Ahmad Adnan

To change the recalcitrant nature of the lignocellulosic material for maximum hydrolysis yield, a comprehensive study was done by using sulphuric acid as an exclusive catalyst for the pretreatment process. The enzymatic digestibility of the biomass [Water Hyacinth: Eichhornia crassipes] after pretreatment was determined by measuring the hydrolysis yield of the pretreated material obtained from twenty four different pretreatment conditions. These included different concentrations of sulphuric acid (0.0, 1.0, 2.0 and 3.0%), at two different temperatures (108 and 121 ºC) for different residence times (1.0, 2.0 and 3.0h).The highest reducing sugar yield (36.65 g/L) from enzymatic hydrolysis was obtained when plant material was pretreated at 121 ºC for 1.0 h residence time using 3.0% (v/v) sulphuric acid and at 1:10 (w/v) solid to liquid ratio. The total reducing sugars obtained from the two-stage process (pretreatment + enzymatic hydrolysis) was 69.6g/L. The resulting sugars were fermented into ethanol by using Saccharomyces cerevisiae. The ethanol yield from the enzymatic hydrolyzate was 95.2% of the theoretical yield (0.51g/g glucose), as determined by GS-MS, and nearly 100% since no reducing sugars were detected in the fermenting media by TLC and DNS analysis.

The current study describes the emphatic use of response surface methodology for the optimized biodiesel production using chemical and enzymatic transesterification of rice bran and sunflower oils. Optimal biodiesel yields were determined to be 65.3 ± 2.0%, 73.4 ± 3.5%, 96.5 ± 1.6%, 89.3 ± 2.0% and 41.7 ± 3.9% for rice bran oil and 65.6 ± 1.2%, 82.1 ± 1.7%, 92.5 ± 2.8%, 72.6 ± 1.6% and 50.4 ± 2.5% for sunflower oil via the transesterification catalyzed by NaOH, KOH and NaOCH₃,NOVOZYME-435 and A.n. Lipase, respectively. Based upon analysis of variance (ANOVA) and Response Surface plots significant impact of reaction parameters under study was ascertained. FTIR spectroscopic and HPLC methods were employed for monitoring the transesterification reaction progress while GC-MS analysis was performed to evaluate the compositional analysis of biodiesel. The fuel properties of both the rice bran and sunflower oil based biodiesel were shown to be technically compatible with the ASTM D6751 and EN 14214 standards. The monitoring of exhaust emission of synthesized biodiesels and their blends revealed a marked reduction in carbon monoxide (CO) and particulate matter (PM) levels, whereas an irregular trend was observed for NO_x emissions.

Background: Pichia pastoris is an established eukaryotic host for the production of recombinant proteins. Most often, protein production is under the control of the strong methanol-inducible aox1 promoter. However, detailed information about the physiological alterations in P. pastoris accompanying the shift from growth on glycerol to methanol-induced protein production under industrial relevant conditions is missing. Here, we provide an analysis of the physiological response of P. pastoris GS115 to methanol-induced high-level production of the Hepatitis B virus surface antigen (HBsAg). High product titers and the retention of the protein in the endoplasmic reticulum (ER) are supposedly of major impact on the host physiology. For a more detailed understanding of the cellular response to methanol-induced HBsAg production, the time-dependent changes in the yeast proteome and ultrastructural cell morphology were analyzed during the production process. Results: The shift from growth on glycerol to growth and HBsAg production on methanol was accompanied by a drastic change in the yeast proteome. In particular, enzymes from the methanol dissimilation pathway started to dominate the proteome while enzymes from the methanol assimilation pathway, e.g. the transketolase DAS1, increased only moderately. The majority of methanol was metabolized via the energy generating dissimilatory pathway leading to a corresponding increase in mitochondrial size and numbers. The methanol-metabolism related generation of reactive oxygen species induced a pronounced oxidative stress response (e.g. strong increase of the peroxiredoxin PMP20). Moreover, the accumulation of HBsAg in the ER resulted in the induction of the unfolded protein response (e.g. strong increase of the ER-resident disulfide isomerase, PDI) and the ER associated degradation (ERAD) pathway (e.g. increase of two cytosolic chaperones and members of the AAA ATPase superfamily) indicating that potential degradation of HBsAg could proceed via the ERAD pathway and through the proteasome. However, the amount of HBsAg did not show any significant decline during the cultivation revealing its general protection from proteolytic degradation. During the methanol fed-batch phase, induction of vacuolar proteases (e.g. strong increase of APR1) and constitutive autophagic processes were observed. Vacuolar enclosures were mainly found around peroxisomes and not close to HBsAg deposits and, thus, were most likely provoked by peroxisomal components damaged by reactive oxygen species generated by methanol oxidation. Conclusions: In the methanol fed-batch phase P. pastoris is exposed to dual stress; stress resulting from methanol degradation and stress resulting from the production of the recombinant protein leading to the induction of oxidative stress and unfolded protein response pathways, respectively. Finally, the modest increase of methanol assimilatory enzymes compared to the strong increase of methanol dissimilatory enzymes suggests here a potential to increase methanol incorporation into biomass/product through metabolic enhancement of the methanol assimilatory pathway.