## 3 Results and Discussion
### PHA Production from Xylan and Agarose
The second sets of experiments were performed to investigate the effect of nitrogen limitation on cells and PHA production. Reports of different researchers suggest that bacteria produce more PHA under the stress of nutrient limitation [59; 60]. Glycogen, polyesters, and polyphosphates are accumulated as energy and carbon reserves [61]. These compounds are useful for cell survival in the case of famine. The first approach of one step culture in nutrient balanced medium for PHA production showed better results than the strategy of two step cell transfer. The PHA content in the cell transfer strategy from agarose was 18.4% and 15.3% of dry cell weight for _Sde_ 2-40 and co-culture, respectively (Figure 10A). PHA concentration was 0.315 and 0.231 g/L for _Sde_ 2-40 and co-culture, respectively (Figure 10B). Similarly, PHA content from xylan was 15.3% and 30.2% of dry cell weight (Figure 10A) and PHA concentration was 0.241 and 0.330 g/L for _Sde_ 2-40 and co-culture, respectively (Figure 10B). Marine bacterium _Sde_ 2-40 is well known for the production of PHA using glucose [13] and starch [14]. The production of PHA by _Sde_ 2-40 was reported to start at exponential growth phase and PHA accumulation of 0.58 g/L at 20 h and 0.53 g/L at 24 h were obtained using 20 g/L glucose and starch in marine broth, respectively [14]. The PHA production exponentially increased after carbon source feeding until fed-batch end and reached 21.3% of dry cell weight (2.71 g/L) using glucose and 17.4% of dry cell weight (2.04 g/L) using starch in marine broth during the fermentation period of 49 h [13]. [PERSON] and [PERSON] [12] reported PHA production by _Sde_ 2-40 up to 1.5 g/L from 4% glucose in marine sea salt minimal media. However, the current study demonstrates the ability of _Sde_ 2-40 to synthesize PHA from agarose and xylan for the first time. The PHA contents from agarose  
Figure 10: Production of PHA in two step culture. Cells grown on nutrient balanced medium were transferred to nitrogen free medium. (**A**) PHA contents of _Sde_ 2-40, _B. cereus_, and co-culture on agarose and xylan; (**B**) PHA concentrations of _Sde_ 2-40, _B. cereus_, and co-culture on agarose and xylan. and xylan were 18.1% and 34.5% of dry cell weight in one step culture and 13.6% and 30.2% of dry cell weight in two step culture, respectively. Depending on the type of strain and conditions for bacterial culture used, PHA accumulation has been shown to be in the range of 1 to over 80% of dry cell weight [62]. Recombinant _Escherichia coli_ was reported to produce 1.1% PHA in nutrient-rich microbial Lennox Broth which was increased to 37% after supplementation of arabin or xylose in the xylan-based media [63]. In the present study, wild strains of the bacteria were used. The present study is the first report to show the highest production of PHA from xylan without pre-treatment by a wild strain of bacteria, to the best of our understanding. Although growth on 0.2% agarose of _Sde_ 2-40 is reported [4; 6], utilization of higher amounts of agarose in media by _Sde_ 2-40 and simultaneous production of PHA has not been attempted yet. This is also the first report of PHA production from agarose. The PHA production by co-culture of _Sde_ 2-40 and _B. cereus_ from xylan in this study was higher (34.5% of dry cell weight) than the earlier report [63]. The results of this study are promising for reducing the production costs of large scale PHA production by utilising abundantly available cheaper carbon sources.