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Ethanol is made via the fermentation of sugar and uses enzymes made from specific yeasts. There are five major sugars; the five-carbon sugars are xylose and arabinose and the six-carbon sugars are glucose, galactose, and mannose (McCoy, 1998). Traditional fermentation utilizes yeasts that convert six-carbon sugars, to ethanol. Glucose is the most used and most preferential fermentation sugar as it contains both carbohydrates and cellulose. Carbohydrates convert to glucose much easier than cellulose; the majority of ethanol currently produced is made from corn, as it makes large quantities of carbohydrates. There is also a large amount of organisms and enzymes for carbohydrate conversion and glucose fermentation on a commercial scale. Cellulose, however, must first be converted to sugar by hydrolysis and then fermented to produce ethanol. Cellulosic feedstock, which contains a mixture of cellulose and hemicelluloses, are more difficult to convert to sugar than are carbohydrates. Two methods are commonly used for the conversion of cellulose to sugar; dilute acid hydrolysis and concentrated acid hydrolysis, both of which use sulfuric acid.
Dilute acid hydrolysis is a two stage process, allowing utilization advantage of the differences between hemi-cellulose and cellulose. The first part is run at low temperature as it maximizes yield from hemicelluloses. The second part is run at a higher temperature, optimizing hydrolysis of the cellulose in the feedstock.
Concentrated acid hydrolysis uses a dilute acid pre-treatment to divide the hemi-cellulose from the cellulose. The biomass is then dried; thereafter concentrated sulfuric acid is added. Water is used to reduce the acids concentration. It is then heated to release the sugars, producing a gel that can be separated from residual solids via column chromatography.
Both procedures (dilute and concentrated acid processes) have drawbacks. Dilute acid hydrolysis of cellulose yield a lot of byproducts. Concentrated acid hydrolysis yields fewer byproducts, but requires the acid is separated and re-concentrated, adding to the complexity of the process. Sulfuric acid is also highly corrosive and difficult to handle. "The concentrated and dilute sulfuric acid processes are performed at high temperatures, which can degrade the sugars, reducing the carbon source and ultimately lowering the ethanol yield (Cooper. 1999).
Enzymatic hydrolysis used on cellulose has the most potential in conversion of biomass to ethanol. It merely replaces the sulfuric acid in the hydrolysis. Cellulase can then be used at lower temperatures, reducing the degradation of the sugars. The process also allows simultaneous saccharification and fermentation, which combines cellulose and fermentating yeasts. This procedure allows the fermentative organisms to convert the sugars to ethanol in the same step (Cooper. 1999).
Fig2. Bio-ethanol process involving cellulose hydrolysis.
Bioconversion of cellulosic residuals to ethanol is more complex than bioconversion of starch residuals and thus requires several steps of processing. The first three steps are biological processes, while the fourth is a chemical engineering process.
First is pretreatment.
Then is the saccharification step, in which, cellulose and hemicelluloses is converted to soluble monomeric sugars by hydrolysis.
Thirdly is the conversion of these monomers to valuable products such as ethanol in a fermentation process.
Finally the separation and purification of the products.
Pretreatment of cellulosic residuals is vital. Hydrolysis is slow if the material is not pretreated, and this leads to a low yield. Some pre-treatment can increase pore size and reduce the crystalline structure of cellulose, making it easier for cellulosic enzymes to work. Since, there is more efficiency with the enzymes, less would be used and thus pre-treatment reduces cost (Mosier et al, 2005). There are three pre-treatment types; chemical (acidic / basic), physical / physiochemical and biological pretreatment, which requires the use of micro-organisms. Biological pretreatment uses selective microbial enzymes for degradation of cellulosic residues; it uses low energy and produces minimum waste. P. chryosporium can attack lignin and has been shown to facilitate the conversion of cotton stalks into ethanol, via solid state cultivation. Phlebia radiata, P. floridensis and Daedalea flavida, degrade lignin selectively in wheat straw and work well on lignocellulosic residues. Ceriporiopsis subvermispora lacks cellulases, but it has manganese peroxide and laccases that selectively delignifies specific wood species (Ferraz, 2003).
Hydrolysis is done after pretreatment. Cellulose and hemi-cellulose are hydrolyzed to their hexose and pentose monomers, using cellulases and hemi-cellulases, respectively. Trichoderma, Penicillium, Aspergillus produce a lot of extracellular cellulases and hemicellulases. The enzymes that are used in hydrolysis are preferred to work at high temperature and low pH, since most pre-treatment uses heat and acidity. Thermostable enzymes also have higher stability and an increased hydrolytic performance. Thermostable enzymes are produced from fungi such as T. emersonii, T. aurantiacus, S. thermophile, Chaetomium thermophilum and Corynascus thermophilus. This indicates good potential for hydrolysis of cellulosic residues (Hahn-Hagerdal, 2006).
The products of the hydrolysis can then be fermented to ethanol. "Among these hydrolytic products, glucose is normally the most abundant, followed by xylose or mannose and other lower concentration sugars. Saccharomyces cerevisiae is the most frequently and traditionally used microorganism for fermenting ethanol from starch-based residues at industrial scales". S. cerevisiae has a high fermentative rate and ethanol tolerance, but it is unable to use xylose or arabinose efficiently as a carbon source, nether can it ferment it to ethanol (Hahn-Hagerdal, 2006). But since xylose is an important economical sugar, recombinant S. cerevisiae have been genetically engeneered to carry xylose-fermenting genes and have been shown to produce yields close to that of optimum. The S. cerevisiae harboring engeneered xylose-fermenting genes have also been modified with arabinose-metabolizing genes from other micro-organisms. "The latest recombinant S. cerevisiae (TMB 3400) has been shown to successfully ferment both xylose and arabinose in addition to glucose" (McMillan and Boynton, 1994).
Inhibiting compounds can also reduce the activity of S. cerevisiae; inhibiting compounds such as phenolics and inorganic compounds can be released during the pre-treatment and hydrolysis procedures. Hence, detoxification of the products must be done, although it increases sugar loss, but, attempts to reduce by-product inhibitions are being made. "Interestingly, the pretreatment with O2 has been shown to be the most efficient in enhancing conversion of the raw material to sugars" (Georgieva et al, 2008) and "ammonia fiber explosion pre-treatment also has been shown to be a good candidate since it does not produce some inhibitory by-products such as furans. However, the disadvantage of the method is that some of the phenolic compounds in lignin may remain on the pre-treated material, which then needs to be washed (Taherzadeh and Karimi, 2008 ).
The final steps of bioconversion of cellulolytic residues to ethanol can be run individually or simultaneously. If using separate hydrolysis and fermentation (or SHF), the hydrolysis products are fermented to ethanol separately allowing individual optimal conditions, but it causes the accumulation of enzyme-inhibiting end-products during the hydrolysis step, reducing efficiency. When using simultaneous saccharification and fermentation (or SSF), the end-products are directly converted to ethanol, by microbial action. This reduces, both, amount of Î²-glucosidase required and ethanol production costs. A shortcoming is that the processes need a compromise (sub-optimal conditions) and neither has its optimal conditions. "Further process integration can be achieved by a process known as consolidated bioprocessing (CBP) which aims to minimize all bioconversion steps into one step in a single reactor using one or more microorganisms. CBP operation featuring cellulase production, cellulose or hemi-cellulose hydrolysis and fermentation of hexose and pentose carbon sugars in one step have shown the potential to provide the lowest cost for biological conversion of cellulosic biomass to fuels, when processes relying on hydrolysis by enzymes and/or microorganisms are used" (Lynd et al, 2005).
Int J Biol Sci ijbsv05p0578g01.jpg
Fig3. Bioconversion of biomass to ethanol.
A wide variety of feed-stocks are available for producing ethanol from cellulosic biomass. Materials are categorized as forest residue, agricultural waste and energy crops. Agricultural waste include crop residues like wheat straw, corn stalks and cobs, rice straw, and sugar cane waste. Forestry waste can include wood that was not utilized or underutilized, logging residues (dead wood that is rough, rotten and salvable) and excess saplings and small trees. Certain energy crops are developed and grown specifically for fuel such as fast-growing trees, shrubs and grasses and switch-grass. MSW also contains some cellulosic materials, such as paper (Segundo and Bruce, 2003).
Feedstock choice is a major cost issue as well as a major environmental issue. Most forest residue is not in large volume, but they have the potential to reduce fire hazard associated with dead wood present in many national forests. Small amounts of forest thinning can be gathered at relatively low costs, but this cost increases as amount increases. Agricultural residue is the source of biomass that can support substantial growth of the ethanol industry as they yield 60 to 100 gallons of ethanol per dry ton. "The available corn stover inventory would be sufficient to support 7 to 12 billion gallons of ethanol production per year, as compared with approximately 1.4 billion gallons of ethanol production from corn in 1998". It is more cost effective than forest thinning, but low crop year affects the availability of corn stover (Wang et al, 1999).
The lower cost is a function of feedstock use and is due to the relatively high density of material available, and does not involve farmland competition. The feedstock is located in the corn-processing belt, located close to existing grain ethanol plants that are expanded to make ethanol from stover (Wang et al, 1999). "About 5% of corn in the world is wasted. If wasted corn could be fully utilized as feedstock for producing bioethanol, then 9:3 GL of bioethanol could be produced, thereby replacing 6:7 GL ofgasoline if bioethanol is used as an alternative vehicle fuel" (Segundo and Bruce, 2003).
About 3.4% of barley in the world is lost as waste. If wasted barley could be fully utilized to produce bio-ethanol, 1.5GL of bio-ethanol can be made globally. This would replace 1.1GL of gasoline if E85 (85% ethanol and 15% gasoline) ethanol is used as fuel for a midsize passenger vehicle. Barley can produce 20.6GL bio-ethanol a year if the waste and barley straw is utilized. Bio-ethanol from barley replaces 1.3% of global gas usage, without utilizing barley from anywhere else. "There is a good opportunity to utilize barley straw as feedstock for producing bio-ethanol in North America" (Segundo and Bruce, 2003).
Potential contribution to ethanol production (billion liter/year)
World Agricultural Wastes1
Trillion grams/year (Tg/y)
Municipal Solid Waste (MSW)
Million metric tons (million MT)
In Canada (2001)
In USA (1995)
Table1. Cellulosic residues and potential for ethanol production
Utilization of oat grain can produce 225ML bio-ethanol, thereby, replacing 161ML gasoline when ethanol is used in E85. Utilization of wasted oat straw and oat grain produce about 3.16GL bio-ethanol. This replaces 2.27GL gasoline if bio-ethanol is used as E85 fuel (Segundo and Bruce, 2003).
Fig4. Biomass fermentation process yielding bio-ethanol.
Micro-organisms in soil present the greatest biodiversity on Earth. Soil habitats provide an optimal medium for microbial growth. Bacteria prefer a thin layer of moisture surrounding clay particles, for growth, while fungi grow better in larger soil pores and are more apt to surviving in dry conditions. Bacteria are usually 0.001 millimeter in diameter and fungal filaments are about 0.005 millimeter. Micro-organisms continually need a supply of food, which is catered for by the surrounding plants and animals. Different microbes eat different plant materials and utilize different animal wastes. Bacteria eat smaller, more soluble compounds, such as especially sugars. Fungi, however, feed on harder organic plant fibers and woody material including cellulose, lignin and plant fibers, which do not decompose as easily. A gram of fertile soil contains up to 3000 million bacteria and 500000 fungi, algae and protozoa. Micro-organisms tend to grow faster in healthy soils that mimic the ideal conditions for plant growth. The soil also needs to maintain an adequate moisture level, as well as good aeration and drainage. Micro-organisms and plants require similar inorganic nutrients, but they are usually heterotrophic and cannot manufacture organic material, via photosynthesis. Fertile soils with vibrant microbial ecosystems are able to break down many organic pollutants (Popelarova et al, 2008).
Carbon dioxide levels in the atmospheric are increasing at a steady rate (Keeling et al., 1995), this is due mainly to the combustion of fossil fuels and to large scale deforestation. Consequently, since there is an increase in atmospheric carbon dioxide levels it is expected that there would be multiple direct and indirect effects on terrestrial ecosystems (Bazzaz, 1990). Among those effects are changes in above-ground primary production which are relatively easy to assess, but changes in carbon allocation below-ground are more difficult to assess. But, this process is thought to be vital for both the correct functioning of terrestrial eco-systems and for carbon sequestering. "Most intriguing is the impact of root growth on soil microorganisms and associated transformation processes. After all, microorganisms play an essential role in the cycling of nutrients associated with primary production and this largely determines the overall ecosystem response" (Paul and Clark, 1989).
Fig5. Interaction between plants and micro-organisms.
The response of the soil microorganisms to the elevated atmospheric carbon dioxide concentrations is a poorly understood phenomenon. "Because only a minority of the microbial community is active, while the majority is dormant and forms a high 'background noise' when measuring soil microbial biomass, responses of soil microorganisms to elevated atmospheric carbon dioxide are hard to measure" (van Ginkel et al., 1999). "Carbon tracers such as 14C or 13C are a good tool to distinguish between already present native-soil organic carbon and incoming plant-derived carbon in the soil and to measure soil microorganisms actively involved in the transformation of either of the carbon sources. To study the metabolic behavior of soil micro-organisms, we calculated the ratio between the 14C-labelled soil microbial biomass and total 14C atmospheric carbon dioxide evolved at the end of incubation with roots and root-derived. What consequences a temperature increase accompanying the raise of atmospheric carbon dioxide will have on microbial behavior and subsequently on decomposition of root material grown at elevated atmospheric carbon dioxide is not clear yet" (Gorissen et al, 1995).
Soil microbiology deals with the study of micro-organisms and their processes in soil. Soil is a heterogeneous medium of solid, gaseous and liquid phases, varying in properties both across the landscape and depth. Soil microbes significantly contribute to the maintenance of the matter and energy turnover in terrestrial environments. Soil is a significant medium for biogeochemical processes, in which mineralization has a significant role. Soil respiration is a process that releases carbon dioxide from soil root respiration and microbial decomposition of organic matter in soil. Carbon, in general, regulates climate change and therefore soil respiration is also relevant to climate change. Mineralization tests are often used to test soil microbial activity. Carbon dioxide production enables the evaluation of mineralization activity of native soil organic matter and the potential activity of soil after the addition of nutrients to the soil (Popelarova et al, 2008).
There are many factors that contribute to climate change, and specifically, global warming. The most major contributors are the nutrient cycles; which include the carbon, nitrogen and phosphorus cycle, all of which require micro-organisms for the cycle to be completed. These processes lead to production of gasses, such as carbon dioxide and methane, which add to the greenhouse effect and global warming (Campbell et al, 1999).
The carbon cycle is the cycle of greatest importance, since it directly effects both the climate change and global warming phenomenon. The production of methane, carbon monoxide and carbon dioxide also contribute to climate change. The atmospheric concentration of carbon dioxide, is produced by micro-organisms via the carbon cycle and also the through anthropogenic activities (such as burning of fossil fuels). This high concentration has an effect on the Earth's heat budget. Most of the solar energy that strikes the Earth is reflected back into space. Water vapor and carbon dioxide are transparent to visible light, but they intercept and absorb much of the reflected infra-red radiation, reflecting it back towards the Earth and retaining some of the solar heat, creating a greenhouse effect. Increase in the level of carbon dioxide is a major concern since it directly affects global warming (Campbell et al, 1999).
Fig6. A typical carbon cycle.
The use of pesticides and depletion of surface- and ground-water, may also have an effect on nutrient cycling. Agriculture, specifically, has a great impact on the nitrogen cycle. Cultivation allows for the rate of degradation of organic matter to be increased, and this releases usable nitrogen that is removed from the ecosystem, when the crops are harvested. Since the plants are removed, not only is the nitrogen lost, but nitrates are leached from the ecosystem. These nitrates are capable of downward leeching and forming toxins in groundwater. Increased nitrogen fixing may also cause an increase in the release of nitrogen compounds, such as nitrogen and nitrogen oxides. Nitrogen oxides have the potential to contribute to global warming and to the climate change. Nitrogen oxides can even cause acid precipitation and further deplete the ozone. Micro-organisms such as Nitrosomonas eutropha and Pseudomonas denitrificans are involved in the nitrogen cycle and lead to production of critical greenhouse gasses, namely nitrous oxide and dinitrate oxide (Prescott et al, 2005).
Fig7. The nitrogen cycle.
Many micro-organisms such as methanogenic archaea are of importance as they produce methane, by the process of methanogensis. Micro-organisms involved in the anaerobic digestion of sewage or waste, form products such as carbon dioxide or acetate. Methanogens act on these byproducts to produce methane (carbon dioxide reducing methanogens and aceticlastic methanogens, respectively). Methane serves as an important resource and is a good energy source, as it is a clean-burning fuel. Methanogensis, however, produces an ecological problem, as methane absorbs infra-red radiation and is thus classified as a greenhouse gas. Hence, methane production contributes greatly to global warming. "There is evidence that atmospheric methane concentrations have risen over the last 200 years" (Prescott et al, 2005). Methanogenic bacteria include Methanospirillium hungatei, Methanovibacter smithii and Methanogenium marisnigri.
Fig8. Sources of atmospheric methane. (Houghton et al. 1996).
The global terrestrial flux of carbon in natural ecosystems is largely influenced by tropical rainforests, but "increased atmospheric carbon dioxide and changes in climate are likely to affect the distribution of carbon pools in the tropics and the rate of cycling through vegetation and soils" (Silver et al, 1998). The dynamics of soil micro-organisms and their communities are driven by plant interaction and are dependant on the purity and amount of carbon dioxide that enters the system. Global climate fluctuations would directly affect plants in terrestrial ecosystems; these changes would include increase in temperatures, alterations in moisture levels and rainfall and may also cause an increase in levels of greenhouse gases, namely carbon dioxide (van Ginkel et al., 1999). Generally, the alterations in microbial processes are in response to plant-driven effects of increase in carbon dioxide levels, but, scientific studies are limited to only examining the net process information (Jones et al 1998). "The effects of elevated CO2 on the diversity and composition of soil microbial communities, as well as on criticalinteractions between functional groups, are still largely unknown" (Hu et al 1999).
Fig9. Illustrats the link between plants and microbial activity in terrestrial ecosystems.
Increased levels of carbon dioxide causes a change in the relationship depicted in figure one. Plant production is often limited to the quantity of nitrogen made available during the decomposition of fresh litter and organic matter present in soil. Plant assimilate the inorganic nitrogen made by the micro-organisms and is present in the form of ammonium ion. Meanwhile, the growth and maintenance of soil micro-organisms is controlled by the amount and type of organic compounds entering the soil via plant litter production. Compound in plant litter, that fuel microbial growth, also fuel a biosynthetic demand for nitrogen to create new nitrogen-conatining compounds. Elevated carbon dioxide levels can alter the amount of energy available for the micro-organisms to grow in soil and can thus cause microbial immobilization and alter the micro-organisms demand for nitrogen and this in turn can cause a deficiency in the amount of inorganic nitrogen available for plant uptake.
Fig10. Utilization of organic and inorganic nitrogen in soil ecosystems.
Interactions with the plant can be governed by stimulation or repression of key microbial groups, which can thus shift the balance between beneficial and harmful interactions with the associated plants. "As the species diversity of soil micro-organisms for most ecosystem functions is very high, it is unlikely that moderate losses of soil microbial diversity will alter soil ecosystem functioning" (van Ginkel et al, 1999).
Moisture content and water availability may limit microbial activity in soil ecosystems, by lowering intracellular water potential and thereby reducing the cells hydration and dropping activity of enzymes. An increase in carbon dioxide levels promotes vegetation growth, since it increases the rate of photosynthesis. Greater plant growth leads to thicker canopy of leaves and, hence, increased transpiration, with less run-off of water to nearby water bodies. But "sometimes rising CO2 has the opposite effect". Carbon dioxide levels also have an effect on the amount of water available to plants and micro-organisms in soil ecosystems. "Studies have shown that increasing carbon dioxide levels are changing how stomata, discharge water. With elevated carbon dioxide levels, leaf pores contract and sometimes close to conserve internal water reserves, thereby increasing water use efficiency and reducing the rate of transpiration." Since these plants release less water, they also take in less water from the environment. Slower uptake of water by plants means more water is available for micro-organisms in the soil and groundwater (Benjamin, 2007).
Soil micro-organisms are highly sensitive to pH; and nutrient availability in soil as well as the nutrient uptake by plants, varies with pH as the majority of the micro-organisms that are involved in organic matter decomposition as well as mineralization, prefer a pH of 5 to 8. Bacteria prefer slightly alkaline pH and soil fungi prefer slightly acidic conditions. Most nutrients that are critical for growth are available within the same pH range as the microorganisms that utilize them. The increase in moisture content, combined with the increase in carbon dioxide content, has the ability to alter the pH of the soil, via formation of carbonic acid, and hence reduce nutrient availability and narrow the niche in which the micro-organisms live (Jackson and Reynolds, 1996).
H20 + CO2 = H2CO3
Fig11. Formation of carbonic acid using carbon dioxide and water.