LIGNOCELLULOSIC BIOMASS

KARAN KUMARAN
(189101022)
DHRUV KHURANA
(189101037)
INTRODUCTION
Before the onset of the petrochemical era, renewable feedstocks supplied a significant
portion of the global chemical and energy needs. In the 1920–1930s the chemurgy
movement in the USA promoted the use of biomass as a source of chemicals, with the
belief that ‘anything that can be made from a hydrocarbon could be made from a
carbohydrate’. The incentive was to find an economic way to use farm surpluses. It was
only in the relatively short period between 1920 and 1950 that we witnessed the
transition to a nonrenewable- based economy that heavily depends on fossil resources.
However, since the first oil crisis in the 1970s, decreasing resources, global warming and
environmental pollution associated with the use of fossil fuels are growing incentives for
the transition to renewable energy such as solar, wind, hydro and biomass. Among the
renewable resources, lignocellulosic biomass is particularly suited as an abundant, low-
cost feedstock for the production of bio-based chemicals, fuels and energy to substitute

fossil resources. A drawback of the growing consumption of biomass for energy is the
increase in price for the biomass feedstock. This conflicts with the biorefinery
requirement for low-cost raw materials. Biomass alone is probably not able to solve the
world’s power needs, but can satisfy the need for the synthesis of carbon- containing raw
materials because its unique composition makes it especially suitable for the extraction
of value-added chemicals and materials, which can replace petrochemicals. The
production of such chemicals from lignocellulosic biomass has a greater added value
compared with the use of biomass as fuel. Therefore, the production of chemicals from
biomass can contribute a great deal to the economy of a society that uses sun and wind
for power. The added value from biomass- derived chemicals can be used to improve the
economic feasibility of biorefineries.

Hybrid processing
Producing fuels and chemicals from lignocellulosic biomass has been traditionally
achieved through two platform technologies. The first is the biochemical process in
which the biomass is converted into reducing sugars through pretreatment and
enzymatic hydrolysis followed by microbial fermentation into fuel products. The
second is the thermochemical process in which biomass is treated by gasification or
pyrolysis for producing intermediates such as syngas or bio-oil, which are further
upgraded into drop-in fuels. As an alternative to these conventional biomass
conversion processes, the combination of the thermochemical and biochemical
processes creates a new conversion platform: hybrid processes that can provide high
carbon conversion efficiency for desired products (Brown, 2007). Hybrid processing
can be any combination of biological, thermal and/or catalytic processes. In this
review, we focus on the sequential thermochemical-biochemical processes.
Depending on the thermochemical process used, the hybrid thermochemical-
biochemical process can either be a fast pyrolysis of biomass into pyrolytic substrates
followed by microbial fermentation, or a gasification of biomass into synthesis gas
(syngas) followed by syngas fermentation.

The hybrid process provides a pathway to advanced biofuels that are functionally
close to petroleum-derived transportation blend stocks. Indeed, the US Department of
Energy has included “Hybrid Biochemical/Thermochemical
Processes” in their roadmap of conversion
technologies for advanced biofuels and identified
the

barrier
areas and
research
activities
needed for such efforts (US Department of Energy,
2011).
The hybrid process captures
the benefits while mitigating
the deficiencies of the

standalone
biochemical
and the
thermochemical
processes. For
example,
thermochemical
process overcomes the recalcitrance of the biomass and
thus eliminates the complex pretreatment step and the need for a
costly enzyme cocktail. It uses the whole biomass irrespective of the type and
composition and converts whole biomass (cellulose, hemicellulose, and lignin) to the
fermentable intermediates. The microbial fermentation is usually completed under
ambient and mild conditions with high scalability. For the pyrolysis-fermentation
process, fast pyrolysis can be performed locally in biomass production sites and
yields crude bio-oil. With proper fractionation, crude bio-oil can be upgraded to drop-
in hydrocarbon fuel, while the unfavorable oxygenated compounds such as sugars
and carboxylic acids can be separated and subjected to microbial fermentation for
production of fuel and chemical. For the gasification-fermentation process, syngas
fermentation is more robust and productive than the Fischer-Tropsch process because
the biocatalysts do not require a strict hydrogen to carbon monoxide ratio, are more
tolerant of the contaminants in syngas, and provide high selectivity of the desirable
products.

Structure & composition of

lignocellulosic biomass
Lignocellulosic biomass is a composite material in which the main constituents are
cellulose (~50% of weight [wt%] dry base [d.b.]), hemicellulose (~25 wt% d.b.) and
lignin

(~25 wt% d.b.). These components are linked together to obtain struc- tural strength
in combination with flexibility. In addition, biomass also contains water and minor
amounts of extractives and inorganic com- pounds (‘ash’). Cellulose is a long- chain
linear polymer that contains predominantly crystalline arrange- ments with smaller
amorphous regions.
Thermochemical-biological hybrid processing uses thermochemical decomposition of
lignocellulosic biomass to produce a variety of intermediate compounds that can be
converted into fuels and chemicals through microbial fermentation. It represents a
unique opportunity for biomass conversion as it mitigates some of the deficiencies of
conventional biochemical (pretreatment-hydrolysis-fermentation) and
thermochemical (pyrolysis or gasification) processing. Thermochemical-biological
hybrid processing includes two pathways: (i) pyrolysis/pyrolytic substrate
fermentation, and (ii) gasification/syngas fermentation. This paper provides a
comprehensive review of these two hybrid processing pathways, including the
characteristics of fermentative substrates produced in the thermochemical stage and
microbial utilization of these compounds in the fermentation stage. The current
challenges of these two biomass conversion pathways include toxicity of the crude
pyrolytic substrates, the inhibition of raw syngas contaminants, and the mass-transfer
limitations in syngas fermentation. Possible approaches for mitigating substrate
toxicities are discussed. The review also provides a summary of the current efforts to
commercialize hybrid processing.

THEORY
Fast pyrolysis produces fermentable pyrolytic sugars, carboxylic acids and lignin
derivatives, which can be utilized by microorganisms to produce advanced
hydrocarbon biofuels.

· Lignocellulosic Thermochemical conversion of biomass for fermentative

substrates

1. Fast pyrolysis of biomass for pyrolytic substrates production

Fast pyrolysis of lignocellulosic biomass is a thermochemical decomposition of the

biomass materials, in the absence of oxygen, that produces an energy rich liquid (bio-
oil), a flammable gas mixture (syngas), and a carbon- and nutrient-rich solid
(biochar). Bio-oil is a liquid mixture approximately containing (based on dry weight
of biomass) 15 wt% carboxylic acids, 25 wt% sugars, 4 wt% alcohols, 10 wt%
aldehydes, 2 wt% esters, 7 wt% furans, 5 wt% ketones and 20 wt% aromatics (Huber
et al., 2006). This composition can vary widely depending on biomass type and
pyrolysis technology. Some of these compounds can potentially be used as substrates
for microbial fermentation; those compounds are summarized below.

2. Pyrolytic sugars
Pyrolytic sugars are derived from depolymerization of cellulose during fast pyrolysis.
The major sugar product of the fast pyrolysis is the anhydrosugar levoglucosan (1,6-
anhydro-β-D-glucopyranose) with small amounts of cellobiosan, xylose, mannose,
and others. The yield of levoglucosan (based on cellulose) can be as high as 60 wt%.
The alkali and alkaline earth metals (AAEM, e.g. K, Ca and Mg) in biomass decrease
levoglucosan yields due to the fragmentation of anhydrosugar rings, which generates
oxygenated compounds such as formic acid, glycolaldehyde, and acetol. Mayes et al.
(2015) elucidated the mechanism from the molecular level that the metal ions can
decrease levoglucosan formation rate by up to 41.6 times, depending on the
adjacency of the cation to the reaction center.

Acid washing to remove AAEM is an effective pretreatment method to increase

anhydrosugar yield. Alternatively, acid infusion to passivate AAEM through
formation of thermally stable salts allows glycosidic bond cleavage to dominate
pyranose and furanose ring fragmentation. Addition of a small amount of oxygen
during fast pyrolysis also appears to be beneficial to sugar yield. Kim et al. (2014)
report sugar yield of 20.62 g/100 g biomass when acid-infused red oak was partially
oxidized with nitrogen sweep gas containing 2.1 vol% oxygen.

3. Acetic acid

Acetic acid is the predominant carboxylic acid produced by deacetylation of

hemicellulose during the fast pyrolysis of lignocellulosic biomass. In general, acetic
acid is recovered in the aqueous phase of bio-oil and is regarded as an undesirable
byproduct because it has low heating value and will cause corrosion during bio-oil
storage, transportation and refining.

4. Lignin derivatives

Lignin is a major component in lignocellulosic biomass (15-30% by dry weight and

20- 40% by energy density) and provides its structural integrity. Lignin valorization
has long been a key shortcoming of traditional biochemical conversion processes,
primarily due to its difficulty in depolymerization and the heterogeneous nature of the
resulting products. During fast pyrolysis, lignin depolymerizes to form phenolic
monomers, attractive as precursors for production of fuels and chemicals, although
the subsequent repolymerization reduces their yield among the pyrolysis products.

5. Gasification of biomass for syngas production

Compared to the fast pyrolysis, gasification of lignocellulosic biomass occurs at

higher temperatures (usually 800-1000°C) with a limited amount of oxygen,
producing syngas as the main product. Syngas is a gas mixture primarily comprised
of carbon monoxide (CO), hydrogen (H2) and carbon dioxide (CO2) with a trace
amount of methane (CH4). Depending on the biomass type and composition, the raw
syngas also contains small amounts of sulfur compounds (H2S, COS), nitrogen
compounds (NH3, HCN), tars (i.e. condensable hydrocarbons), alkali metals, and
chlorine (Woolcock and Brown, 2013).

Traditionally, biomass-derived syngas can be upgraded to alcohols and liquid

hydrocarbon fuels through the Fischer-Tropsch (FT) process, in which iron, cobalt, or
ruthenium are used as catalysts for converting gaseous compounds into liquid
products under 200-350°C (Dry, 2002). The FT process usually requires a strict H2 to
CO ratio and is vulnerable to CO2; the metal catalysts have lower selectivity and are
very sensitive to the impurities (e.g. sulfur) contained in the syngas.

· Biological conversion of fermentative substrates into fuels and chemicals

1. Fermentation of pyrolytic substrates

2. Pyrolytic sugars

Levoglucosan as a fermentative substrate can be hydrolyzed into glucose or directly

metabolized. Acid-hydrolyzed levoglucosan has been used as a substrate for yeasts
and fungi to produce ethanol or lipids. This method, however, can result in some
sugar loss during the neutralization of the acid hydrolysate. Some microorganisms
can utilize levoglucosan naturally as the sole carbon and energy source Levoglucosan
kinase (lgk) is the key enzyme for these microorganisms to convert levoglucosan into
glucose-6-phosphate (G6P), which then enters the glycolysis pathway. The yield of
the products based on levoglucosan is similar to that based on glucose, indicating that
levoglucosan to G6P is not a rate-limiting step.

Some industrial workhorse strains, such as E. coli, can be engineered for direct
utilization of levoglucosan. For example, Zhuang and Zhang (2002) expressed a
cDNA library of lgk from fungus A. niger CBX-209 in E. coli, although the resulting
enzyme activity was only one-third of that in the wild strain. (Dai et al. 2009)
transformed lgk gene from yeast Lipomyces starkeyi YZ-215 into E. coli BL21 and
found that the recombinant E. coli could grow on levoglucosan when induced with
isopropyl-β-D- thiogalactopyranoside. More recently, ethanologenic E. coli KO11
was engineered by expressing lgk from Lipomyces starkeyi YZ-215; the engineered
strain can use levoglucosan as a sole carbon source for ethanol production without
additional antibiotics or inducers The LGK enzyme has recently been subjected to a
rigorous structural analysis (Bacik et al., 2015).
Cellobiosan (1,6-anhydro-β-cellobiose) is another anhydrosugar in bio-oil. Compared
to levoglucosan, there has been a lack of studies on direct microbial fermentation of
cellobiosan. However, cellobiosan can be hydrolyzed into reducing sugars including
glucose, levoglucosan and/or cellobios that can be potentially utilized as fermentative
substrates. Most microorganisms cannot efficiently utilize cellobiose due to the lack
of cellobiose transporter(s) and cellobiose-hydrolyzing enzyme (β-glucosidase).
Many studies have been conducted to enable cellobiose utilization by
microorganisms. For example, Galazka et al. (2010) expressed a high-affinity
cellobiose transporter from the fungus Neurospora crassa in Saccharomyces
cerevisiae to assimilate cellobiose and β-glucosidase for intracellular cellobiose
hydrolysis. Several approaches have also been attempted to increase the cell growth
rate when using cellobiose, including direct evolution of the cellodextrin transporters,
optimizing the cellobiose phosphorylase pathway, and regulating suboptimal non-
glucose sugar fermentation.

3. Acetic acid

Acetic acid is the predominant carboxylic acid in bio-oil produced from deacetylation
of hemicellulose. Some microorganisms can metabolize acetic acid to produce acetyl-
CoA, a central intermediate for fatty acids biosynthesis (Ratledge, 2004). A number
of oleaginous yeasts (Cryptococcus albidus, Cryptococcus curvatus, Yarrowia
lipolytica) and microalgae (Chlorella protothecoides) have been reported to produce
lipids by utilizing acetic acid as the sole carbon source with lipid content as high as
55% (g/g biomass). The main lipids produced by these oleaginous strains are C16-
C18 fatty acids ready for biodiesel production. However, there are only a few studies
on lipid production using pyrolysis-derived acetic acid. Unlike pyrolytic sugar
fermentation based on anaerobic glycolysis pathway, lipid biosynthesis from acetate
is an aerobic process. Therefore, maintaining an appropriate dissolved oxygen level is
important for the carbon metabolism. Important engineering issues such as aeration
efficiency and power consumption should be considered.

4. Lignin derivatives

Fast pyrolysis of lignin produces phenolic monomers which can be utilized by

aromatic-catabolizing microorganisms to produce the central intermediates (e.g.,
catechol and procatechuate) via aerobic peripheral pathway. These central
intermediates are then subjected to O2-dependent aromatic-ring cleavages by
dioxygenase enzymes found in many bacteria and fungi. This aromatic catabolism
serves as a “biological funnel” to reduce the heterogeneity of the lignin derivatives.
Linger et al. (2014) reported the utilization of a natural aromatic-catabolizing
bacterium, Pseudomonas putida KT2440, to convert the lignin-derived phenolic
liquor into the medium chain-length polyhydroxyalkanoates (mcl-PHAs). The
research team demonstrated that P.putida KT2440 could be used as a biological
platform to produce muconate (13.5 g/L) from diverse lignin-derived aromatic
monomers (e.g. phenol, vanillin, ferulic acid) via protocatechuate and catechol
branches of the β-ketoadipate pathway. The muconic acid was recovered with high
efficiency (74%) and high purity (>97%) and then subjected to catalytic
hydrogenation to produce adipic acid, the most commercially important dicarboxylic
acid. The aromatic- catabolizing pathways in P. putida KT2440 can be further
modulated to optimize the yield of the desired product via the central intermediates
(pyruvate, succinate, acetyl-CoA). For example,

Johnson and Beckham (2015) attained a 5-fold increase in pyruvate production by

replacing the protocatechuate ortho pathway in P. putida KT2440 with a xenogeneic
meta-cleavage pathway from Sphingobium sp. SYK-6. These advances in biological
utilization of lignin derivatives enhance economic viability of hybrid process
(Ragauskas et al., 2014). Most recently, Salvachúa et al. (2015) reported that the
bacterial consortium of P. putida strains, Amycolatopsis sp., Acinetobacter ADP1 and
Rhodococcus jostii were able to depolymerize lignin with high molecular weight and
catabolize aromatic monomers simultaneously. This may enable biological utilization
of lignin-derived phenolic oligomers in bio-oil, thus alleviating the aforementioned
monomer-repolymerization issues. However, since biological upgrading of lignin
derivatives depends on aromatic catabolism with oxygen consumption, developing
cost-efficient aeration will be crucial for economic viability and scalability of lignin
valorization.

· Fermentation of syngas

1. Metabolic pathway in syngas fermentation

A variety of microorganisms are capable of performing syngas fermentation. Based

on the end products, those microorganisms can be hydrogenogens (Table 3) and
acetogens (Table 4). The hydrogenogens produce H2 from proton reduction coupled
with CO oxidation to CO2, which is also referred to as the biological water-gas shift
reactions (Do et al., 2007):

CO + H2O → CO2 + 2H+ + 2e- → CO2 + H2

The above reactions are catalyzed by two key enzymes: nickel-CO dehydrogenase
(Ni-CODH) and CO-induced hydrogenase.
Acetogens are facultative autotrophs capable of CO/H2/CO2 metabolism via the
Wood- Ljungdahl (WL) pathway. The overall stoichiometry of the pathway is
presented as:

2CO2 + 4H2 + ATP → Acetyl-CoA + 2H2O + nATP

where n is the ATP conservation coefficient; acetyl-CoA serves as a central
intermediate and an ATP source, which is utilized to produce metabolites such as
acetate and ethanol, with the supply of electron donors (H2 or CO).

2. Metabolic engineering of acetogens in syngas fermentation

Most acetogens produce acetate as the sole end product. Some organisms are also
capable of converting syngas to other products such as ethanol, butanol, butyrate, and
2,3- butanediol but the yields are usually low. Native acetogens have been engineered
to divert carbon flow to the desired end products with improved yields. Table 5
summarizes the recent advances of genetic manipulation of acetogens. Clostridium
ljungdahlii has been widely used as a platform for heterologous gene expression. For
example, six butanol pathway genes from C. acetobutylicum were expressed in C.
ljungdahlii via a pIMP1 plasmid-based shuttle vector, with up to 2 mM butanol
produced by the recombinant C. ljungdahlii. However, the plasmid transformation
efficiency was low by this method. Recently Leang et al. (2013) reported a more
efficient electroporation protocol to perform chromosomal gene deletion for C.
ljungdahlii via double-crossover homologous recombination with suicide vector.
With this toolkit, Banerjee et al. (2014) adapted the bgaR-PbgaL plasmid-based
lactose-inducible system originally developed for C. perfringens to C. ljungdahlii for
acetone production. The inducible system redirected the carbon and electron flow for
biosynthesis of the desired products other than acetate, producing 13 mM acetone
with 25% carbon yield based on syngas input. The authors also determined that
adhE1, not adhE2, is the gene responsible for ethanol production (Banerjee

et al., 2014). The same lab later transformed 8 genes (thl, crt, bcd, etfA, etfB, hbd, ptb
and buk) encoding the key enzymes for butyrate pathway from C. acetobutylicum to
C. ljungdahlii. The authors inactivated pta-dependent acetate, adhE1-dependent
ethanol, and ctf- dependent fatty acid synthesis pathways to enhance the butyrate
production to 17 mM with 68% of carbon yield and 73% of electron yields.

Genetic manipulation of other acetogens has also been reported . For example,
LanzaTech claimed that up to 25.6 mM butanol was produced from steel mill waste
gas (mainly CO) by overexpression of butanol synthesis pathway genes from C.
acetobutylicum in C. autoethanogenum. In addition, these researchers identified two
key enzymes, 2,3-butanediol dehydrogenase (2,3-BDH) and NADPH-dependent
primary-secondary alcohol dehydrogenase (CaADH), contributing to 2,3-butanediol
production during C. autoethanogenum syngas fermentation. CaADH was
demonstrated in vitro to convert acetoin, acetone and butanone to 2,3-butanediol,
isopropanol and 2-butanol, respectively. This indicates the potential of using C.
autoethanogenum as a platform for producing higher alcohols (C3, C4). Straub et al.
(2014) improved acetate titer (51 g/L) using Acetobacterium woodii by
overexpression of the native genes encoding the four THF-dependent enzymes,
phosphotransacetylase and acetate kinase.

The development of metabolic engineering of acetogens is still in its infancy. For

example, the butanol titer was still very low (2 mM) using the engineered pathway in
C. ljungdahlii; by the end of the batch fermentation, all butanol disappeared and was
possibly consumed by the cells to produce butyrate (Kopke et al., 2010). In another
research, Ueki et al (2014) developed the genetic knockout system for C. ljungdahlii
to interrupt acetate kinase and CoA transferase. However, this attempt did not
completely eliminate acetate and ethanol

production; the majority of carbon flux in the WL pathway still flowed to acetate
from syngas, indicating the existence of other unidentified genes controlling their
metabolism. Further efforts on metabolic engineering of acetogens are needed.

It should be noted that a series of articles were published recently by Tyurin and his
colleagues on Clostridium metabolic engineering for producing ethanol, acetone,
butanol, methanol, and 2,3-butanediol by eliminating acetate formation. Those
results, however, have been refuted by other researchers. For example, Bengelsdorf et
al. (2013) questioned the very high level of the recombinant products while no
mutant strains were available in public cell collections and no 16S rDNA data were
available in public database for other researchers to reproduce the results. Latif et al.
(2014) pointed out that complete elimination of the acetate synthesis by deletion of
the pathway genes prevents the concurrent ATP synthesis, which may impact the
growth energetics. As a matter of fact, in many experiments reported by other groups,
acetate was still the primary product in syngas fermentation even though the
recombinant Clostridium strain was reconstructed to produce other metabolites.

SUMMARY
This research paper provides a comprehensive review of two thermochemical-
biochemical hybrid processes: pyrolysis-fermentation, gasification-fermentation,
including their features, challenges and mitigating strategies. The hybrid processing
provides an alternative platform for efficient production of biofuels and commodities
from lignocellulosic biomass.

Fast pyrolysis produces fermentable pyrolytic sugars, carboxylic acids and lignin
derivatives, which can be utilized by microorganisms to produce advanced
hydrocarbon biofuels. Currently, the inhibition from the toxic compounds is the
biggest hurdle for the viability of pyrolytic substrate fermentation. Further research
should focus on the development of more efficient detoxification methods for less
toxic substrate and robust strains highly tolerant to toxicity. It is important to use both
of the two strategies in order to enhance the economic feasibility of the pyrolytic
substrate fermentation process. Moreover, it is necessary to elucidate the mechanism
of toxicity so that a reverse engineering of evolved strains can be developed for an
expanded genetic toolbox to be applied to other microorganisms.

Gasification-syngas fermentation is more advanced in terms of commercial

development. However, this platform is also faced with obstacles. First, as the most
prominent product of syngas fermentation, ethanol is not an optimal fuel due to its
low energy density and the “blend wall” limitation. Even with advancement of
genetic manipulation, the productivity of more advanced C4-based fuels is still low
even due to the intrinsic stoichiometry and energy conservation restriction.
Mixotrophic cultivation of acetogens may solve this but the influence of sugar
addition needs to be carefully evaluated. The engineering issues in syngas
fermentation such as gas contaminants and mass transfer will also need to be
addressed.

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