BIOCHEMICAL CONVERSION PROCESS

Introduction

Today, there are four conversion technologies currently available, each

appropriate for specific biomass types and resulting in specific energy products:

1. Thermal conversion is the use of that, with or without the presence of oxygen, to
convert biomass materials or feedstocks into other forms of energy. Thermal
conversion technologies include direct combustion, pyrolysis, and torrefaction.
2. Thermochemical conversion is the application of heat and chemical processes in
the production of energy products from biomass. A key thermochemical
conversion process if gasification.
3. Biochemical conversion involves use of enzymes, bacterial or other
microorganisms to break down biomass into liquid fuels, and includes anaerobic
digestion and fermentation.
4. Chemical conversion involves use of chemical agents to convert biomass into
liquid fuels.

Biochemical conversion of biomass involves use of bacteria, microorganisms and

enzymes to breakdown biomass into gaseous or liquid fuels, such as biogas or
bioethanol. The most popular biochemical technologies are anaerobic digestion (or
biomethanation) and fermentation. Anaerobic digestion is a series of chemical
reactions during which organic material is decomposed through the metabolic
pathways of naturally occurring microorganisms in an oxygen depleted environment.
Biomass wastes can also yield liquid fuels, such as cellulosic ethanol, which can be
used to replace petroleum based fuels. The use of microorganisms for the
production of ethanol is an ancient art. However, in more recent times such
organisms have become regarded as biochemical factories for the treatment and
conversion of biological materials. Fermentation technologies, with the assistance of
biological engineering, are leading to breakthrough processes for creating fuels and
fertilizer, and products useful in agriculture.
Anaerobic Digestion

Anaerobic digestion is the use of microorganisms in oxygen free environments

to break down organic material. Anaerobic digestion is widely used for the production of
methane- and carbon rich biogas from crop residues, food scraps, and manure
(human and animal). Anaerobic digestion is frequently used in the treatment of
wastewater and to reduce emission from landfills.

Anaerobic digestion involves a multi stage process. First, bacteria are used in
hydrolysis to break down carbohydrates, for example, into forms digestible by other
bacteria. The second set of bacteria converts the resulting sugars and amino acids into
carbon dioxide, hydrogen, ammonia and organic acids. Finally, still other bacteria
convert these products into methane and carbon dioxide. Mixed bacterial cultures are
characterized by temperature range, for example, above 0 oC and up to 60oC. When
functioning well, the bacteria convert about 90% of the biomass feedstock into biogas
(containing about 55% methane), which is a readily useable energy source.

Solid remnants of the original biomass input are left over after the digestion process.
This by product, or digestate, has many potential uses. Potential uses include fertilizer
(although it should be chemically assessed for toxicity and growth inhibiting factors
first), animal bedding and low grade building products like fiber board.

Biofuel Production

A variety of fuels can be produced from waste resources including liquid fuels,
such as ethanol, methanol, biodiesel, Fischer Tropsch diesel, and gaseous fuels such
as hydrogen and methane. The resource base for biofuel production is composed of a
wide variety of forestry and agricultural resources, industrial processing residues, and
municipal solid and urban wood residues. Globally, biofuels are most commonly used to
power vehicles, heat homes, and for cooking.

The largest potential feedstock for ethanol is lignocellulosic biomass wastes,

which includes materials such as agricultural residues (corn stover, crop straws, and
bagasse), herbaceous crops, short rotation woody crops, forestry residues, waste
paper, and other wastes. Bioethanol production from these feedstocks could be an
attractive alternative for disposal of these residues. Importantly, lignocellulosic
feedstocks do not interfere with food security.

Ethanol from lignocellulosic biomass is produced mainly via biochemical routes.

The three major steps involved are pretreatment, enzymatic hydrolysis, and
fermentation. Biomass is pretreated to improve the accessibility of enzymes. After
pretreatment, biomass undergoes enzymatic hydrolysis for conversion of
polysaccharides into monomer sugars, such as glucose and xylose. Subsequently,
sugars are fermented to ethanol by the use of different microorganisms.

Polysaccharides Hydrolysis

Biomass contains complex sugars or polysaccharides such as cellulose.

Polysaccharides are composed of monosaccharides linked together by glycosidic
bonds. These bonds can be cleaved by hydrolysis. The hydrolysis of polysaccharides to
soluble sugars is called saccharification. In this process, enzymes are used to
hydrolyze the glycosidic bonds of a polysaccharide. These enzymes are called
glycoside hydrolases or glycosidases.

Malt made from barley is used as a source of -amylase to break down starch
into the disaccharide maltose, which can be used by yeast to produce beer. Other
amylase enzymes may convert starch to glucose or to oligosaccharides. Cellulose is
first hydrolyzed to cellobiose by cellulase and then cellobiose is further hydrolyzed to
glucose by beta-glucosidase. Animals such as cows (ruminants) are able to hydrolyze
cellulose into cellobiose and then glucose because of symbiotic bacteria that produce
cellulases.

Chemistry of Pretreatment

Because of the recalcitrant nature of cellulose that is embedded in the plant cell
wall structure, a pretreatment is a key step in the utilization of biomass. Pretreatment
greatly increases the rates and extents of enzyme action in breaking down cellulose to
fermentable sugars by improving the accessibility of structural carbohydrates in the cell
wall. Yields of fermentable sugars from untreated lignocellulosic materials are low
because of the highly packed cellulose structure from acid or enzymatic hydrolysis.

Maximizing the use of all lignocellulosic material that is capable of yielding simple
sugars is essential for improving yield, for example in the production of ethanol, and
lowering the cost of its production. Hence, pretreatment of lignocellulosic material is
required to improve the hydrolytic efficiency of cellulose by removing and hydrolyzing
hemicellulose, by separating the cellulose from the lignin, and by loosening the structure
of cellulose and thereby increasing its porosity.

Among the various pretreatment methods, hydrothermolysis with steam or water

has been shown to be effective in removing and solubilizing hemicellulose and thus in
improving hydrolytic efficiency. Hot-water pretreatment of lignocellulosic biomass at a
controlled pH effectively dissolves hemicellulose and some of the lignin and minimizes
the formation of monosaccharides and other coproducts that could interfere with
biological processes downstream. For example, monosaccharides inhibit cellulase in
the hydrolysis of cellulose downstream. The sugars could degrade further to form such
toxic substances as furfural during the pretreatment step. Other pretreatments are
similarly effective, and they use acid, bases, ammonia, or other materials. Several of the
promising pretreatment methods have been demonstrated on a pilot scale, but the
lowest-cost approach is yet to be determined.

Chemical Degradation & Inhibitors formed during Pretreatment

While the aims of pretreatment are to uncover the cellulose for enzymatic
saccharification and fractionating the main components of the feedstock, pretreatment
often involves side reactions resulting in lignocellulose-derived by-products that are
inhibitory to downstream biochemical processes. Inhibition problems become more
significant as the by-products accumulate as a result of recirculation of process water,
or as their concentration increases when high solids loadings are used to achieve
concentrated sugar streams and high product titers..
 Since the formation of inhibitory substances is much dependent on the
pretreatment process, this review includes a brief discussion on the most commonly
used pretreatment techniques.

Pretreatment
Main Effect Used Chemicals By Product
Methods
Hydrolysis of Aliphatic carboxylic
Acid based H2SO4, SO2, HCl,
hemicelluloses to acids, phenylic
methods H3PO4
monosaccharides compunds, furans
Solubilization of
Acetic acid, minor
Hydrothermal hemicelluloses
No additives amounts of furan
processing without complete
aldehydes
hydrolysis
Acetic acid,
Removal of lignin hydroxyl acid,
Mild alkaline NaOH, Ca(OH)2,
and a minor part of dicarboxylic acids,
methods NH3
hemicelluloses phenolic
compounds
Aldonic and aldaric
Removal of lignin
acids, furoic acid,
Oxidative methods and part of H2O2, O2, O3
phenolic acid, acetic
hemicelluloses
acid
Methods that target Kraft pulping, sulfite
Chemical Pulping lignin and to some pulping, soda
Aliphatic acids
Process extent pulping, organosolv
hemicelluloses pulping
Dissolution of
specific Dependent on
Alternative solvents lignocellulosic Ionic liquids solvent and
components of the conditions
whole biomass
Acid based methods
Acid hydrolysis is one of the most promising pretreatment methods with
respect to industrial implementation. It is usually performed with mineral acids,
 but organic acids and sulfur dioxide are other options. Dilute sulfuric acid
pretreatment has been studied for a wide range of lignocellulosic biomass. It
results in high recovery of the hemicellulosic sugars in the pretreatment liquid,
and in a solid cellulose fraction with enhanced enzymatic convertibility. Acid
pretreatment has also some drawbacks, such as high cost of the materials used
for construction of the reactors, gypsum formation during neutralization after
treatment with sulfuric acid, and formation of inhibitory by-products.
Hydrothermal processing
Hydrothermal processing is an approach in which water in liquid phase or
in vapor phase is used to pretreat lignocellulosics biomass. It is a relatively mild
pretreatment method that does not require any catalysts and does not cause
significant corrosion problems. Under high pressure water penetrates into the
biomass, hydrates cellulose, and removes most of the hemicelluloses and a
minor part of lignin. The solubilization of hemicelluloses is catalyzed by
hydronium ions resulting from water auto-ionization. Controlling the pH around
neutral values minimizes the formation of fermentation inhibitors.
Mild Alkaline methods
Alkaline treatment can be used for removing lignin and thereby increasing
the digestibility of cellulose. Compared to acid and hydrothermal processes, mild
alkaline pretreatments lead to less solubilization of hemicelluloses and less
formation of inhibitory compounds, and they can be operated at lower
temperatures. Sodium hydroxide and potassium hydroxide are the most
commonly used forms of alkali, but their cost is a serious limitation. Other
suitable forms of alkali are calcium hydroxide and ammonia, which can be used
in processes such as lime pretreatment, ammonia recycled percolation (ARP)
and ammonia fiber expansion (AFEX).
Oxidative methods
The use of oxidants for pretreating lignocellulosic biomass allows the
reduction of cellulose crystallinity and disruption of association between
carbohydrates and lignin. These methods include alkaline peroxide pretreatment,
ozonolysis, and wet oxidation. Wet oxidation is achieved by treating biomass with
water and air or oxygen at high temperatures for relatively short times.
Hemicelluloses are extensively solubilized, and recovered mostly as
 oligosaccharides. Lignin is fragmentized and oxidized to aliphatic carboxylic
acids and phenolic compounds. The combination of wet oxidation with alkaline
compounds minimizes the formation of furan and phenolic aldehydes.
Chemical Pulping processes
Although pulping processes are primarily used for manufacturing of paper
and cellulose derivatives, the integration of ethanol production to pulp mills has
been demonstrated at commercial scale. Chemical pulping can be applied to
both softwoods and hardwoods, and the major technologies used are Kraft and
sulfite pulping. In the Kraft process, which is based on NaOH and Na2S, lignin
and parts of the hemicelluloses are degraded into black liquor, which is typically
used for energy purposes. In sulfite pulping, which is based on an aqueous
mixture of bisulfite (HSO3) and sulfite (SO32), the hemicelluloses are
hydrolyzed and removed to the spent sulfite liquor (SSL), while the cellulose is
maintained almost intact. SSLs from softwood are rich in hexoses, and can be
fermented to ethanol by Saccharomyces cerevisiae, whereas those from
hardwoods are more difficult to ferment because of their high pentose content.
Soda pulping is typically used for pulping of non-wood plants with higher
contents of inorganic material than wood. A difference compared to Kraft and
sulfite pulping is that it does not require sulfur-containing chemicals.
In organosolv pretreatment, which was initially investigated as an
alternative to conventional chemical pulping processes, organic solvents are
used for solubilizing lignin. The pretreatment is typically performed at around 200
C, but if acid catalysts are used the process can be run at lower temperatures.
The solvents must be removed from the system to avoid inhibition of enzymatic
hydrolysis and fermentation, and should be recycled to reduce operational costs.
The most economic option is to use low-molecular weight alcohols, but the risk of
operations with volatile and flammable solvents has motivated the interest for
nonvolatile organic compounds.
Alternative solvent pretreatment
The use of ionic liquids (ILs), is another alternative for pretreatment of
lignocellulosics materials. ILs disrupt the non-covalent interactions between
lignocellulose components without leading to significant degradation. Cellulose
regenerated from IL solutions has increased enzymatic convertibility. The
 development of energy-efficient recycling methods, and the implementation of
effective strategies for recovery of hemicelluloses and lignin from pretreatment
liquids is required for the industrial application of ILs. Even though the formation
of inhibitors is limited, the minor amounts of ILs remaining in the pretreated
materials are potentially toxic to enzymes and fermentative microorganisms.

Impact of Formed Inhibitor

1. Inhibition of microorganisms
By-products of pretreatment of lignocellulose under acidic conditions can
be divided into groups on basis of chemical functionality, origin, and effects on
the fermenting microorganism. Carbohydrate degradation products such as the
common aliphatic carboxylic acids acetic acid, formic acid, and levulinic acid, and
the furan aldehydes furfural and HMF exhibit relatively low toxicity, but can be
present in high concentrations depending on the pretreatment conditions and the
feedstock.
A researcher studied the concentrations and effects of acetic acid, formic
acid, and levulinic acid in acid hydrolysates of Norway spruce and in
fermentations with S. cerevisiae. They found that concentrations of around 100
mM were required to observe inhibitory effects. Due to the low acetyl content,
softwood hydrolysates have relatively low concentrations of acetic acid.
Formation of formic acid and levulinic acid occurs at the expense of sugars and it
is therefore desirable to use pretreatment conditions in which the formation of
these acids is minimized. For these reasons the concentrations of aliphatic
carboxylic acids in pretreated softwood may be low enough to stimulate rather
than inhibit ethanol formation. This is the effect of increased demand for ATP
and/or less efficient production of ATP due to uncoupling of the respiratory chain
and the oxidative phosphorylation of ADP, which leads to increased ATP-
generating glycolytic activity at the expense of biomass formation. The use of
hardwood and agricultural residues with high acetyl content as feedstocks as well
as the development of high-solid processes contribute to making inhibition by
aliphatic carboxylic acids more important.
 Aromatic carboxylic acids are found within the group of phenylic
compounds, which include both phenolic aromatic carboxylic acids, such as for
example ferulic acid and 4-hydroxybenzoic acid, and non-phenolic aromatic
carboxylic acids, such as cinnamic acid.
2. Inhibition of cellulolytic enzymes
The catalytic action of cellulolytic enzymes can be inhibited by non-
productive binding to constituents of the solid fraction, such as lignins and
residual hemicelluloses. The positive effect on enzymatic hydrolysis of cellulose
achieved by adding bovine serum albumin could be attributed to prevention of
unproductive binding of cellulase onto lignin.
Inhibition of cellulases is also caused by soluble carbohydrates and
aromatic substances in the pretreatment liquid. Product inhibition of cellulolytic
enzymes by monosaccharides, such as glucose, and disaccharides, such as
cellobiose, is a well-known problem. More recently, the inhibitory effects of
oligosaccharides derived from xylan and mannan have been investigate. The
presence of such oligosaccharides is dependent on the pretreatment method,
and also on the potential inclusion of enzymes that degrade hemicellulose-
derived oligosaccharides in the enzyme preparation.
References:

Martin, C., Jonsson, L.J., Pretreatment of lignocellulose: Formation of inhibitory by-

products and strategies for minimizing their effects. DOI:
https://doi.org/10.1016/j.biortech.2015.10.009

https://www.bioenergyconsult.com/biochemical-conversion-technologies/

https://www.nap.edu/read/12620/chapter/6#124

http://www.wgbn.wisc.edu/conversion/bioenergy-conversion-technologies