32.4: Part 2 - Biofuels A - Corn and Sugar Cane Ethanol

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Learning Goals (ChaptGPT+ o1) 1/16/25)

1. Comprehend the Carbon Cycle and Energy Fluxes:

   • Explain the major carbon reservoirs (atmosphere, oceans, lithosphere, biosphere) and the fluxes between them, including the significance of stocks (in GtC) and fluxes (GtC/yr).

2. Apply Stoichiometry and Dimensional Analysis:

   • Use unit conversions (e.g., from GtC to ppm and from percentage to ppm) and stoichiometric relationships to quantify carbon in the atmosphere and calculate net changes from fossil fuel emissions.

3. Understand Bioethanol Production Chemistry:

   • Describe the key reactions involved in the production of bioethanol—from photosynthesis to fermentation and combustion—and how these reactions theoretically support a carbon-neutral model.

4. Evaluate Feedstock Sources for Bioethanol:

   • Differentiate between first-, second-, and higher-generation bioethanol feedstocks (e.g., corn vs. sugar cane vs. cellulosic biomass) and their respective impacts on land use, energy yield, and environmental sustainability.

5. Interpret Life Cycle Analyses (LCA) for Biofuels:

   • Critically assess the methodology and metrics of LCAs, including global warming potential (GWP), carbon intensity, and other environmental impact categories, to evaluate the net CO₂ emissions of bioethanol production.

6. Analyze Energy Density and Efficiency:

   • Compare the energy values (ΔHc°) of various fuels, understand how ethanol’s energy output compares to gasoline, and assess how energy density influences the overall efficiency of biofuels.

7. Integrate Biochemical Enzymology in Feedstock Processing:

   • Explain the roles of enzymes (such as alpha-amylase, beta-amylase, and invertase) in the saccharification of starch and sucrose during bioethanol production, including the structural and catalytic aspects of these enzymes.

8. Relate Isotopic and Chemical Data to Climate Impact:

   • Understand how isotopic analyses and stoichiometric balances help verify claims about carbon neutrality and clarify the actual environmental impact of bioethanol versus fossil fuels.

9. Critique the Socio-Economic and Environmental Trade-offs:

   • Evaluate the broader impacts of bioethanol production—including land use changes, food security, and pollution—by comparing the benefits and drawbacks against those of fossil fuel use and other renewable energy sources like solar and wind.

10. Examine Policy Implications and Global Energy Strategies:

    • Discuss the role of governmental policies, such as renewable fuel standards, in shaping bioethanol production and assess how changes in fossil fuel subsidies and incentives for renewable energy affect overall CO₂ emissions and climate change mitigation strategies.

These learning goals are designed to help biochemistry majors synthesize their knowledge of chemical reactions, enzyme mechanisms, and quantitative analysis with environmental and socio-economic factors, leading to a comprehensive understanding of bioethanol production and its role within the global carbon cycle.

Introduction

The world has a great need for energy. We have invested vast sums of money in finding and using fossil fuels. At first glance, fossil fuels appear to be an ideal energy source since they are highly reduced, easily stored, energy-dense, and abundant. Yet we now know the immense cost of their use: pollution that shortens lives and climate change. We have dramatically increased our bioethanol production from corn and sugar cane to remove our reliance on fossil fuels for transportation. Ethanol is partially oxidized as it has one oxygen atom in the two-carbon molecule. Hence, the energy released per gram is about 63% (by mass) and 70% (by volume) compared to gasoline. The energy values for various fuels are shown in Table  \(\PageIndex{1}\) below, where ΔH_c° is the standard enthalpy of combustion.

 

Table  \(\PageIndex{1}\):  Energy values for a variety of fuelsData source:  https://www.engineeringtoolbox.com/s...nt-d_1987.html

Name	Formula	State	-ΔHc° kJ/mol	-ΔHc° kJ/g or MJ/kg	-ΔHc°  kcal/kg
Ammonia	NH3	gas	383	22.48	5369
Butane	C4H10	gas	2878	49.50	11823
Carbon (graphite)	C	cry	394	32.81	7836
Carbon monoxide	CO	gas	283	10.10	2413
Ethanol	C2H6O	liq	1367	29.67	7086
Hydrogen	H2	gas	286	141.58	33817
Methane	CH4	gas	891	55.51	13259
Methanol	CH4O	liq	726	22.65	5410
Naphthalene	C10H8	cry	5157	40.23	9609
Octane	C8H18	liq	5470	47.87	11434
Propane	C3H8	gas	2220	50.33	12021
wood (red oak)				14.8	3540
coal (lignite)				15	3590
coal (anthracite)				27	4060
methyl stearate (biodiesel)	(CH3(CH2)16(CO)CH3			40	9560

Nevertheless, ethanol is readily made and is a valuable biofuel. A glance at the table suggests that H₂ would be the best possible fuel, given that it has the highest energy release per gram and contains no carbon. It can't be produced at the needed scale, and it isn't easy to store and transport. The critical infrastructure for its widespread use is lacking. Yet these factors could be solved. We'll explore biohydrogen in a separate chapter. 

In theory, bioethanol is carbon neutral since each carbon in the ethanol is fixed from atmospheric CO₂ during photosynthesis. The combustion of ethanol returns the CO₂ to the atmosphere in a net zero emission fashion, as shown in the reactions below.

Eq 1:  6CO₂ (g)  +  6H₂O (l)  →  C₆H₁₂O₆ (s)  + 6O₂ (photosynthesis)

Eq 2:  C₆H₁₂O₆ (s) → 2 CH₃CH₂OH (l)  +  2CO₂ (g) (anaerobic ethanol biosynthesis)

Eq 3:  2CH₃CH₂OH (l)  +  6O₂   →  4CO₂ (g)  +  6H₂O (g) (combustion of ethanol)

Six CO₂s in, six out! It seems simple, but it's not since we have to manufacture bioethanol, which is energy and land-intensive. We'll explain more later. First, let's explore how ethanol is synthesized for its two major uses: drinking and use as a biofuel.

Ethanol Production Overview

The scale of worldwide ethanol production is quite staggering. Let's first consider the production of ethanol by yeast for alcoholic beverages. About 100 billion US gallons/yr (BGY) of beer, 7 BGY of wine, and 6 BGY of spirits are produced yearly. Assuming beer, wine, and spirits are about 5%, 12%, and 40% percent ethanol by volume, respectively, the volume of actual ethanol/year made by yeast in these alcoholic beverages is about 5 BGY (beer), 0.85 BGY (wine) and 2.4 BGY (spirits). This sums to about 8.3 billion gallons of ethanol produced by these microorganisms. Compare this to fuel ethanol production each year, shown in Figure \(\PageIndex{1}\).

Figure \(\PageIndex{1}\): US Fuel Ethanol Production.  Data from U.S. Bioenergy Statistics

Note that the y-axis is in units of 1000s gallons of ethanol. Peak US production was in 2018, when 16 billion gallons were produced, about 1/10 of the gasoline used yearly in the US. The year Renewable Fuel Standards (RFS) were introduced in the USA (2005) is also shown. This dip in 2020 is attributed to the COVID-19 pandemic.

The US and Brazil produce about 85% of fuel ethanol, as shown below in Figure \(\PageIndex{2}\).

Figure \(\PageIndex{2}\):  Fuel ethanol production (billions of gallons or BG) around the world per year. https://afdc.energy.gov/data/10331

Almost all US ethanol is made from corn, while Brazil's primary source is sugar cane. 

Now, the world produces 3x more ethanol for driving than for drinking. These statistics show that the world can quickly respond when it meets our needs.

An overview of ethanol biosynthesis

Whether ethanol is made for the beverage or biofuel industries, yeast plays a major role, as we explored in Chapter 14.2: Fates of Pyruvate under Anaerobic Conditions- Fermentation.  Yeast contains all the enzymes necessary to convert glucose (6C), made from various "feedstocks", to pyruvate (3C) through the glycolytic pathway.  This is followed by converting pyruvate to ethanol using two key yeast enzymes.  First, pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase, which uses TPP as a cofactor. Acetaldehyde is then reduced to ethanol by ethanol dehydrogenase, using NADH as a substrate, in a process that regenerates NAD⁺, allowing glycolysis to continue.  Figure \(\PageIndex{3}\) shows these combined anaerobic reactions, known as fermentation.  

Figure \(\PageIndex{3}\): Summary of Ethanol Fermentation in Yeast

Yeast are facultative (not obligate) anaerobes in that they can produce energy by glycolysis and ethanol fermentation without oxygen. Of course, in the presence of oxygen, the pyruvate made from glycolysis in yeast is preferentially converted to acetyl-CoA, which enters the citric acid cycle and oxidative phosphorylation pathways to maximize ATP production. Yeast is abundant, so all that is needed is a significant source of glucose.

An abundant source of glucose for bioethanol production is plants that contain starch (for example, corn) or abundant simple sugars (for example, sucrose in sugar cane). Starch, an α (1,4) glucose polymer with α (1,6) branches, can easily be broken down in an industrial setting with amylases to form glucose.  A significant problem with this "first" generation source of glucose is that food crops (corn and, to a lesser degree, sugar cane) are used for biofuel consumption instead of for food. "Second" generation sources of glucose are crop and wood waste products that contain cellulose, a β (1,4) glucose polymer found with another carbohydrate polymer, lignin.  A significant problem with the use of cellulose is the high chemical stability of the β (1,4) glycosidic bond. Fungi and bacteria are sources of β-glycosidases to liberate free glucose from cellulose.   "Third" generation sources of glucose use algae, which does not displace cropland for bioethanol production.  A "fourth" generation source of glucose uses genetically engineered organisms, which might become future sources of bioethanol.  Figure \(\PageIndex{4}\) summarizes the different generations of feedstock sources for bioethanol production.

Figure \(\PageIndex{4}\):  Generation feedstock sources for bioethanol production.   Tse, T.J.; Wiens, D.J.; Reaney, M.J.T.  Fermentation 2021, 7, 268. https://doi.org/10.3390/fermentation7040268.  Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

We will discuss first-generation sources, such as corn and sugar cane, which are used to produce most of the world's bioethanol, in this chapter section and the other two in subsequent sections.  

Corn Bioethanol

Corn is a significant source of starch, an α (1,4) polymer of glucose with α (1,6) branches. Hence, glucosidases are used to hydrolyze starch to glucose. First, the dry corn is ground in a mill, breaking the outer coat of the corn kernel and increasing access to the starch. Heated water is added to form a mash or slurry. Cooking at greater than 85 ^oC helps hydrolyze some glycosidic bonds and lowers the viscosity of the slurry. In the process of liquefaction, the pH is adjusted to around 6.0. Different α-amylases (endoglycosidases) are added, which cleave the α (1,4) glycosidic bonds to produce shorter dextrins (containing branched glucose units not cleaved by alpha-amylases), and α (1,4) linked glucose oligosaccharides of lengths from 2 glucose units (called maltose) up to 7-8. β-amylase, an exoamylase, is also used, which successively cleaves maltose units, Glc α (1,4)Glc, from the nonreducing ends of the chains

Alpha-amylases

A mixed-rendered structure of the human pancreatic alpha-amylase is shown below in Figure \(\PageIndex{5}\).

Figure \(\PageIndex{5}\): Surface representation of the active site of HPA (5TD4)  https://pdb101.rcsb.org/global-healt...ha-glucosidase.   CC-BY-4.0 license. Attribution: David S. Goodsell and the RCSB PDB.

The surface view highlights the deep C-shaped groove into which the substrate, in this case, octaose, is bound. Consistent with substrate numbering for proteases, the starch substrate is numbered ..-2, -1, +1, +2, with cleavage occurring between the -1 and the +1 bound alpha-glucose residues. The protein has three domains (orange, blue, and pink). This particular structure had an active site mutant (Asp300Asn, D300N). The enzyme has bound calcium and chloride ions. Ca²⁺ maintains the necessary structure, while Cl^-, bound in the C domain, is an allosteric activator.  

The octaose binding site is between the A and B domains.   Asp197, Glu233, and Asp300 are critical catalytic residues, with Asp 197 acting as a nucleophile to produce a glycosylated intermediate hydrolyzed in the next step. Asp197 and Glu233 act as general acids/bases. We will explore similar mechanisms for the action of beta-amylase (below) and cellulase (next chapter section).

Figure \(\PageIndex{6}\) shows an interactive iCn3D model of starch binding sites on the Human pancreatic alpha-amylase D300N variant complexed with an octaose substrate (5TD4)

Figure \(\PageIndex{6}\): Starch binding sites on the Human pancreatic alpha-amylase D300N variant complexed with an octaose substrate (5TD4). (Copyright; author via source). Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...W29jf4yAc1JEq9

The domains in the enzyme are colored-coded, as in Figure 4. Key active site residues for binding and catalysis are shown as sticks and labeled.

Beta amylase

β-amylase (also called β-1,4-maltosidase) is a key enzyme in the saccharification process, in which starch and cellulose are broken down into monosaccharides. β-amylase is abundant in crops (wheat, barley, soybeans, etc.), other higher plants, bacilli, and fungi. It is used in making beer and caramel (malt syrup). As an exo-glycosidase, it cleaves Glc α(1,4) Glc (maltose) units from the nonreducing end of starch. It is called β-amylase since the hydrolysis proceeds with the inversion of configuration at the reducing end of the freed maltose. It can't cleave at α-1,6 branches, so if used alone, this enzyme produces free maltose and large β-limit dextrins. When fruits ripen, the enzyme cleaves starch to produce sweet maltose. It is also used in seed germination.

Malting

Plants have to sprout, which requires energy and free sugars. Maltose is produced on activation of β-amylase during seed germination and sprouting. Although maltose is less sweet than sucrose and fructose, it is used in hard candies, given its tolerance to the heat needed in candy production. Malting of grains is accomplished by adding water to sprout them, leading to maltose and other sugars forming. This is followed by drying, with the malted grains used as sweeteners in the food industry.    Malted grains are used to produce beer, whisky, some baked goods, and drinks. Barley is the most commonly malted grain used in cereals. 

Huge amounts of amylases are needed for corn ethanol production, and they must withstand the conditions necessary for the industrial production of ethanol. Much effort has been devoted to finding and characterizing microbial β-amylases. We'll describe one, AmyBa, from B. aryabhattai.   Figure \(\PageIndex{7}\) shows sequence similarities among various bacterial β-amylases.

Figure \(\PageIndex{7}\):  Sequence and structure analysis of AmyBa. .  Duan, X., Zhu, Q., Zhang, X. et al. Expression, biochemical, and structural characterization of high-specific-activity β-amylase from Bacillus aryabhattai GEL-09 for application in starch hydrolysis. Microb Cell Fact 20, 182 (2021). https://doi.org/10.1186/s12934-021-01649-5.  Creative Commons Attribution 4.0 International License. Creative Commons Attribution 4.0 International License. http://creativecommons.org/licenses/by/4.0/.

Panel A shows multiple sequence alignments of β-amylases. The strictly conserved residues are displayed on a red background, and the highly conserved residues are shown on a yellow background. The secondary structure elements are shown for B. cereus β-amylase (PDB ID: 5BCA). The signal-peptide-cleavage site and two catalytic residues (E) are indicated by black triangles (black inverted triangles). Conservation of the flexible loop motif (HXCGGNVGD) is noted. β-amylase accession numbers are as follows: B. aryabhattai (WP_033580731.1), B. cereus (P36924.2), B. flexus (RIV10038.1), B. firmus (P96513.1), B. circulans (P06547.1), T. thermosulfurigenes (P19584.1). 

A comparison of the structures of B. aryabhattai β-amylase with soybean β-amylases is shown in Figure \(\PageIndex{8}\).

Figure \(\PageIndex{8}\) B Three-dimensional molecular model of B. aryabhattai β-amylase (AmyBa). C Superimposition of AmyBa (Blue) and soybean β-amylases (PDB ID: 1Q6C) (gray) and D (PDB ID: 1Q6C) (gray). The C-terminal SBD in microbial β-amylases (box, purple) and the C-terminal loop in plants (box, red).  Duan, X, et al., ibid.

The AmyBa has an additional starch binding domain at the carboxy terminus (Panel B) compared to soybean β-amylases (panel D).

Since no structures of (AmyBa are publically available, we present Figure \(\PageIndex{9}\), which shows an interactive iCn3D model of beta-amylase from Bacillus cereus var. mycoides in complex with maltose (1J0Z)

Figure \(\PageIndex{9}\): Beta-amylase from Bacillus cereus var. mycoides in complex with maltose (1J0Z). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...S2aWbL3z3jPnW8

Like alpha-amylase, β-amylase has an N-terminal catalytic domain with a beta-barrel, a connecting second domain, and a third C-terminal domain, primarily antiparallel β-sheets.   Two key catalytic side chains, Glu 172 and Glu 367, are in the beta-barrel.  

In Chapter 20.6, we discussed starch synthesis (not its hydrolysis) in detail. We showed that the reaction, which uses a NDP-sugar as a glycan donor, could proceed either with retention or inversion of the anomeric carbon of the donor NDP-sugar. This is illustrated for the reaction of a C1 α-NDP donor monosaccharide with a monosaccharide acceptor to produce the α(1,4) link with retention of configuration or the β(1,4) link with inversion as shown in Figure \(\PageIndex{10}\) below.

Figure \(\PageIndex{10}\): Reaction of a donor NDP-monosaccharide and an acceptor monosaccharide with retention or inversion of configuration at the anomeric carbon of the donor

The same stereochemical outcomes are possible for the hydrolysis of acetal bonds by glycosyl hydrolases. Alpha-amylases cleave the α (1,4) glycosidic bonds to produce shorter dextrins (containing branched glucose units not cleaved by alpha-amylases), and α (1,4) linked glucose oligosaccharides of lengths from 2 glucose units (maltose) up to 7-8. This reaction, hence, proceeds with the retention of configuration. In contrast, beta-amylases cleave starch to produce maltose with an inversion of configuration at the anomeric-reducing end of the maltose. We explore the chemistry of retention and inversion more in the next section on cellulase, which cleaves the β (1,4) acetal link in cellulose, but in general, reactions that proceed with inversion react in an S_N2 response, similar to the nucleophilic attack on alkyl halides. For the glycosyl transferases that proceed with inversion, the attacking nucleophile on the acceptor is made more nucleophilic by general base catalysis by a deprotonated glutamic or aspartic acid.

Figure \(\PageIndex{11}\) shows the results of in silicon docking studies of a small glycan, maltotetraose, to AmyBa.

Figure \(\PageIndex{11}\): Molecular docking of AmyBa with maltotetraose. The overall structure and substrate binding pocket analysis of AmyBa are shown. 

AmyBa displayed very high amylase activity compared to other microbial β-amylases, and its enzymatic activity was much closer to that of sweet potato β-amylase. Molecular dynamic and docking programs can be used to calculate binding energies for substrates. The binding energy and enzymatic activities for bacteria and sweet potato β-amylase were highly correlated, suggesting that the extensive interactions of AmyBa and maltotetraose help drive catalysis by using the energy released on binding to lower activation energies for the reaction. 

Saccharification

To enter glycolysis and fermentation, maltose must be converted to the monosaccharide glucose.   The conversion of a polysaccharide to its monomers is called saccharification. To complete the conversion of starch to glucose, another enzyme, glucoamylase (also called amyloglucosidase and γ-amylase), is added. It is an exoglucosidase that cleaves both α (1,4) in amylose, amylopectin, and maltose and α (1,6) branches to form free glucose.    It is a member of the glycoside hydrolase family 15 in fungi, glycoside hydrolase family 31 of human maltase-glucoamylase, and glycoside hydrolase family 97 of bacterial forms.

Fermentation

Glucose (C₆H₁₂O₆) can now enter the glycolytic pathway and continue to ethanol after conversion of pyruvate to acetaldehyde by pyruvate decarboxylase and final conversion of acetaldehyde to ethanol by alcohol dehydrogenase:

C₆H₁₂O₆ (s) → 2 CH₃CH₂OH (l)  +  2CO₂ (g) (anaerobic ethanol biosynthesis)

The yeast Saccharomyces cerevisiae catalyzes this entire pathway.  

The final fermentation process yields a 12-15% ethanol solution, which is distilled to form a 95% ethanol/5% water azeotrope. The water is removed by adding zeolites (molecular sieves), which adsorb water but not ethanol. 

Life Cycle Analysis of Bioethanol:  Is it better than fossil fuels?

We reiterate the promise of bioethanol to address global warming and climate change by presenting again the chemical equations that suggest that its use as a fuel is carbon neutral:

6CO₂ (g)  +  6H₂O (l)  →  C₆H₁₂O₆ (s)  + 6O₂ (photosynthesis)

C₆H₁₂O₆ (s) → 2 CH₃CH₂OH (l)  +  2CO₂ (g) (anaerobic ethanol biosynthesis)

2CH₃CH₂OH (l)  +  6O₂   →  4CO₂ (g)  +  6H₂O (g) (combustion of ethanol)

If only these three equations, this simple model for production and use of corn bioethanol, were the only factors influencing net CO₂ emission on bioethanol burning, there would be no controversy about its use. Yet the actual CO₂ emissions depend on many more hidden from view by these simple equations. A life cycle analysis (LCA) is needed to determine corn ethanol's environmental impact (in this case, net CO2 emissions) through every phase of its existence, from the cradle to the grave, starting with planting corn to using bioethanol for transportation.  

All models must be tested. It's easiest to start with the simplest model. If the data fit the model, great, you're done. If not, new, more expansive models must be developed and tested. Those vociferously supporting bioethanol use often use the simple stoichiometry evident in the three equations to state that bioethanol is carbon neutral. Most would want a detailed life cycle analysis (LCA) before jumping to an immediate conclusion.

LCAs are very challenging, and data on a global scale is required. Some measurements at the worldwide scale have significant uncertainties and are estimates, at best. A recent study looked at the impact of a specific event, the adoption of the US Renewable Fuel Standards (RFS) that regulate biofuels in the US (which produces about half of all the world's biofuels), on CO₂ emission from the significant increase in corn plant and corn ethanol the followed the adoption of the standard. .  Using LCA based on a series of economic and environmental metrics, the model shows that bioethanol is not a panacea for CO₂ emissions and may be more detrimental than fossil fuels use for vehicles.

The study calculated the carbon intensity changes for corn ethanol in the time frame after the standards were adopted. Scientists have used other events that led to immediate changes (9/11) and 1-2 year changes (Covid pandemic) on environmental parameters like CO₂ emissions.

Carbon intensity measures how much CO₂ or CO₂ equivalents are emitted per some unit of activity.  The unit activity can be kWh of electricity generated, megajoule of energy (MJ) in produced fuel,  amount of oil produced, bushels of a particular food produced, or dollars generated (GDP). Ideally, policies should be implemented to decrease carbon intensity. Use of green energy derived from solar and wind lowers carbon intensity. Energy intensity, the total energy production per GDP, is a similar metric. 

Figure \(\PageIndex{12}\) shows carbon intensity per GDP per country over the last 30 years (data from Our World in Data).

Figure \(\PageIndex{12}\):  Consumption-based carbon intensity from 1990 to 2018. Our World in Data.

The world is generally moving toward more efficient energy production, but our energy consumption is still dramatically increasing.

The LCA model showed that the renewable fuel standards (RFS) policy led to these interrelated outcomes. It:

• increased corn prices by 30% and the prices of other crops by 20%
• increased US corn cultivation by 2.8 Mha (8.7%) and total cropland by 2.1 Mha (2.4%) in the years following policy enactment (2008 to 2016). (1 hectare is an area of a square with  100 meters sides, equivalent to 10,000 m²
• increased annual nationwide fertilizer use by 3 to 8%
• increased water quality degradation by 3 to 5%
• increased emissions from domestic land use changes

According to the study, these combined produced a carbon intensity of corn ethanol that was "no less than gasoline and likely at least 24% higher. " 

The changes in the metric are visually described in Figure \(\PageIndex{13}\).

Figure \(\PageIndex{13}\):  Changes due to the RFS. (A) Corn planted area. (B) Cropland area. (C) Carbon emissions. (D) Nitrogen applications. (E) Nitrous oxide emissions. (F) Nitrate leaching. (G) Phosphorus applications. (H) Soil erosion. (I) Phosphorus runoff. Positive numbers indicate an increase due to the RFS. Field-level results were aggregated at the county level for enumeration and visualization.   Tyler J. Lark  et al. PNAS. 119, 2022 (https://doi.org/10.1073/pnas.2101084119) Creative Commons Attribution License 4.0 (CC BY).

Land use changes include farming land that was retired or designated for conservation programs. Tilling additional land releases carbon stored in the soil. The increased farming significantly increased fertilizer production, which leads to N₂O emissions.   In addition, more of the existing cropland was planted with corn. These findings contrast with a USDA study that shows that corn ethanol has a 39% lower corn ethanol intensity than gasoline, which was stated to derive from carbon captures from the newly planted crops. However, that study did not account for emissions from land use changes.

LCA can identify aspects of production that lead to the most negative consequences, which, for the sake of this chapter, is greenhouse gas emissions.  For example, the LCA for corn ethanol might improve if the CO₂ released on its production during anaerobic ethanol biosynthesis could be captured. Outcomes would also change if renewable energy sources were used for stages of production that require fossil fuel use.  These studies also don't include the hazardous pollution released in bioethanol production.  

This rigorous LCA did not address the moral question of using land that could be used to feed people to produce bioethanol for use in our ever-bigger vehicles.  In addition, opponents of solar energy installations suggest they require so much land that they would remove land for agricultural purposes.  What is missing from their argument is the vast amount of land used now for corn ethanol.  Farmers planted 90 million acres of corn in 2022 in the US, a land area about 90% the size of the entire state of California.  44% of that corn went to biofuels, and only 12% went to human consumption.  In addition, approximately 44% percent was used to feed animals for human consumption, an inefficient and unsustainable use of cropland and resources. 

Figure \(\PageIndex{13a}\) below shows how little corn grown in the USA is for human consumption.

Figure \(\PageIndex{13a}\): Corn and Other Feed Grains - Feed Grains Sector at a Glance. Updated 1/5/2025.  

Hannah Ritchie at Our World in Data (the source of many interactive graphs in this chapter) has written eloquently and persuasively about why we should use solar to power electric vehicles than corn bioethanol. Here are some conclusions she draws:

• 20 m² of solar panels produce 1 MWh of electricity per year.  This implies that the 25 million hectares of land now used for corn bioethanol (which covers just 10% of our driving fuel), if covered with solar cells, would produce 12,500 TWh.  That is about 3 times the USA's annual electricity consumption in 2023.
• Considering just transportation, the main use of biofuels, 1 hectare of solar panels, would allow electric vehicles to travel 3 million km (1.87 million) miles. In contrast, 1 hectare of corn for bioethanol allows cars to travel 22,000 km (13,700 miles).  
• People drive approximately 5 trillion km = 3.1 trillion miles annually in the USA. 1.5 million hectares of solar panels would fulfill that energy need (just 6% of the 25 million hectares of land used to produce corn bioethanol, which powers just 10% of our driving needs.

Using agricultural land for crops and solar panels would be a win for all. 

Production of sucrose and bioethanol from sugarcane

Like corn, sugar cane, a tropical, perennial grass, is used (mainly in Brazil) to produce ethanol. Sugar cane is a C4 plant with a high ability to fix carbon. It is a perennial and does not need replanting yearly, making it a more ideal feedstock than corn for bioethanol production. In 2020, sugar cane was by far the most-produced crop or livestock product in the world (1.87 billion metric tons), followed by corn (1.16 billion metric tons). The production by country for both corn and sugar cane is shown in Figure \(\PageIndex{14}\).

Figure \(\PageIndex{14}\):  Corn and sugar cane production by country. Graphs from Our World in Data. https://ourworldindata.org/agricultural-production#

It might come as a surprise that sugar cane production is so high compared to grain crops that provide nutrition (not just "sweet" calories), but given our addiction to sweet foods, it shouldn't. 

Sugar cane is often harvested manually in developing countries. It is then cut, milled, and mixed with water to extract the soluble sucrose (table sugar). Figure \(\PageIndex{15}\) shows the sugar cane components during extraction.

Figure \(\PageIndex{15}\): Components of Sugar Cane (after Larissa Canilha et al. J Biomed Biotechnol. 2012; 2012: 989572. doi: 10.1155/2012/989572

Sucrose is a nonreducing disaccharide (O-α-D-glucopyranosyl-(1,2)-β-D-fructofuranoside). Its structure is shown in Figure \(\PageIndex{16}\).

Figure \(\PageIndex{16}\): Structure of fructose

Sucrose, Caramel and Molasses

Sucrose decomposes at 186 °C (367 °F) instead of melting (a feared event for organic chemistry students who wish to record melting temperatures in the lab) to form caramel. Molasses is a viscous liquid product derived from refining sugar cane or sugar beets. It is used as a sweetener with its taste properties, and it's a component of brown sugar as well. On a sweetness scale, if sucrose is assigned a value of 100, fructose is 140, high fructose corn syrup is 120-160, and glucose is 70-80.

For bioethanol production, sucrose is degraded by the enzyme invertase to form monomeric glucose and fructose. Invertases are activated during the milling and liquefication of sugar cane, so if sucrose is the desired commercial product, an additional clarification step (heating to 115°C and treating with lime and sulfuric acid) is necessary to prevent hydrolytic cleavage of sucrose.

Bioethanol production from sugar cane sucrose

Bioethanol can be made from either the fibrous lignocellulose remains of the sugar cane, called bagasse, or from water-soluble sucrose. We will describe the production of cellulosic ethanol from field crop stalks, called stover, and leaves, straw, wood chips, and sawdust (all "waste" biomass) in Chapter 31.5. The same principles apply to bioethanol production from bagasse (the solid remains after the juice extraction from sugar cane).   Note that bagasse is often burned to provide energy for sugar cane processing). 

Sugar cane, sugar beets, and sweet sorghum, a C4 plant similar to sugar cane, are also used to produce bioethanol. Sweet sorghum is a very efficient plant for producing biomass through photosynthesis. It grows in temperate and tropical climates, has a short growing period, and is resistant to drought and cold. Its stalks contain free sugars and lignocellulose.

This chapter will focus on bioethanol production from sugar cane sucrose. Again, this is accomplished using yeast (Saccharomyces cerevisiae), which has the enzyme invertase 2 (beta-fructofuranosidase 2 or Saccharase) needed to convert sucrose into sucrose fructose and glucose, which can enter glycolytic and fermentation pathways.  

Invertase, shown in 1842 to invert the stereochemistry of sugars, was first isolated from yeast in 1860. It has a secreted glycosylated homooctameric form and an intracellular form, all products of the same gene. It's a member of Family 32 of the glycoside hydrolases. The structure of the Saccharomyces invertase (SInv) octamer structure is shown in Figure \(\PageIndex{17}\) below.

Figure \(\PageIndex{17}\): Structure of octameric SInv. M.Angela Sainz-Polo et al. JBC, 288, 9755-9766 (2013).  DOI:https://doi.org/10.1074/jbc.M112.446435. Creative Commons Attribution (CC BY 4.0)

Panel a shows a view of the SInv octamer in ribbon (left) and solvent-accessible surface (right) representations, showing each subunit colored differently.

Panel b shows that the octamer is rotated 90°, illustrating that it can be best described as a tetramer of two different kinds of dimers, AB/CD and EF/HG, which are compared by superimposing subunit F on subunit B in c

Even though all eight subunits in the octamer are identical (58.5K, 512 aa), the quaternary structure of the 8-mer can best be viewed as a tetramer of dimers (i.e. 4 dimers pack to form two packed tetramers giving the octamer). The AB and CD  dimers pack in a "closed form" with a narrow active site pocket, allowing a glycan of 3-4 monomers. The EF and GH dimers pack in an "open form" with a wide active site pocket for longer glycans. Of course, our main interest here is in the binding of sucrose.

Figure \(\PageIndex{18}\) shows an interactive iCn3D model of Saccharomyces cerevisiae invertase (4EQV)

Figure \(\PageIndex{18}\): Saccharomyces cerevisiae invertase (4EQV). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...ysQW8YSrvPkus9

The color coding of the subunits is the same as shown in the right top image of Panel A, Figure 16 above.

The GH32 enzymes, including invertase, have a catalytic domain consisting of a 5-bladed β-propeller. Each blade has four antiparallel beta-strands. The blades surround an active site enriched in carboxyl side chains. 

The "closed" form active site of the AB and CD dimers has Phe 388 and Phe 296 at its base, which provide hydrophobic interactions. The "open" form active site of the EF and GH dimers also has a salt bridge between Asp 45 and Lys 385. These are shown in Figure \(\PageIndex{19}\), along with a bound 1-kestose, which is a trisaccharide"sucrose analog" found in vegetables.  It consists of a β-D-fructofuranose connected to β-D-fructofuranosyl and α-D-glucopyranosyl residue at the 1- and 2-positions.

Figure \(\PageIndex{19}\):  Dimer interface at the active site. The octameric SInv active site interfaces are detailed, keeping the same color pattern as above with one subunit being shown in ribbon representation for clarity. Angela Sainz-Polo et al. JBC, 288, 9755-9766 (2013). DOI:https://doi.org/10.1074/jbc.M112.446435. Creative Commons Attribution (CC BY 4.0)

Panel A shows that the AB/CD dimers have tight interactions between the catalytic and β-sandwich domains. Hydrophobic interactions around found at the base of the catalytic pocket through Phe-388 and Phe-296. 

Panel b, by contrast, shows that the EF/GH dimers interact only through their β-sandwich domains. In addition, the catalytic pocket is also paved by a new salt bridge formed between Asp-45 and Lys-385 from the β-sandwich domain, which lines the cavity. A putative 1-kestose molecule is shown in a spherical representation.  

The hydrolysis of sucrose by invertase proceeds with the retention of configuration at the anomeric carbon. An active site, Aspartate 22, acts as a nucleophile to form a glycosylated intermediate (fructose-Asp). This is followed by hydrolysis of the intermediate. An active site, Glutamate 203, acts as a general acid/base. The fructose could also be transferred to another glycan in a transglycosylation reaction. The hydrophobic side chains Phe-388 and Phe-296 line the base of the active site pocket.

Figure \(\PageIndex{20}\) shows an interactive iCn3D model of the AB dimer of Saccharomyces cerevisiae invertase with key active site residues (4EQV)

Figure \(\PageIndex{20}\): AB dimer of Saccharomyces cerevisiae invertase with key active site residues (4EQV). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...hX9FbxLLip1f97

 

Figure \(\PageIndex{21}\) shows an interactive iCn3D model of the EF dimer of Saccharomyces cerevisiae invertase with key active site residues (4EQV). It has an additional salt bridge between Asp-45 and Lys-385.

 

 

Figure \(\PageIndex{21}\): EF dimer of Saccharomyces cerevisiae invertase with key active site residues (4EQV). (Copyright; author via source).
Click the image for a popup or use this external link: https://structure.ncbi.nlm.nih.gov/i...Dh33BtT5c8GXK6

Life Cycle Analysis of Sugar Cane Ethanol

Does the production of bioethanol from sugar cane lead to lower net CO₂ emissions than bioethanol produced from corn? The answer would depend on whether sucrose (first generation) or lignocellulose (second generation) from bagasse is the feedstock.  

A recent LCA has been performed on the first-generation (feedstock is sucrose) production of bioethanol from sugar cane in Ecuador. This sugar-based feedstock has a lower production cost. It requires just grinding and the addition of yeast for fermentation; it does not require a saccharification step. 

Figure \(\PageIndex{22}\) shows the various stages and processes used to perform LCA on the bioethanol production from sugar cane sucrose.  It's presented to show the complexity of such analyses, so look at the details only if you are especially interested. 

Figure \(\PageIndex{22}\): Anhydrous ethanol life cycle system boundaries and main product flow quantification for 2018.  Arcentales-Bastidas, D.; Silva, C.; Ramirez, A.D. The Environmental Profile of Ethanol Derived from Sugarcane in Ecuador: A Life Cycle Assessment Including the Effect of Cogeneration of Electricity in a Sugar Industrial Complex. Energies 2022, 15, 5421. https://doi.org/10.3390/en15155421.   Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)

The study analyzed four stages, agricultural, milling, distillation, and electricity generation (presumably by burning the byproduct bagasse), for impacts. They defined two functional units:

• 1 ton of sugarcane "at the farm gate” for the agricultural stage;
• 1 L of ethanol "at the plant (factory) gate”.

The key results of the study are as follows:

• The global warming potential (GWP) impact at the "farm gate" was 53.6 kg of carbon dioxide equivalent (kg CO₂ equiv) per ton of sugarcane produced. This arises mainly from N₂O (34%), a potent greenhouse gas released in the process, and diesel fuel used in agricultural machinery (24%). 
• The GWP for 1 L of ethanol produced at the plant gate was 0.60 kg CO₂ equiv, with the distillation phase contributing the most.

Before proceeding further, let's explain the key term, global warming potential (GWP), widely used in LCA. It adds the contribution of other greenhouse gases like methane (CH₄) and nitrous oxide (N₂O), each with unique IR absorption spectra and atmospheric half-lives.  The IPCC uses a 100-year time frame for the calculation of the GWP and uses this formula:

CO₂ Equivalent kg = CO₂ kg + (CH₄ kg x 28) + (N₂O kg x 265)

\begin{equation}
\mathrm{CO}_2 \text { equivalent } \mathrm{kg}=\mathrm{CO}_2 \mathrm{~kg}+\left(\mathrm{CH}_4 \mathrm{~kg} \times 28\right)+\left(\mathrm{N}_2 \mathrm{O} ~k g \times 265\right)
\end{equation}

• CO₂ has a GWP of 1 by definition since it is the reference.  Its time frame in the atmosphere (100s  to 1000 years) doesn't matter since it is the reference.
• CH₄ has a GWP of around 27-30 over 100 years, reflecting its higher IR absorbance but lower lifetime (around 12 years).  
• N₂O has a GWP of around 265-273 over a 100-year timescale and a lifetime of around 109 years.

The equation can be amended by adding other greenhouse gases released in manufacturing and the use of refrigerants.  These include Freon-12 (Dichlorodifluoromethane) (CFC-12, with a lifetime of 100 years and a GWP100 of 10,200, and SF₆ (used in the electricity industry to keep networks running safely and reliably) with a lifetime of 3200 years and GWP00 of 23,500!

Nitrous Oxide - a potent greenhouse gas but not a laughing matter

N₂O (laughing gas) is an overlooked source of greenhouse gases, but it leads to about 7% of the warming effect of the greenhouse gases with a long lifetime and a high GWP100.  Agricultural practices lead to about 65% of its total emissions.   It is a component of the soil and atmosphere nitrogen cycle.  In soil, its concentration depends on soil microbes that engage in nitrification and denitrification processes.  These depend on the amount of fixed nitrogen, oxygen levels, and metabolically available carbon sources.  The nitrification reaction, which occurs in aerated and moist soils, involves the oxidation of NH₃↔NH₄⁺ to NO₂ and NO₃^-, with some N₂O release. The major source of N₂O occurs under anaerobic conditions.  These general reactions are shown below.

• Nitrification (aerobic, oxidation):  N₂ →  (NH₃ ↔ NH₄⁺) → NO₂ → NO₃^- 
• Denitrification (anaerobic, reduction):  NO₃⁻ → NO₂⁻ → NO → N₂O → N₂

In anaerobic sites, NO is an electron receptor during microbial respiration.  N₂O is produced when excess nitrogen is available (past the needs of plants and microorganisms), so excess use of fertilizers and manure increases production.  Nitrifying and denitrifying bacteria are most active in producing N₂O in environments with abundant N relative to assimilatory demands by other microorganisms or plants (Firestone and Davidson, 1989), as is often the case following soil amendment of fertilizers, manure, or crop residues.  Physical processing of the soil (such as tillage) also affects N₂O emissions by introducing crop residues in the soil, changing soil particle size and surface area, and changing the porosity of the soil.  These affect soil substrate/product availability and their aqueous and gas diffusion rates.

Let's use dimensional analysis from introductory chemistry to convert the GWP from the farm gate/agricultural stage (53.6 kg CO₂ equiv/ton of sugar cane) to kg CO₂ equiv/1L of ethanol (EtOH) so we can add it to the GWP from the plant gate, which is expressed in kg CO₂ equiv/L ethanol produced.  The dimensional conversion is shown in Table  \(\PageIndex{2}\) below.  

53.6 kg CO2 equiv	1 ton SC	1 L Juice	=	0.1 kg CO2 equiv
1 ton SC	800 L juice	0.7 L EtOH		1 L EtOH produced

Table  \(\PageIndex{2}\):  Conversion of 53.6 Kg CO₂ equiv/ton of sugar cane from the farm gate (left-hand column) to 0.1 Kg CO₂ equiv/1L of ethanol (EtOH).

Now, add this to the reported 0.60 kg CO₂ equiv/1 L of ethanol from the plant (factor) gate, and you will get a total of 0.7  kg CO2 equiv/1 L of ethanol produced from sugar cane.  This compares to a reported value of 2.3 kg CO₂ equiv/1 L of gasoline produced, so based on this study, sugar cane-derived bioethanol is significantly better as its manufacturing emits less CO_{2, equiv} than gasoline manufacturing. 

But remember that ethanol is partly oxidized and produces less energy on combustion. Burning 1 L of gasoline releases 33.6 MJ of energy while 21.2 MJ is released on burning ethanol, so we should multiply the 0.7 kg CO2 equiv /1 L of ethanol by 33.6/21.2 = 1.6 to give a normalized carbon intensity = 0.95. This uses the equivalent amount of ethanol (1.6 L) to give the same energy as 1 L of gasoline.  Still, burning sugar can bioethanol is better than burning gasoline based on the release of CO₂ equiv.

 

The promise of bioethanol is based on the notion that 1 C atom was used to create it, and 1 C atom would be released during its combustion, as illustrated in the equations we saw above (and presented again below).

Eq 1:  6CO₂ (g)  +  6H₂O (l)  →  C₆H₁₂O₆ (s)  + 6O₂ (photosynthesis)

Eq 2:  C₆H₁₂O₆ (s) → 2 CH₃CH₂OH (l)  +  2CO₂ (g) (anaerobic ethanol biosynthesis)

Eq 3:  2CH₃CH₂OH (l)  +  6O₂   →  4CO₂ (g)  +  6H₂O (g) (combustion of ethanol)

Now add to this series of net zero CO₂ reactions the release of CO₂ from the manufacture of bioethanol, and you can see that the notion of net 0 for biofuels is misleading.  Based on the analyses above, it appears that burning bioethanol from sugar cane produces significantly less CO₂ equiv than gasoline, but it's nowhere near net 0.  Biofuel use can play a big part in reducing emissions when they replace gasoline, but their use must be coupled with other ways to reduce emissions (such as solar and wind) to produce the magnitude of the reductions we need.  It also appears that the jury is still out about the environmental benefit of using corn bioethanol.

The LCA analysis described above reflects the global warming potential for using sugar cane sucrose for bioethanol production.  However, bioethanol production from sugar cane juice has other negative impacts, as listed in Table  \(\PageIndex{4}\) below.

Table  \(\PageIndex{4}\): Impact categories included in the LCA 

Impact Category	Characterization Factor	Reference Unit
Climate change	Climate change—GWP100	kg CO2eq.
Freshwater eutrophication	Freshwater eutrophication potential—FEP	kg Peq.
Marine eutrophication	Marine eutrophication potential—MEP	kg Neq.
Abiotic depletion	Metal depletion—MDP	kg Feeq.
Photo oxidant formation	Photochemical oxidant formation potential—POFP	kg NMVOCeq.
Particulate matter emissions	Particulate matter formation potential—PMFP	kg PM10eq.
Terrestrial acidification	Terrestrial acidification potential—TAP100	kg SO2eq.

 

Summary

This chapter examines the intricate relationship between the global carbon cycle and bioenergy production, with a focus on bioethanol as a renewable fuel alternative. It begins by contrasting the advantages of fossil fuels—energy-dense, abundant, and easily stored—with their severe drawbacks, including air pollution and climate change. The text then introduces bioethanol, highlighting that although it is derived from renewable sources and is theoretically carbon neutral (when considering photosynthesis and combustion reactions), its production is energy- and land-intensive, and it does not completely offset CO₂ emissions.

Key points include:

• Fuel Energy Comparisons:
  A table compares the standard enthalpies of combustion (ΔH₍c₎°) for various fuels, illustrating that while hydrogen offers the highest energy release per gram, its storage and production challenges make ethanol a more viable option in the near term.

• The Concept of Carbon Neutrality:
  The chapter outlines the biochemical pathway for bioethanol production—from the fixation of CO₂ via photosynthesis to the fermentation of sugars by yeast, culminating in ethanol combustion that theoretically returns the same CO₂ to the atmosphere. However, practical challenges, such as energy input during production and land use impacts, complicate the net carbon balance.

• Quantitative Analysis of the Carbon Cycle:
  Detailed discussions cover the quantification of carbon stocks (in gigatons of carbon, GtC) and the calculation of fluxes (GtC/yr) between the atmosphere, oceans, and terrestrial systems. Conversion factors are provided to interconvert between GtC, gigatons of CO₂, and atmospheric parts per million (ppm), which are essential for modeling the global carbon budget.

• Bioethanol Production Processes:
  The production of ethanol from both corn and sugar cane is explored. For corn, the chapter explains how starch is hydrolyzed through enzymatic liquefaction and saccharification, while for sugar cane, the focus is on the extraction of sucrose followed by fermentation. The role of yeast in anaerobic ethanol biosynthesis is detailed, along with structural insights into the key enzymes involved.

• Enzymology and Structural Insights:
  The chapter includes discussions of enzymes such as alpha-amylase, beta-amylase, and invertase, providing structural representations and interactive models. These enzymes are critical for breaking down complex carbohydrates into fermentable sugars, illustrating the biochemical underpinnings of bioethanol production.

• Life Cycle Analysis (LCA) and Environmental Impacts:
  A comprehensive LCA is presented for sugar cane-derived bioethanol, examining not only the direct CO₂ emissions from fermentation and combustion but also the hidden emissions from agricultural practices, land use changes, and processing. The concept of global warming potential (GWP) is introduced to compare the environmental impacts of bioethanol and gasoline, revealing that although sugar cane ethanol has a lower carbon intensity, it is not entirely carbon neutral.

• Broader Socio-Economic and Energy Considerations:
  The chapter concludes by addressing the societal implications of bioethanol production, including the competition for arable land between fuel and food production, and contrasts bioethanol with alternative renewable energy sources such as solar power. It highlights the need for integrated energy strategies that reduce reliance on fossil fuels while considering economic, environmental, and social factors.

Overall, this chapter provides junior and senior biochemistry majors with a multidisciplinary understanding of the carbon cycle, the chemistry and biochemistry of bioethanol production, and the complexities involved in evaluating its environmental impact through quantitative and life cycle analyses.