Corn Fractionation

The germ, pericarp fiber, and endosperm fiber can be recovered as coproducts in addition to ethanol and DDGS through dry-grinding ethanol process or a modified dry-grind process (E-Mill)

Removal of the suspended solids improves both the fermentation rate and ethanol productivity because such solids interfere with the enzyme kinetics, heat transfer and mixing of the mash. In addition, other benefits can be obtained as follows:

   • Recovery of germ as valuable coproducts that can increase plant capacity and protein content of DDGS

   • Recovery of coarse fiber as valuable coproducts that can be used for the production of corn fiber oil,corn fiber gum, as well as corn ethanol, resulting in an increase in capacity of plant,protein content of DDGS, and reduction in fiber content of DDGS.

  • In Enzymatic Milling Process, recovery of pericarp and endosperm fiber as valuable coproducts can increase plant capacity and protein content of DDGS as well as reduce significantly fiber content in DDGS. In addition, the use of GSH enzymes during E-milling can results in a synergy effects, i.e. improving the fractionation process and converting starch into dextrins and sugars for fermentation.

Granular Starch Hydrolysis

Granular Starch Hydrolysis with external enzymes or corn with expressed enzymes:


• Use granular starch-hydrolyzing (GSH) enzymes (e.g., Stargen from Genencor or BPX from Novozymes) to convert starch into dextrins at temperatures lower than 48 C and hydrolyze dextrins into fermentable sugars during SSF


• Not require heating of the corn slurry to high temperatures for cooking or liquefaction; therefore, GSH enzymes reduce the overall utility requirements of the dry grind process.


• With GSH enzyme, the liquefaction, saccharification, and fermentation steps can all be combined into one single step. Compared to conventional enzyme treatments,
      o No increase in viscosity of the corn slurry that occurs during gelatinization and cooking; Therefore, higher concentrations of solids can be used in corn slurries, which allows the fermentation to reach increased final ethanol concentrations
     o Glucose concentrations with GSH enzymes are typically lower but the final ethanol concentrations and ethanol yields remain similar.
     o glycerol concentrations are lower for GSH treatment so that yeast cells are thus subjected to a low osmotic stress; thus improving the overall productivity by lowering the production of glycerol


• The cost of GSH enzymes is approximately double that of conventional enzymes; but overall material, capital, and operational cost may be advantageous.


In addition, if using enzyme expressed corn seeds as feedstocks, the cost of enzyme will be signifcantly low.

High Gravity Fermentation

A high final ethanol concentration during ethanol production will improve the plant profitability by reducing water usage, increasing the plant capacity, and decreasing the downstream processing costs, thereby improving plant efficiency. High final ethanol concentrations (i.e., >14 %) can be achieved by high-gravity fermentation. At a high initial dry solids content (~33 %), it is particularly important to ensure that the nutritional needs of the yeast are appropriately met as a preventive measure to avoid stuck or sluggish fermentations.

It has been found that temperature staging (33 C during early stage and 28 C or lower during later stages) is also important to complete a high-gravity mash; The addition of proteases, urea, Mg2+, yeast extracts, and amino acids, have all been reported to provide useful nutrients to yeasts, thereby increasing fermentation rates and final ethanol concentrations in high-gravity mashes.

Dry-mill corn ethanol process

 In a conventional dry-grind process, corn is ground and mixed with water to produce slurry. The slurry is cooked; starch in the slurry is liquefied, saccharified, and fermented to produce ethanol. The remaining nonfermentables in corn (germ, fiber, and protein) are recovered together at the end of the dry-grind process as an animal food coproduct called distiller dried grains with solubles(DDGS). Due to its lower capital and production investments than wet-mills, it is still the dominant process for corn ethanol production nowadays. Conventionally, the process involves the following steps:

Milling. The feedstock (Corn) passes through a hammer mill to break down the corn kernel into a fine powder.

Cooking. The fine powder flour is mixed with water to obtain ground corn slurry with ~27-37% solids content. The pH of the ground corn slurry can be adjusted with anhydrous ammonia to stabilize within the range 5.5 to 6.5. The slurry is then heated to 85 C (185_F) for 30-45 min, and subsequently cooked with high pressure steam in a jet cooker at 104 C (220 _F). After cooling to 85 C (185 F) and a holding period of 30–45 min, the slurry is reheated and cooked to gelatinize the starch and break down its crystalline structure. The resulting mixture of amorphous starch is called the ‘mash.’

Liquefaction. A unit operation involving an enzymatic digestion of the starch molecules in the mash into imonoeric sugar and oligosaccharides. In this step, alpha-amylase (an endoenzyme ) applied randomly hydrolyzes a-1,4-glucosidic bonds to reduce the viscosity of gelatinized starch, producing soluble dextrins and oligosaccharides.

       • The total alpha-amylase dose:0.2 to 0.4 kg enzyme per metric ton ofvdry solids (0.02–0.04 %, w/w).

       • The common practice: add one-third of the alpha-amylase prior to cooking and the remainder after the jet cooker step. The resulting liquefied corn mash has a typical composition as: maltotetraose and other soluble dextrins 24–35%; maltotriose 1.7–3.3%; maltose 0.4–2.7%; and glucose 0.1–1.3 %; i.e. a starch hydrolysate that has a dextrose equivalent ranging from 10.4 to 19.1 (The dextrose equivalent is a measure of the percentages of glucosidic bonds that are hydrolyzed during liquefaction.)

Saccharification. After the corn mash cools to 32 C, a second enzyme, glucoamylase (,an exoenzyme) is added (0.05–0.08 %, w/w) to catalyze the release of successive glucose units from the non reducing ends of soluble dextrins by hydrolyzing both linear a-1,4-glucosidic and branched a-1,6-glucosidic linkages, resulting in the fermentable sugars, mainly glucose.

Fermentation. The glucose generated by glucoamylase during saccharification step will be fermented into ethanol by yeasts (typically Saccharomyces cerevisiae) and carbondioxide as a by-product. Saccharomyces cerevisiae is the yeast species commonly selected because of its quick, efficient production of alcohol and its ability to withstand heat, osmotic stress and high alcohol concentrations. The fermentation process generally takes about 50 to 60 hours with a final ethanol concentrations of14%- 20% (v/v) depending on the corn slurry solids contents from improved fermentation process. Batch or continuous fermentation systems may be used, although batch processing is more common due to its simple, easily cleaning, and flexibility. Some new fermentation systems are designed to minimize dilution water, which reduces the evaporation requirements in the feed processing stages after fermentation.

Distillation. Distillation is the process of separating the ethanol from the solids and water in the mash. The fermented mash, now called “beer”, is pumped to the continuous flow, multi-column distillation system where the ethanol is removed from the solids and the water. Conventional distillation/rectification methods can produce 95% pure (190 proof) ethanol because the ethanol and water form an azeotrope so that further separation by heat cannot occur. Therefore, the ethanol leaves the top of the final column in the distillation syetem at about 95% concentration, and the residue mash (stillage), is transferred from the base of the column to the co-product processing area.

Dehydration. The ethanol from the top of the column passes through a dehydration system to remove remaining water to obtain anhydrous ethanol (pure, without water, and approximately 200 proof). Most modern dry-grind ethanol plants use a molecular sieve system to produce pure ethanol.

Stillage Processing. The solid and liquid fraction remaining after distillation is referred to as “whole stillage”, which includes fiber, oil and protein components of the grain, and the non-fermented starch.

      The “thin stillage” is first separated from the insoluble solid fraction using centrifuges or presses/extruders and then sent to evaporator units to remove excess water to obtain the thick, viscous syrup, which is mixed back with the solids to create a feed product known as Wet Distillers Grains with Solubles (WDGS) with about 65% moisture.

     Due to its low shelf life and high transportation cost, WDGS is usually dried to 10 to 12% moisture to produce a product known as Dried Distillers Grain with Solubles (DDGS). Although drying distillers grains is energy-intensive, consuming about one-third of the energy requirements of the entire dry-grind plant, , it is essential to produce a uniform, stable, high-quality feed co-product to the profitability of the plant, resulting in most plants producing DDGS rather than WDGS.

Typical Yield from a Bushel of Corn from a Dry-Mill Ethanol Plant

• 2.7 gallons of ethanol
• 17.5 pounds of distillers dried grains
• 17 pounds of carbon dioxide

Inhibitors

Inhibitors based on their chemical functional groups can be grouped as aldehydes, ketones, phenols, and organic acids.

  • Aldehyde inhibitors: compounds with one or more functional aldehyde groups, regardless of the base structure of a furan ring, a benzene ring or a phenol-related structure, including
      o furfural and HMF
           Cell walls and membranes of yeast cells grown under furfural and HMF-challenged conditions appear damaged when compared to those of controls grown in the absence of any inhibitor, resulting in the delay of cell growth and reduction of ethanol productivity
           Furfural and HMF inhibit cell growth and ethanol production rates at lower concentrations. Individual strains have been isolated that retain their ability to produce ethanol in the presence of 10 to 79 mM of either furfural or HMF, including strains of these species: Saccharomyces cerevisiae, Pichia stipitis, Candida shehatae, Corynebacterium glutamicum, Zymomonas mobilis, and Escherichia coli.
      o 4-hydroxybenzaldehyde, vanillin, syringaldehyde, isovanillin,ortho-vanillin, Cinnamaldehyde
         more inhibitory than those derived from sugar dehydration

• Ketone inhibitors:4-hydroxyacetopheone, acetovanillone and acetocsyringone
         exert a greater inhibitory effect on bacteria such as Thermoanaerobacter mathranii than on yeasts, in terms of reduced growth and ethanol yield

• Organic acid inhibitors: sharing a common carboxylic acid functional group, all contain a carboxyl functional group such as
       o acetic acid,formic acid, levulinic acid, caproic acid,furoic acid, 4-hydroxybenzoic acid, 3-hydroxybenzoic acid, 2-hydroxybenzoic acid, 2,5-dihydroxybenzoic acid, protocatechic acid, vanillic acid, gallic acid, syringic acid, 4-hydroxycinnamic acid, homovanillic acid, guaiaclyglycolic acid, and sinapic acid. These inhibitors are thought to be exert their inhibitory actions via their carboxyl functional groups.
             more toxic to isolates of bacteria than yeasts at low concentration
             The toxicity has been correlated with their degree of hydrophobicity, suggesting the involvement of a hydrophobic target such as the cell membrane

• phenol-based inhibitors: phenol, benzene-1,2-diol (catechol) etc.
      o cause increased membrane fluidity and affect membrane permeability, which may enhance synergistic inhibition when combined.
      o Phenols such as cathecol, hydroquinone, and coniferyl alcohol almost completely inhibit E. coli , but are relatively less toxic to yeast. However, eugenol and isoeugenol are inhibitory to yeasts at low concentrations.
     o The three main phenol structure building blocks in lignin show the order of inhibitory effects, ranked from strong to weak, as (1) hydroxyphenol, (2) guaiacyl, and (3) syringyl

In general, aldehydes and phenols are more toxic than organic acids.

In addition, some inhibitions will be amplified through process. A typical example is that fermentative microorganisms will be inhibited by concentrated non-volatiles such as lignin derivatives and extractives after concentrating although low initial inhibitor concentration in hydrolyzate.

Van Krevlen diagram: A useful means of comparing biomass and fossil fuels for their heating values

H/C and O/C ratio for several solid fuels (Van Krevelen diagram).

The lower the respective H/C and O/C ratios,  the greater the energy content of the material, because the lower energy contained in carbon–oxygen and carbon–hydrogen bonds, than in carbon–carbon bonds.

On the way to green fuels

We need make a sound judgement before making a decision.
Read, listen, consult, think, judge, plan, and act!

Important factors that can not be overlooked by using biomass as renewable energy sources

It is believed that burning biomass will not contribute carbon dioxide to the atmosphere because replanting harvested biomass ensures that CO2 is absorbed and returned for a cycle of new growth, i.e biomass emits roughly the same amount of carbon during conversion as is taken up during plant growth. But people have to realize that there is the time lag between the instantaneous release of CO2 from burning biomass energy and its eventual uptake as biomass, which can take many years. Therefore, there is a need to recognize this time delay and take appropriate action to mitigate against the lag period. Most imortantly, the action of consuming biomass resources for fuel  and replacement planting needs to occur concurrently so that the overall resultant from the use of biomass does not contribute to a build up of CO2 in the atmosphere from a long term.

Unforfunately, not amny people realize the time lag and many countries have not implement a programme of replacement planting when they already started harvesting the biomass.

Analytical Methods for Mass Balances

Biomass compositional data is essential to beginning any study, especially the carbohydrate contents (both soluble and  structural carbohydrates) that is the base to calculate theoretical ethanol and sugars yields, to close carbohydrate mass balances, to determine the enzyme loadings, and to calculate the process efficiencies.

1. Waxes and fats are extracted into either ether or hexane using sonication.
2. The soluble sugars are subsequently extracted in 80% (v/v) ethanol, a concentration which is selective for monosaccharides
3. Wateris then used to extract any water-soluble polysaccharides (e.g., fructans)
4. Starch is then removed by using amylase in acetate buffer

The final residual material is essentially pure plant cell walls, which is called "structural carbohydrates", including hemicellulose and cellulose. The structural carbohydrates composition will be analysized following two-step acid hydrolysis using H2SO4.

As a complete compositional analysis is labor-intensive, it is often more practical to measure selected components only. Soluble sugars can be extracted by sonication in water if the samples are not to be processed for determining starch contents. For total carbohydrate analysis (including soluble sugars), a two-stage acid hydrolysis procedure similar to NREL protocol can be directly applied to native samples

Cellulosic biomass pretreatment reactor

A news from Biorefiningmagazine.com reported that  a bench-scale horizontal reactor designed and manufactured by AdvanceBio Systems LLC will be in use by Penn State University for their cellulosic pretreatment research. This is great! If looking at the reactors that have been used in bench top studies on biomass pretreatment, most of them can not be use to run the large particle size (un-milled biomass) with good high shear mixing at high solids content.

Structural features lead to two-phase enzymatic hydrolysis for mild pretreated biomass

The limiting factors that affect enzymatic hydrolysis of biomass have been traditionally divided into two groups: substrate-based and enzyme based. The substrate-based factors mainly involve chemical structural features such as the compositions of cellulose, hemicellulose, lignin, and side groups bound to hemicellulose and physical structural features that consist of accessible surface area, crystallinity, the physical distribution of lignin in the biomass matrix, degree of polymerization, pore volume, and biomass particle size.

For mild pretreated biomass, the inital rate of enzymatic hydrolysis will be influenced by the transport of enzymes and the dominant hydrolysis will be on amorphous cellulose. At this phase, the hydrolysis of hemicellulose will correlate with the slow increase of glucose yield. When the hemicellulose removal to some point, say 50% removal, a slight hemicellulose hydrolysis will lead to significant increase in cellulose hydrolysis, i.e. crystal cellulose hydrolysis: A transition point occurs, demonstrating the main or 2nd phase hydrolysis.

At 1st phase, lignin content and side groups in hemicellulose play an important role. Any treatments to remove lignin and peel off side groups and break down hemicellulose will improve enzyme transport though opened channels; At the 2nd phase,cellulose crystallility become the limiting factor, any destruction of cellulose crystallility will speed up the hydrolysis and increase the yield.

Whole slurry hydrolysis: an approach to produce cheap sugar

Almost all of currently reported  biomass pretreatments are those reqyiring post- liquor/solid separation or washing,  or nutralization, or even detoxification before further processing (hydrolysis), which will add additional cost due to the more step unit operations. A cost-effective pretreatment should be low demand of post- pretreatment processing. A "whole slurry" processing or hydrolysis will be ideal case. Therefore, the pretreatment should not be to acidic or too basic, or too harsher that generate too much toxic compounds. I am working on a process that really can realize this goal.

Ionic liquids pretreatment of biomass

Recently more research are conducted on Ionic liquids (< 140 C) pretreatment of biomass, demonstrating

• Converts Cellulose I to cellulose II, improving cellulose digestibility
• Deferulylation of biomass, removal of lignin
•  Cellulose swelling, destruction of cellulose crystal
•  Work for mixed feedstocks-flexibility on biomass resources

Recovering sugars from ILs has been alos investigated. More questions remain on IL pretreatment:

• Is it effective on un-mill biomass such as wood chips and 1-2 inche agricultural residues?
• What about the moisture of wet biomass (in reality, it is hard to dry the biomass with moisture content < 5%) on IL stability?
• The recovery yield? Can be great than 99%?
• Any inhibition or toxic on enzymes and fementation microorganisms?

Benefits from Surfactants (Tween and BSA) During Enzymic Hydrolysis

It appears that Tween improves biomass enaymatic hydrolysis through three effects: enzyme stabilizer, lignocellulose disrupter, and enzyme effector. BSA treatment can improve both cellulase and beta-glucosidase activity due to the non-specific competitive, irreversible adsorption of BSA on lignin.

However, all of the research were based on the current leading pretreatment methods. Scince these pretreatments are really cost non-competitive. Any slight improvements in hydrolysis yield and reduction in enzyme loading is negligible compared with the cost of pretreatment and large scale enzyme production.

A cost effective pretreatment must be developed, which does not require harsher conditions (very acidic and very alkaline, and very high temperature). Is it possible? Yes, if we think the chemistry carefully!

When reading the most recently published review paper on pretreatments, nothing new included. I do not understand that there are so many people working in this field, but most of them just copy the ideas and focus on those so-called leading pretreatments.

I think more education on biomass chemistry is really need to train people/students to develop new approches/solve problems before fundemetal understanding of the problems.