*4.3.2 Suppressing glycogen synthesis pathway*

The 3PG intermediate is utilized for both glycogen and PHB polymer production. The productivity of glycogen is high and quicker than that of PHB in nitrogen deprivation conditions (30% PHB and 60% glycogen (dcw) is produced) [92]. Assimilation of CO2 through ribulose-1,5-biphosphate carboxylation by the Rubisco produces 3PG which is directed to glycogen biosynthesis more than PHB accumulation. Grundel


#### **Table 2.**

*Genetic manipulations to increase PHB biosynthesis.*

et al. reported that there is no influence on growth under continuous light conditions while the biosynthesis pathway of glycogen was impaired in *Synechocystis sp.* PCC 6803 [93]. In the study conducted by Wu et. al, [94], an increase in PHB accumulation from 8–13% was observed in knockout mutants unable to produce glycogen and did not turn into dormant mode and was unable to recover from nitrogen scarcity. However, PHB-deficient mutants produced the same level of glycogen as the wild-type and recovered from scarcity once replenished with nutrients. A deficiency of growth was observed in the mutants with the knockout of genes involved in both polymer syntheses. Thus, it is important to improve the synthesis of PHB yield with robust PHB production and suppressed glycogen pathway.

#### *4.3.3 Exploitation of metabolic inhibitors to increase cyanobacterial PHA*

As the imbalance of C: N and NADPH: ATP ratios are contributing factors in stimulating PHB production many studies were carried out on the effect of the metabolic inhibitor on PHB production. Upon supplementing *N. muscorum* with carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD), the PHB pool was increased to 21% and 17% from 8.5%, respectively, were reported [55]. The addition of monofluoroacetate increased the PHB pool up to 19% (dcw), while Lmethionine-DL-sulfoximine (MSX) and azaserine addition also enhanced PHB production. Treatment with metabolic inhibitors such as DCCD, CCCP, and [3-(3,4-di chlorophenyl)-1,1- dimethylurea influenced the NADPH: NADP ratio along with PHB accumulation in *Synechocystis* PCC 6803 were reported [95]. This strategy of using metabolic inhibitors could help to enhance PHA accumulation in both wild-type and recombinant cyanobacteria.

#### *4.3.4 Mixed consortium*

The mixed consortium of cyanobacteria, bacteria, and algae is a feast-famine strategy where a sequencing batch reactor (SBE) without aeration is used for the cultivation [96, 97]. The concept of the consortium is developed to increase the system efficiency by enhancing productivity and accessibility of resources, community stability, efficient nutrient cycling, and partitioning, and distribution of carbon or energy source in a non-competitive manner. The oxygen produced by the algae cells during the famine phase is used to consume the NADPH reserves of the cells leading to around a 20% (dcw) increase in PHA accumulation [97]. A permanent feast regime under high light intensity conditions promoted PHA production to a maximum of 60% (dcw) in photosynthesis mixed culture. The famine phase can be eliminated using axenic dark feast conditions increasing productivity by up to 60% (dcw) by facilitating the acetate uptake [96, 97].

#### *4.3.5 Two-Stage cultivation*

The two-stage cultivation strategy is exploited for high biomass production and increased concentration of PHA thermoplastics. The cells are grown in optimal nutritional conditions in the first or growth stage to achieve high biomass concentration. The cells are recultivated in fresh media with the limitation of a specific nutrient (nitrogen and/or phosphorous) in the second or accumulation stage to induce stress and produce PHA. A study conducted on *Chlorogloea fritschii* TISTR 8527 in two-stage cultivation shows a maximum PHB accumulation of 25% (dcw) using acetate as

substrate with 51 7% (w/w) of conversion efficiency [83]. As the first stage produces maximum biomass this strategy appears to be potentially viable for large-scale production, but the shear forces experienced by the cells during recultivated give rise to a new lag phase. Two-stage cultivation of *Synechocystis cf. salina* PCC 6909 operated in a single stage without recultivation of biomass produced about 90 mg.L<sup>1</sup> of PHAs in 14 days. The cyanobacterium was grown in an optimized media such that the phosphorous and nitrogen were almost utilized by 7–8 days with a maximum biomass production of up to 2 g.L<sup>1</sup> (dcw) and thereby entered the accumulation stage due to nutrient starvation without harvesting and transfer of biomass [98]. The overall production cost of PHA production can be reduced using such type of two-stage cultivation strategy.

### **5. Problems**

Currently, the major bottleneck is the non-existence of an economical mass cultivation strategy for the commercial production of cyanobacterial PHAs. The two commercial-scale mass cultivation approaches as **(i)** closed photobioreactors and **(ii)** conventional open pond culture systems. The close photobioreactors are effective for monoculture cultivation as they are of more controlled types. An ideal photobioreactor should be flexible to all system requirements for different strains and specific growth environments for the production of the product of interest [99]. Open pond culture system is cheaper compared to photobioreactor which requires high construction, operation, and maintenance cost.

Biomass harvesting from the water on a commercial scale is still a major issue partly due to the low concentration (0.2–2 g.L<sup>1</sup> ), small size, and colloidal stability [100]. Filtration, flocculation, gravity settling, and centrifugation are some of the techniques exploited for harvesting cyanobacterial biomass. Flocculation is costeffective and energy efficient compared to centrifugation and it can also handle a huge volume of culture. Addition of inorganic salts such as AlCl3, Al2(SO4)3, FeCl3, and so on, cationic starch and chitosan are used for the flocculation of biomass [101, 102]. Several research efforts are being carried out for developing cost-effective and efficient cyanobacterial biomass harvesting technologies. For example, the settling velocity distribution of flocculated microalgal/cyanobacterial biomass is a critical parameter for developing cost-effective gravity settlers for biomass recovery.

The drying of biomass is essential for further downstream processing and storage. Around 20% of the overall cost of PHA production from *Spirulina* is contributed to the drying process. The high-energy input process of drying is only required for PHA extraction. Air drying is quite feasible, but it requires a large area and a longer time. Solar or wind energy utilization for the drying process could overcome these limitations [6].
