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Review of Literature

The introduction of energy obtained from fossil fuels facilitated a social and

technological revolution. Developmental progress in the last century is unparalleled by anything

else in history, all aided by new sources of generated power. Despite the incredible advances

brought about by the utilization of this new energy source, there is a huge flaw. Fossil fuels,

which are being consumed at greater rates every year, are nonrenewable and harmful to the

environment. Supplies are quickly depleting and the environmental issues surrounding global

warming are worsening, assisted by the detrimental effects of fossil fuel consumption

(Environmental Problems, n.d.). Corn based ethanol, also known as first generation biofuel, is

currently one of the main alternatives to traditional fossil fuels. However due to increasing

population growth, this is inefficient and unsustainable as well. Coined the food vs fuel dilemma,

as global population grows decisions will have to be made about the allocation of the limited

number of resources on earth. In the case of necessity, it is highly likely that corn will be used as

food for an increasingly large and urban society than as an alternate fuel source ("Food Versus,"

2016). A third alternative has been discussed in the science community. It includes making a

bioethanol from agricultural waste, known as second generation biofuel. This option is not

environmentally harmful. Since it is produced from waste products, it does not compete with

food, has the potential to be very efficient, and answers the problems arising from fossil fuels.

Solutions remain to be be found for a few key issues surrounding the production of second

generation biofuel, the main one concerning the development of a pretreatment that is ideally

environmentally friendly, economically viable, and one that produces a high fuel yield (Saritha,

Arora, & Lata, 2012).


The heat produced by the burning of fossil fuels, namely coal, oil, and gas, has been the

prevalent source of energy since the 19th century and the industrial revolution. This

nonrenewable energy source has remained at the forefront of energy production today despite the

constant deterioration of the environment. The rapid increase in carbon emissions in the last

century due to the burning of fossil fuels is a large concern that has reached beyond the scientific

community. For years there has been more and more attention paid to air pollution and shifting

weather patterns, affecting agriculture and general quality of living especially in highly

urbanized areas. The concentration of atmospheric carbon dioxide before industrialization was

found to be >280 ppm and by the 80s had risen to be 350 ppm. It is speculated that levels will

double as early as 2030 (Environmental Problems, n.d.). Despite the difficulty in changing the

infrastructure of society to accommodate more green power, it is necessary to move away from

the unsustainability and devastation of fossil fuels towards a sustainable diesel fuel alternative.

Ever since the oil crises of the 1970s, first generation biofuel made from sugar rich

substrates such as molasses or cane juice or starch rich ones such as corn has been one of the

main alternatives to traditional fuels. Alcohol is made by breaking down the starch in corn

kernels into simple sugars, which are then fermented into alcohol using yeast. This is a step in

the right direction, but still contains some major flaws. For one, corn based ethanol is rarely used

as a stand alone fuel and is often mixed with gasoline, which diminishes its environmental

advantages (Mosier & Ileleji, 2006). However, the major pitfall of first generation biofuel is that

it is made from only edible substances, which causes it to compete with food production. This

competition makes them unable to meet the increasing fuel demand and negate first generation

biofuels as a viable permanent alternative. In fact, increased cultivation of these raw materials


are damaging to biodiversity and may lead to more frequent deforestation for farmland (Saini,

Saini, & Tewari, 2015).

Agricultural residue is a much more attractive alternative. Known as second generation

biofuel, crop waste such as sugar cane bagasse, corn stover, wheat and rice straw, and many other

lignocellulosic materials could be a large source of untapped energy potential. The plant biomass

required is renewable, cheap, and not competing as a food source. It also incredibly abundant, as

seen in figure 1, giving it incredible fuel potential. Farmers typically throw away or burn large

amounts of crop residue and waste after the harvest. Utilizing agricultural residue as source of

fuel would increase land use efficiency, give developing and largely agricultural countries a

measure of self reliance for energy because material can be grown locally, be renewable, and not

contribute significantly to pollution (Saini, Saini, & Tewari, 2015).

Figure 1 (Saini, Saini, & Tewari, 2015)

Most agricultural residue is classified as lignocellulosic material. This means that it is

primarily made up of three main components: lignin, cellulose, and hemicellulose.

Lignocellulosic material has thick and sturdy cell walls, designed to be structurally sound for the


health of the plant. As shown in figure 2, the lignin structure within the cell wall resembles a

matrix surrounding the cellulose. Hemicellulose coats the cellulose and links it to the lignin

("Plant Cell," n.d.). It essentially creates a barrier against cellulolytic enzymes from various

microorganisms that want to access the sugar in cellulose. While this offers a crucial degree of

protection to the plant, it makes lignocellulosic material incredibly unyielding to direct enzyme

hydrolysis. Coined the lignin challenge, working around the obstacle of lignin recalcitrance to

maximize product recovery has been one of the main objects in second generation biofuel


Figure 2 ("Plant Cell," n.d.)

Other obstacles remain, including the high crystallinity of cellulose, which makes

enzyme hydrolysis more difficult, and the accessible surface area of the substrate. It important to

mention that fuel from lignocellulosic biomass is completely attainable. However, economically,

production is expensive. Enzymes and pretreatments that are required for a high sugar yield and

therefore a high fuel yield are costly (Saritha, Arora, & Lata, 2012). Much of the research

continuing to be done on second generation biofuel is working on alternative methods of


production to reduce the impact and cost of production while simultaneously raising the

production yield so that this energy source is feasible on a large scale.

One notable exception is Iogen Corporation. With many investors, a team of scientists

and engineers have been working together to create a commercial process for creating second

generation biofuel out of cellulosic plant material. Located in Ottawa, Canada, they also partner

with Raizen Energia in Brazil whose sugarcane mills they use as a source of material. 2015 saw

the official opening of Iogens (and the worlds) first commercial facility producing second

generation biofuel. In order to maximize product recovery, Iogen uses a pretreatment involving

physical processing to reduce particle size and then a combination of high pressure steam and

acid hydrolysis. This pretreatment allows enzymatic hydrolysis to facilitate saccharification.

Then the components are separated, processed, and fermented into usable fuel ("Cellulosic

Ethanol," 2015).

Many other pretreatments have been developed with the objective to break the lignin-

cellulose-hemicellulose matrix, overcome the issue of lignin recalcitrance, and decrease cellulose

crystallinity to allow for greater enzymatic hydrolysis. These can be separated into four broad

categories: physical, chemical, physico-chemical, and biological. Physical pretreatment is

included in all most all processes. Grinding or milling the biomass reduces particle size and

cellulose crystallinity and the degree of polymerization, all of which facilitates easier hydrolysis

and saccharification by allowing more substrate to access the cellulase. This is usually followed

up by chemical pretreatment. Alkali pretreatments are effective by disrupting the lignin structure

by enlarging the internal surface area of the cellulose. This also decreases its crystallinity and

degree of polymerization. By disrupting the lignin structure this also provides more cellulose

accessibility to enzymatic hydrolysis. Acid hydrolysis takes a more indirect approach and makes


the hemicellulosic component soluble, providing more cellulose accessibility. This method can

either be used with concentrated or diluted acids. While it is effective in hydrolysis, it tends to

create inhibiting compounds and toxic byproducts that make it unfavorable. Other procedures

include physico-chemical processes such as steam explosion and liquid hot water pretreatment,

but they often have high energy demands and are thus not practical on a larger scale (Saritha,

Arora, & Lata, 2012).

The first three pretreatments discussed above are all manufactured processes. Biological

pretreatments differ in that they utilize organisms that produce enzymes that specifically attack

lignocellulosic structure. These extracellular enzymes are known as ligninases. Especially

effective enzymes in this group include laccase, also know as phenol oxidase, and peroxidases.

Fungi, such as white rot and brown roti, are able to anaerobically break down components using

these enzymes. White rot fungi in particular produce lignocellulolytic enzymes that are effective

at breaking down lignin. They are often seen in nature growing on the lignin content in wood

(Dashtban, Schraft, Syed, & Qin, 2010). The main goal is finding a fungus that attacks lignin

effectively while minimizing cellulose loss so that the process is effective in producing a high

product yield. Some white rot fungi attack all three lignocellulose components while others are

lignin selective, making them desirable for a potential biological pretreatment (Dashtban,

Schraft, Syed, & Qin, 2010). Many benefits are gained by a biological pretreatment compared to

other methods. The process is mild, has low energy consumption, is not impactful

environmentally and avoids the unwanted byproducts and inhibitors of chemical pretreatments. A

concern is time length, which can range from several days to several weeks depending on the

fungus and the material, in order to produce the desired output (Saritha, Arora, & Lata, 2012).

Pretreatments account for the most expensive and energy consuming part of the fuel process


(Sindhu, Binod, & Pandey, 2015). It is therefore necessary to find a pretreatment that maximizes

efficiency. Ideally a fungi, time frame, and substrate combination would be found that is able to

degrade lignin while not inhibiting cellulose hydrolysis in order to gain the highest yield


Research conducted by Taylor Maher in 2014 at Pennsylvania State University examined

the effects of two different white rot species on different lignocellulosic materials. He decided to

use P. chrysosporium, a fungus that degrades both lignin and cellulose, and C. subvermispora,

which selectively degrades lignin. His research examined growth rates of the fungi and

composition of the treated substrates. Ultimately he was able to determine that biological

pretreatment would be possible on a large scale for second generation biofuel production.

Numerous other studies have examined the effects of different fungi on different types of

lignocellulosic biomass. Their findings are summarized in figure 3, provided by an overview

written by researchers via the Association of Microbial Biologists of India. As shown, different

species of fungi and different types of biomass incubated for different lengths of time result in

varying degrees of delignification. For example, Ceriporiopsis Subvermispora incubation for an

additional week on corn stover resulted in a 7.61% difference in the extent of delignification.

Another note is the effectiveness of different substrates. Bamboo culms incubated with Trametes

versicolor G20 for 120 days only resulted in 12% delignification while the same substrate

incubated with 9 Echinodontium taxodii 2538 for 120 days achieved 29.14% delignification.

This table shows the extent of potential that lies with biological pretreatment as an effective

degrader of lignin, as well as the necessity to find the right substrate, fungus, and time length

combination in order for optimum results.


Figure 3 (Saritha, Arora, & Lata, 2012)


The search for an alternative fuel source becomes more pressing every day. The negative

environmental effects of fossil fuels have been worsening. Air pollution and carbon emission

levels has risen beyond anything recorded in the past. In addition, fossil fuels are a nonrenewable

resource and are being depleted (Environmental Problems, n.d.). Corn based bioethanol

production competes with agricultural resources used as food, which places a natural limit on

how much it is able to be produced. This makes it unable to ever fully replace fossil fuel

consumption, and thus it is not a sustainable option, although it is a greener alternative (Sindhu,

Binod, & Pandey, 2015). Accessing the simple sugars in lignocellulosic biomass for second

generation biofuel would solve many problems. It is manufactured from unused agricultural

waste and residue and does not compete with food, as well as being environmentally friendly. It

is extremely abundant and would open new markets in agriculturally dominated countries for

energy production. Several issues with second generation biofuel production still need to be

addressed. Presently the process is not economically viable, nor does it maximize efficiency due

to lignin recalcitrance and cellulose crystallinity. Pretreatments to make the sugars more

accessible run into the obstacles of large energy consumption or undesirable by products. Using a


biological pretreatment that utilizes the correct fungus-substrate-time combination has the

potential to avoid these issues, maximize efficiency, and operate on a large scale basis. Second

generation biofuel production research grows in importance and necessity on a daily basis as the

global environment continues to decline.



If ceriporiopsis subvermispora is applied to wheat straw it will be able to degrade the

lignin enough to produce a higher glucose yield than the control (no pretreatment) because it is a

white rot fungi that selectively degrades lignin by producing laccase and manganese peroxidase

(Dashtban, Schraft, Syed, & Qin, 2010). Both of those enzymes are able to effectively degrade

lignin. Because it is selective, it also will not produce any enzymes that would significantly harm

the cellulose yield. The longer the fungus is allowed to grow on the wheat straw, the higher the

glucose yield will be because more lignin will have degraded, leaving more cellulose accessible

(Saritha, Arora, & Lata, 2012) .

If an acid pretreatment is used on wheat straw it will have a higher glucose yield than the

control because the acid hydrolysis will be able to effectively disrupt the recalcitrant

lignocellulosic structure to allow cellulose access. It will be less effective than the biological

pretreatment because acid hydrolysis produces inhibiting by products that will limit the glucose

yield (Saritha, Arora, & Lata, 2012).


Cellulosic Ethanol Process. (2015). Retrieved November 6, 2016, from Iogen

Corporation website:


Dashtban, M., Schraft, H., Syed, T. A., & Qin, W. (2010, May 23). Fungal

Biodegradation and Enzymatic Modification of Lignin. Retrieved November 6, 2016,

from NCBI website:

Environmental Problems with Coal, Oil, and Gas. (n.d.). Retrieved from

Food versus Fuel. (2016, September 9). Retrieved October 31, 2016, from European

Biofuels Technology Platform website:

Maher, T. (2014). Application of two Different Fungal Species for Biological

Pretreatment in an Integrated Lignocellulosic Biofuels Paradigm. Unpublished working

paper, Pennsylvania State University, Pennsylvania, PA.

Mosier, N. S., & Ileleji, K. (2006, December). How Fuel Ethanol is Made from Corn.

Retrieved November 6, 2016, from Purdue Extension website:

Plant Cell Walls. (n.d.). Retrieved November 6, 2016, from Complex Carbohydrate

Research Center website:

Saini, J. K., Saini, R., & Tewari, L. (2015). Lignocellulosic Agriculture Wastes as

Biomass Feedstocks for Second-Generation Bioethanol Production: Concepts and Recent

Developments. Biotech, 5(4), 337-353.

Saritha, M., Arora, A., & Lata. (2012). Biological Pretreatment of Lignocellulosic Substrates for

Enhanced Delignification and Enzymatic Digestibility. Indian J Microbiol, 122-130.

Sindhu, R., Binod, P., & Pandey, A. (2015). Biological Pretreatment of

Lignocellulosic Biomass- An Overview. Bioresource Technology, 1-6.