was produced by C. necator DSM 545 (Fukui and Doi 1998). 

Maltose as the major product after hydrolysis of starch was utilized by Braunegg et al. (1999) for cultivation of three different strains of A. latus (DSM 1122, 1123 and 1124) in 10-1 bioreactors. Compared with the results on glucose, specific rates for growth and product formation were lower using starch hydrolysate, but the yields for production of biomass and PHA were comparable. 

Apply to over 90% of PHA containing cells Employ a feast and famine selection process to find a robust PHA production strain The use of robust PHA production strains, better imder a nonsterUization condition The manipulation of the N-terminus of PHA synthase The development of low-cost technology for production of low-cost PHA biofuels or fuel additives Plant molecular biology... 

Figure 3.6 Strain and process deveiopment for industrial production of PHAs. (Ref. [60], reprinted with permission of ACS Pubiications.)...

---