However, since lipids in the vegetative tissues of transgenic energycane are present in lower amounts and are heterogenious in composition [1], appropriate and efficient bioprocessing technologies are needed to deconstruct the lignocellulosic structure and recover vegetative lipids without their degradation along with cellulosic sugars from the feedstock. Chemical-free hydrothermal pretreatment has been shown to maintain the lipid profile during processing and enrich biomass residues with vegetative lipids that can be recovered at the end of the process [4]. We present a successful bioprocessing approach for transgenic energycane at a pilot scale to demonstrate its potential commercialization as an alternative renewable feedstock for cellulosic sugars and renewable diesel production. After pretreatment, a major fraction of vegetative lipids remained in the biomass residues which we recover at the back end of the process. The lipid recovery efficiency of the total process for untreated biomass was calculated to be 75.9% which showed approximately 17% improvement for pretreated biomass residues (88.7%). In addition, enzymatic saccharification of pretreated biomass residues recovered > 90% of cellulosic sugars using purified commercial cellulases. Based on the average biomass yield of transgenic energycane ~17 tons/ha, the calculated potential and actual lipid yields of transgenic energycane were 0.42 tons/ha and 0.32 tons/ha, respectively.
The cost of pure cellulase is one of the primary challenges that limit the use of lignocellulosic biomass in Industrial Biotechnology to produce bio-based products and the commercialization of 2nd generation biofuels. Through this work, we present a technology to facilitate the large-scale production of cellulase. We present an illustration of ‘Plant as factories’ for the production of transgenic bacterial cellulases in tobacco leaves followed by its extraction in active form for the production of cellulosic sugars from bioenergy crops. The capacity of tobacco plants to hyperaccumulate transgenic proteins in plastids has been utilized for the stable expression of bacterial cellulases under field conditions [5]. We present the use of recombinant cellulases in crude leaf extract of transgenic tobacco for saccharification of transgenic energycane bagasse pretreated at a pilot scale.
The crude transgenic tobacco leaf extract poermitted the recovery of ~ 30% w/w of glucose from lignocellulosic biomass. However, the recovery of glucose from lignocellulosic biomass increased aproximately3-fold by supplementation of 25% pure commercial enzyme. The observation indicates the potential of ~75% reduction of cellulase cost in industries such as paper and pulp, detergent, cosmetics, etc. Since there is no detrimental effect on biomass yields and other physiological parameters due to metabolic engineering [5]. Crude leaf extract containing cellulase obtained from a hectare of tobacco can saccharify approximately 5 tonnes of lignocellulosic biomass. In addition, a leaf area index of six m2 of leaf per m2 of land translates to 170 kg of transgenic cellulase per hectare. This has the potential to lower the total cost of cellulase production as low as 4.5 USD/KG, which is competitive with the submerged fermentation process of cellulase production [6]. Furthermore, the cellulase cost can be reduced significantly by increasing the expression levels to 70% of total soluble protein which is the theoretical maximum. Biomanufacturing platform development via synergistic efforts of synthetic biology and bioprocessing technology will be discussed.
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