**5. Integrated biorefinery applications to lignocellulosic biomass**

Processing of cellulosic biomass using the biorefinery approach has often had its roots in the processing of first generation biomass. Typical first generation biomass such as corn, sugarcane and cassava (mostly in Africa and Asia) are still the most preferred feedstock in commercial fermentation processes. The biorefinery way of processing corn by microbial fermentation often yields ethanol, citric acid, lactic acid or lysine as the main product depending the primary product goal of the biorefinery [62]. Conventionally, the starch fraction of the corn is processed to dextrose via enzymatic pathways before microbial fermentation to the desired product. Corn fibre, gluten meal and corn steep liquor are the usual by-products obtained in a corn biorefinery which are of enormous value. Corn fibre which is lignocellulosic in nature can be further hydrolysed to obtain glucose, xylose and other monomeric

sugars which can be further fermented to products such as ethanol, xylitol and acetate [63]. Gluten meal from corn which is very high in proteins can be used as feed for livestock and poultry or as substrates for various pharmaceutical products and commercial polymers [64]. Corn steep liquor which is also high in proteins is often used as a nitrogen source in various fermentation processes [65].

Sugarcane biorefineries are usually very interesting due to the unique composition of sugarcane which is usually 11–16% sucrose, 70–75% water and 10–16% fibre [63]. Sugarcane processing begins with the extraction of cane juice which immediately leads to the generation of solid residue in the form of sugarcane bagasse. Sugarcane bagasse is very high in fibre and is considered a lignocellulosic biomass. This bagasse can be valorised in a relatively more complex pathway to ethanol and other chemicals using microbial fermentation technologies or simply used as fuel in boilers for the generation of steam and electricity. The latter is the predominant process application of bagasse in industry currently. Sugarcane alone as a single biomass can be processed to obtain first generation ethanol from the cane juice and second generation ethanol from the bagasse. Additionally, sugar processing plants which use sugarcane obtain molasses as a sucrose-rich by-product which can also be used as substrate for ethanol production [63].

The potential co-production of ethanol and xylitol from sugarcane bagasse was examined in a study by Unrean and Ketsub [66]. In the study, cellulose and hemicellose fractions of the sugarcane bagasse were separated using sulphuric acid and enzymatic hydrolysis processes. The pretreated cellulose was used as substrate for the recovery of ethanol using *Saccharyomyces cerevisiae* as the fermenting organism while hemicellulose hydrolysate was used as substrate for the recovery of xylitol with *Candida tropicalis* as the fermenting organism. The product recoveries reported from the use of the bagasse was 0.44 g/g total glucose for ethanol and 0.50 g/g total xylose for xylitol. An economic analysis within the same study revealed a 2.3 fold increase in profitability for the integrated ethanol and xylitol production process over standalone cellulosic ethanol production [66].

Cellulosic pulp and paper mill waste in the form of primary sludge was examined in a study as a substrate for the production of bioethanol and biolipids [67]. In the integrated study, bioethanol and biolipids were both obtained from the hydrolysates of the primary sludge at yields of 9% and 37.8%, respectively. *S. cerevisiae* was used as the fermenting organism for bioethanol while the oleaginous yeast *Cutaneotrichosporon oleaginosum* was used as the organism for the biolipids production. A unique addition to the biorefinery process was the use of the unhydrolysed primary sludge as a cement additive or fibre reinforcement material in comparison with conventional Portland cement. The comparison of the compression load between the two materials indicated that the unhydrolysed paper mill material had 102% higher compressive strength than the Portland cement [67]. This unique application of fermentation based and non-fermentation based processes to harness the use of the pulp and paper residual biomass in a zero waste approach can be explored for other lignocellulosic biomass.

Dairy manure, a nitrogen rich cellulosic biomass has been examined as a substrate for the co-production of fumaric acid and chitin [68]. Fumaric acid is commonly used in food flavouring and preservation while chitin is a natural biopolymer with applications in the water treatment and pharmaceutical industries. In the study by Liao et al. [68], *Rhizopus oryzae* ATCC 20344 was applied as fermenting organism in a one-pot fermentation process to obtain fumaric acid in the liquid medium of the broth while chitin was found in the resulting fungal biomass formed in the broth. A maximum fumaric acid yield of 31% and a chitin yield of 0.21 g/g fungal biomass (from 11.5 g/l fungal biomass concentration) was obtained [68].

*Integrated Biorefinery Approach to Lignocellulosic and Algal Biomass Fermentation Processes DOI: http://dx.doi.org/10.5772/intechopen.97590*

Wheat straw and corn stover have been studied as substrates for the co-production of hydrogen and ethanol [69]. In the study, genetically engineered *Escherichia coli* were applied in a dark fermentation process as means to maximise the simultaneous production of the two products. The engineered strained of *E. coli* produced a 30% increase in the co-production yield of hydrogen and ethanol. The yields obtained were 323 ml H2/g total reducing sugars (TRS) and 3.5 g ethanol/g TRS for wheat straw and; 337.1 ml H2/g TRS and 2.9 g ethanol/g TRS for corn stover [69].

Lignin utilisation has been a very important of part of the goal to maximise the use of lignocellulosic biomass in a biorefinery context. Considerably large quantities of lignin-rich by-products are generated from the conversion of cellulosic biomass to biofuels and various organic compounds. The efficient use of the lignin can improve the overall economics of the commercial use of lignocellulosic biomass [70]. A wide range of polymeric materials which can be used as precursors for even more valuable products have successfully been derived from lignin. They include polyesters, epoxy and phenolic resins, hydrogels, graft polymers, vanillin and polyamides. Vanillin in particular is an important compound used as flavouring agent in the food and pharmaceutical industries. It has also been considered as a precursor to hydrogels, polyester epoxide and polyethylene. Direct lignin recovery from lignocellulosic biomass can be effected using the Kraft process, lignosulfonates process, organosolv process, steam explosion or using ligninolytic enzymes such as lignin peroxidase, manganese peroxidase and laccase [70].

### **6. Integrated biorefinery applications to algal biomass**

Several studies have used the integrated biorefinery approach to maximise the use of algal biomass and improve both their economic and process sustainability. This approach was used in the processing of the green seaweed, *C. linum* to coproduce bioethanol and biogas in a single study [41]. A bioethanol yield of 0.41 g/g reducing sugar (0.093 g/g pretreated seaweed) was obtained after the pretreatment, enzymatic hydrolysis and fermentation of the seaweed biomass. The enzymatic hydrolysis was done with a crude enzyme from *Aspergillus awamori* at 45°C and pH 5 for 30 hours while the fermentation was done with *S. cerevisiae* at 28°C for 48 hours while shaking at 150 rpm. The fermentation broth was then distilled to recover the ethanol while the residue referred to as vinasse was used as the feed for anaerobic digestion. The anaerobic digestion of the vinasse which was done at 38°C in a 0.5 l digester for 30 days yielded 0.26 l/g VS of biomethane [41]. The final waste generated was 0.3 g/g biomass which represents a substrate utilisation of up to 70%. This approach did indeed enhance the use of the substrate.

Ashokkumar *et al.* [71] also made a similar attempt with the biorefinery approach. They considered the integrated conversion of the brown seaweed *Padina tetrastromatica* to both biodiesel and bioethanol. The crude lipids content was first extracted from the biomass using various solvents to obtain a yield of 8.15% w/w biomass. This was processed further through transesterification (the process of exchanging the organic group R' of an ester with the organic group R' of an alcohol) to obtain a final biodiesel yield of 78 mg/g biomass. The residual biomass after lipids extraction was hydrolysed and fermented using baker's yeast to obtain a bioethanol yield of 161 mg/g residual biomass [71]. This study demonstrated that the integration of biodiesel and bioethanol production processes on a single seaweed biomass can efficiently harness both the lipid and carbohydrate fraction which could form up to 70% of the entire biomass.

A unique application of the biorefinery approach was used by Xu *et al.* [72]. In their study, mannitol was first removed from the brown seaweed *L. japonica* leaving behind an alginate rich suspension. The alginate suspension was used as substrate for volatile fatty acid (VFA) production via fermentation. The VFAs produced were recombined with the mannitol to produce lipids through fermentation with the oleaginous yeast, *Cryptococcus curvatus*. During the alginate fermentation process several by-products were obtained including; acetate, succinate, lactate, formate, propionate, butyrate and ethanol. A maximum lipids yield of 48.3% was achieved. The lipids obtained were very high in oleic acid (48.7%), palmitic acid (18.2%) and linoleic acid (17.5%) which indicates a fatty acids composition similar to vegetable oil [72]. The lipids can therefore be used for a myriad of applications including culinary processes and biodiesel production.

Dong *et al.* [50] were able to effectively hydrolyze the microalgae *S. acutus* to obtain reducing sugars while making the lipids more easily extractable. An ethanol concentration of 22.7 g/l was obtained from the algae while the recovery of lipids was in the range of 82–87% of total lipids after ethanol removal [50]. There was no adverse effect observed on lipids recovery due to either the acid pretreatment or the fermentation of soluble sugar processes which preceded the lipids extraction. The fatty acid methyl esters concentration was also found to be high for the lipids recovered which makes it a good substrate for biodiesel production. Lee *et al*. [73] also recovered similar products of lipids and ethanol from the microalgae, *D. tertiolecta*. In their study, 48 g lipids were extracted from 220 g of the microalgae while the residual biomass after lipid extraction was found to have a carbohydrates content of 51.9%. Upon fermentation with *S. cerevisiae*, 0.14 g ethanol/g residual biomass (0.44 g ethanol/g glucose) was obtained from the residual biomass [73]. The successful demonstration of potential biodiesel and bioethanol co-production from microalgae indicatives high potential improvements in the economic feasibility of microalgal biorefineries.

The red macroalgae, *Gracilaria verrucosa* was used a substrate for the coproduction of agar (a hydrocolloid) and ethanol [74]. In the study, 33% agar was extracted from the biomass while the residual pulp was enzymatically hydrolysed to obtain 0.87 g reducing sugars/g cellulose. The hydrolysate obtained from the pulp was fermented with *S. cerevisiae* to produce ethanol with a yield of 0.43 g/g reducing sugars. A mass balance assessment in the study indicated that for every 1000 kg of dried algal biomass, 280 kg of agar can be obtained together with 38 kg of ethanol. Additionally, 20 and 25 kg of lipid and protein, respectively can be obtained from the residual pulp after agar extraction [74]. In a similar approach, the hydrocolloid, carrageenan was first extracted from the seaweed *E. cottonii* before the application of the residual pulp in ethanol production [3]. The carrageenan extraction led to an increase in the cellulose fraction to 64% in the residual seaweed pulp. Ethanol yields of 0.25–0.27 g/g residual seaweed pulp were obtained using *S. cerevisiae* as the fermenting organism [3].

Co-production of biosolar hydrogen and biogas was explored on the microalgae *C. reinhardtii* as a means to evaluate the integrated biorefinery approach to processing the biomass [75]. Hydrogen was first produced using the sulphur deprivation method. This method involves the cultivation of algal cells in a sulphur-containing medium until the cells reach the stationary growth phase. Cell pellets are then harvested and re-suspended in sulphur-free medium followed by incubation in light at 600 μmol/m2 /s under room temperature. The production of hydrogen prior to anaerobic digestion of the microalage resulted in a 123% increase in biogas generation from an initial 587 ml biogas/g volatile solids with 66% CH4 content [75].

In another biorefinery process, the microalgae *Nannochloropsis* sp. was used as substrate for the recovery of three different valuable products [76]. Supercritical CO2 was used to extract 45 g lipids/100 g dry biomass and 70% of pigments which were mainly carotenoids. The residual microalgal biomass after extraction was used as an efficient substrate to produce hydrogen at a yield of 60.6 ml/g dry biomass through dark fermentation with *Enterobacter aerogenes* [76]. Harnessing these valuable products from a single biomass shows high economic prospects for microalgal biorefineries.
