World Journal of Microbiology and Biotechnology (2021) 37:134

https://doi.org/10.1007/s11274-021-03102-6

REVIEW

Woody biomass as a potential feedstock for fermentative gaseous

biofuel production
Suren L. J. Wijeyekoon1  · Alankar A. Vaidya1

Received: 30 April 2021 / Accepted: 1 July 2021 / Published online: 14 July 2021
© The Author(s), under exclusive licence to Springer Nature B.V. 2021

Abstract
Biogas and biohydrogen are compatible gaseous biofuels that can be blended with natural gas for reticulated fuel supply to
reduce greenhouse gas emissions. Sustainably grown woody biomass is emerging as a potential feedstock in the production
of biofuels. Woody biomass is widely available, uses non-arable land for plantation, does not require synthetic fertilisers to
grow and acts as a carbon sink. The cellulose and hemicellulose fractions of wood are renewable sources of sugars that can
be used for fermentative production of gaseous biofuels. However, widespread use of wood as a gaseous biofuel feedstock is
constrained due to the recalcitrant nature of wood to enzymatic hydrolysis. Pretreatment makes cellulose and hemicellulose
accessible to microbial enzymes to produce fermentable sugars. Here we review wood composition, its structure and differ-
ent pretreatment techniques in the context of their effects on deconstruction of wood to improve hydrolysis and fermentative
gaseous fuel production. The anaerobic digestion of pretreated wood for biogas and dark fermentation for biohydrogen pro-
duction are discussed with reference to gas yields. Key advancements in lab-scale research are described for pretreatments
and for pure, co- and mixed culture fermentations. Limitations to yield improvements are identified and future perspectives
and prospects of gaseous biofuel production from woody biomass are discussed, with reference to new developments in
engineered biocatalysts and process integration.

Keywords Anaerobic digestion · Biogas · Biohydrogen · Biomethane · Dark fermentation · Pretreatment · Woody biomass

Introduction promising alternative, with an estimated potential generation

Owing to surging world population, rapid industrialisation
and economic growth, global energy demand is predicted to
increase from 5.5 × 105 PJ in 2010 to 6.6 × 105 PJ in 2020.
It is estimated that this demand will reach 8.6 × 105 PJ in
2040 (Guo et al. 2015). World energy supply is dominated
by non-renewable fossil fuels, which are limited in supply.
At current consumption rates, supplies of petroleum, natural
gas, and coal will only last for another 45, 60, and 120 years,
respectively (International Energy Agency 2020). The
dwindling supply and volatile prices of fossil fuels, along
with global climate change, necessitate development and
utilization of renewable energy alternatives. Biofuels are a
* Alankar A. Vaidya
Alankar.vaidya@scionresearch.com
Suren L. J. Wijeyekoon
Suren.wijeyekoon@scionresearch.com
1
Scion, Te Papa Tipu Innovation Park, 49 Sala Street,
Rotorua 3046, New Zealand
of up to 25% of global energy requirements by 2035 (Kopetz
2013). Second generation biofuels based on lignocellulosic
biomass are under development. These feedstocks are abun-
dant, carbon neutral and do not compete with food sources
(Balat and Balat 2009).
From different lignocellulosic options, wood repre-
sents an abundant, non-food resource which is intensively
researched as a feedstock for renewable energy. As of 2020,
the worldwide growing stock of forests, a primary source of
wood, is about 557 billion m3 and from this stock approxi-
mately 4.3 billion m3 of wood is harvested annually (FAO
2020). Almost 75% of a tree is utilised as sawn timber, with
the remaining sawdust, fine particles, slabs, edgings and
off-cuts being classed as wood waste. This waste wood is
a potential resource for biofuel production that can have
a significant impact on profitability throughout the forest
value chain (Klitkou et al. 2019). A complimentary source of
wood residue also comes from thinning and cutting needed
to increase the production of quality timber as well as the
thinning of forest stands to reduce the danger of fire. The

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amount of dry wood that could be recovered from forest
thinning is significant. Production of bioenergy from under-
utilised woody feedstock will help to diversify the product
portfolio of the forestry industry, offsetting the rapid decline
of traditional wood-based industries such as pulp and paper
(Kalyani et al. 2017).
The large-scale combustion of wood to extract energy
is no longer deemed to be an efficient and environmentally
desirable choice and other pathways for bioenergy and bio-
fuels must be developed (Ingersoll 2020). Two very promis-
ing pathways for renewable gaseous biofuel production from
wood are microbial fermentation to produce biohydrogen
and biomethane by dark fermentation (Levin et al. 2006;
Xiao et al. 2010; Monlau et al. 2011) and anaerobic diges-
tion (AD) (Odhner et al. 2012; Salehian et al. 2013; Shafiei
et al. 2014; Mulat et al. 2018; Li et al. 2019) respectively.
This process is shown schematically in Fig. 1.
In order to expedite microbial fermentation of wood,
appropriate pretreatment is necessary as a first step to facili-
tate generation of C5 and C6 sugars by enzymatic hydroly-
sis. These simple sugars are transformed by AD to meth-
ane (CH4) and carbon dioxide (CO2). After stripping CO2
from this biogas, the biomethane can be used to partially
replace natural gas, with no requirement to upgrade existing
physical and logistical infrastructures (Tasca et al. 2019).
The AD process can be switched toward dark fermentation
to produce hydrogen (H2) instead of CH4, by controlling
operational parameters such as pH and retention time to
inhibit methanogenesis (Monlau et al. 2011). Biohydrogen
can be used in fuel cells and internal combustion engines to
Fig. 1 Schematic diagram of wood to gaseous biofuel
13
World Journal of Microbiology and Biotechnology (2021) 37:134
produce electricity and mechanical energy. Hydrogen has
several advantages as a fuel that make it of great interest
as an emerging energy carrier, such as zero carbon, high
energy content and good combustion kinetics (Koroneos
et al. 2004).
Although there are many reviews on lignocellulosic bio-
mass conversion to gaseous biofuels, there were no reviews
dedicated to woody biomass, which is one of the major
lignocellulosic feedstocks currently used for commercial-
scale liquid biofuel production. This manuscript seeks to:
(1) review the potential of woody biomass as a fermentation
feedstock for gaseous biofuel production; (2) discuss pre-
treatment technologies and low-energy-intensive fermenta-
tion processes for conversion of wood-derived substrates and
the challenges involved; (3) identify future research needs
for sustainable gaseous biofuel production from woody
biomass.
Wood composition
The main components of wood are cellulose, hemicellu-
lose, lignin, minerals, extractives and water. Their percent-
age vary with the type of wood as shown in the Table 1.
Cellulose is the largest component in wood. It is a linear
polysaccharide of glucose made up of cellobiose units. The
cellulose chains are packed by hydrogen bonds in so-called
‘elementary microfibrils’. These fibrils are attached to each
other by hemicelluloses, amorphous polymers of different
sugars, as well as other polymers such as pectin, and covered
World Journal of Microbiology and Biotechnology (2021) 37:134
Table 1 Major constituent Type of wood Cellulose
compositions (wt%) in different
types of wood Softwood 38–52
Hardwood 40–55
Bamboo 38–51
Palm 32–65
Larch 38–53
by lignin. The microfibrils are often associated in the form
of bundles or ‘macrofibrils’. This special and complicated
structure makes cellulose in wood resistant to both biologi-
cal and chemical treatments (Taherzadeh and Karimi 2008).
Hemicellulose is the second largest component, consisting
18–30% of dry wood. It is made up of different monomeric
sugars, namely xylose, mannose, galactose, rhamnose, and
arabinose (Monlau et al. 2011). Lignin is the polyaromatic,
hydrophobic and biologically recalcitrant component of the
wood. The phenolic composition of lignin differs markedly
between softwoods and hardwoods and is responsible for the
higher calorific value of softwood (Demirbaş and Demirbaş
2004; Monlau et al. 2011).
The chemical composition of wood varies from species to
species. On average, wood is made up of carbon (C) (50%),
oxygen (O) (42%), hydrogen (H) (6%) and nitrogen (N) (1%)
with the remaining 1% containing macro-elements (calcium,
potassium, sodium, magnesium, iron, and manganese) and
micro-elements (sulfur, chlorine, silicon, phosphorus and
others) (Ingersoll 2020). The C:N ratio of 50 makes wood a
nitrogen-limited feedstock for biological processing (a ratio
of around 30 is required for AD). Wood is heavily oxygen-
ated, with a C:H ratio of around 8 compared to the ratio of
3 for CH4, suggesting it is also a hydrogen-poor feedstock.
However, in biological processing, wood fractions are hydro-
lysed to reduce its C:H ratio.
Wood has moderate calorific values (HHV) ranging from
17 to 26 MJ/kg depending on composition and oxygen con-
tent; cellulose has an HHV of 18 MJ/kg and lignin ~ 25 MJ/
kg (Demirbaş and Demirbaş 2004). Wood may also con-
tain up to 5% of low molecular weight compounds such as
extractives. Extractives are organic compounds (fatty acids,
terpenes, resins, waxes and tannins), none of which are read-
ily biodegradable. Fresh wood contains almost 60% bound
water, making direct combustion of wood for energy produc-
tion inefficient and requiring wood to be dried to reduce its
water content. However, for biohydrogen and biogas produc-
tion the wood need not be dried; in fact, wood-associated
water is required for microbial fermentation, which elimi-
nates the cost of drying the wood when fermentation is the
preferred process. In biogas fermentation both cellulose and
hemicellulose are metabolized by mixed microbial cultures
(Xiao et al. 2010; Monlau et al. 2011).
Page 3 of 15 134
Hemicellulose Lignin Extractives References
15–28 25–39 1–24 (Sella Kapu and Trajano 2014)
24–40 18–25 0.1–7.7 (Chandra and Saddler 2012)
20–29 21–31 6–13 (Sella Kapu and Trajano 2014)
9–33 13–48 1–9 (Li et al. 2020)
15–36 22–26 3–5.6 (Bergstedt and Lyck 2007)
Wood pretreatment
Wood is highly resistant to microbial degradation due to
the crystallinity of cellulose, its accessible surface area
and protection by lignin and hemicellulose, the degree
of cellulose polymerization and degree of acetylation of
hemicelluloses (Taherzadeh and Karimi 2008). In fact,
there is no known single microorganism that can directly
decompose wood in its natural form and extract its energy
content. In order to expedite microbial fermentation of
wood, appropriate pretreatment is necessary as a first
step to facilitate the action of enzymes on cellulose and
hemicellulose to generate simple C5 and C6 sugars. These
simple sugars are assimilated and transformed through
microbial AD to produce CH4 and CO2. To make wood
amenable to hydrolysis, various pretreatments are required
to remove or alter lignin, increase the pore volume and
internal surface area of lignocellulosic substrates and
reduce the degree of polymerization and crystallinity of
the cellulose. To be effective and economical, pretreatment
should: (a) produce reactive cellulose for enzymatic attack
(b) reduce destruction of hemicellulose and cellulose, (c)
avoid the formation of inhibitors of hydrolytic enzymes
and fermenting microorganisms (d) minimize energy
demand (e) reduce the cost of size reduction of feedstocks
(f) require minimal input of chemicals (Taherzadeh and
Karimi 2008; Chen et al. 2013).
Pretreatment will be effective only when it achieves a
high yield of reducing sugars after enzymatic hydrolysis
(Wyman et al. 2005). The type and concentration of reducing
sugars (monosaccharides and oligosaccharides) produced by
enzymatic hydrolysis are different for different feedstocks
and pretreatment methods, and this ultimately affects the rate
and yield of biogas production. Under severe pretreatment
conditions, some monosaccharides and soluble lignin frag-
ments are degraded or transformed into inhibitor compounds
such as 2-furaldehyde (furfural, F) and 5-hydroxymethyl-
2-furaldehyde (hydoxymethyl furfural, 5HMF), aliphatic
acids such as formic, acetic and propionic and phenolic
compounds. These inhibitors can interfere with the functions
or osmotic pressure of microbial cells, directly inhibiting
the fermentation pathway (Chen et al. 2013). Fine tuning of
pretreatment conditions is required to achieve the highest
biogas and biohydrogen productivity.
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134 Page 4 of 15
Pretreatment methods can be divided into mechanical,
biological, thermal and physico-chemical methods. A com-
bination of two or more different pretreatments are often
used for woody biomass to make the overall process effi-
cient and economical (Kim et al. 2016). The efficiency of
pretreatment depends on the source and type of biomass
and there is no universal standard pretreatment method that
can be applied to every or any type of feedstock. The major
pretreatment methods applied to wood in the production of
gaseous biofuels are summarised in Fig. 2.
Mechanical pretreatments
Mechanical pretreatment such as chipping, grinding, shred-
ding or milling is the first pretreatment applied to woody
biomass. The focus here is to reduce the particle size, fibril-
late fibres to increase the surface area of the biomass and
reduce the degree of crystallinity and polymerization of
Fig. 2 Major pretreatment methods applied to wood in the production
of gaseous biofuels
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World Journal of Microbiology and Biotechnology (2021) 37:134
cellulose and hemicellulose. Generally, these pretreatments
do not generate any compounds that are toxic or inhibitory
to enzymatic hydrolysis or fermentation. However, milling
is a very energy intensive process and difficult to implement
on an industrial scale. For woody biomass, wet ball milling
is the most effective method (Vaidya et al. 2016). When a
combination of mechanical refining and thermal/chemical
pretreatment is used, the aim of mechanical pretreatment is
to decrease the severity of the following thermal/chemical
pretreatment.
Thermal pretreatments
Hot water (hydrothermal, solvolysis, hydrothermolysis,
aqueous fractionation, aquasolv)
Wood at about 1–8% of dry matter is heated in the presence
of water to a high-temperature (160–230 °C) for a few min-
utes to hours. Under high pressure, water in liquid form pen-
etrates the biomass, increasing the internal surface area and
causing hydrolysis of hemicellulose and lignin (35–60%)
with some cellulose losses (4–22%) (Monlau et al. 2011).
Three reactor configurations are possible depending on the
direction of the flow of hot water on the biomass: co-current,
counter-current and flow-through. These pretreatments do
not require washing or neutralization steps as no chemicals
are used. However, it is an energy and water intensive pro-
cess with a large loss of solubilized reducing sugars and
hydrolysed hemicellulose and lignin in the liquid phase. The
recovery of waste heat from the pretreatment reactor is criti-
cal for energy efficiency of the whole process.
Steam/steam explosion (SE)/autohydrolysis
Biomass, after mechanical size reduction, is heated rapidly
with saturated steam to a high temperature (160–260 °C)
and pressure (0.7–4.8 MPa) and held under these conditions
for several seconds to a few minutes to promote hemicel-
lulose hydrolysis and transformation of lignin. A varia-
tion of steam pretreatment is a hydrothermal pretreatment
which involves initial steaming of wood chips at 173 °C for
3–72 min without addition of chemicals. The steamed chips
are then passed through a compression screw to squeeze out
water-solubilised hemicelluloses. The pressed chips are then
disc-refined and ball-milled to produce easily hydrolysable
substrate (Suckling et al. 2017).
In SE, the pressure is suddenly released, allowing water
molecules to escape in an explosive manner. This opens the
wood cell walls and increases the surface area and hence
enzymatic hydrolysis of the biomass. Sometimes, incom-
plete disruption of the lignin-carbohydrate complex can lead
to reduced enzymatic hydrolysis. This limitation has been
overcome for Pinus radiata by a combined pretreatment
World Journal of Microbiology and Biotechnology (2021) 37:134
of SE followed by fungal (Trametes versicolor) exposure
resulting in higher (almost 25%) conversion of cellulose to
glucose compared to SE treatment alone. The combined SE
and fungal pretreatment increased the relative porosity of the
substrate and reduced its lignin content (Singh et al. 2016).
Physico‑chemical pretreatments
Acidic pretreatment employs inorganic acids such as hydro-
chloric (HCl), sulphuric (H2SO4), phosphoric (H3PO4) and
nitric (HNO3), or organic acids such as citric, malic and
oxalic acids. Alkaline pretreatment uses sodium hydroxide
(NaOH), potassium hydroxide (KOH), calcium hydroxide
(Ca(OH)2), ammonium hydroxide (NH4OH) and anhydrous
ammonia (NH3).
Alkaline—ammonia fibre explosion (AFEX)/ammonia
recycle percolation (ARP) and soaking aqueous ammonia
Alkaline pretreatments can be carried out at high pressure
and low temperature (~ 90 °C) for up to 10–60 days. At
higher temperatures (150–190 °C) the effect of ammonia
is rapid and the duration of pretreatment can be reduced to
10–90 min. AFEX causes swelling and phase change of cel-
lulose causing reduction in cellulose crystallinity. A small
amount of hemicellulose is solubilized in an oligomeric form
(Mosier et al. 2005) in addition to the alteration and removal
of lignin. Overall, this pretreatment increases the reactivity
of the carbohydrates. The residual nitrogen remains in the
substrate at the end of pretreatment and can be used as a
N source in anaerobic and dark fermentations. Safety and
environmental concerns with ammonia have prevented these
pretreatments from progressing to industrial scale.
The use of NaOH, KOH, and Ca(OH)2 lead to cleavage
of ester bonds in phenolic-carbohydrate complexes, swelling
of fibers and an increase in pore size, facilitating the dif-
fusion of hydrolytic enzymes into the substrate. Generally,
alkaline pretreatments are effective on herbaceous plants,
agricultural residues, municipal solid waste and hardwoods
but less effective on biomass with a high lignin content such
as softwoods.
Acid
Dilute H2SO4 (0.4–2%) is the acid of choice at temperatures
of 160–220 °C and treatment times of a few minutes. This
pretreatment releases oligomeric and monomeric sugars
from carbohydrates and the resulting product is more ame-
nable to enzymatic hydrolysis. Improvement in hydrolysis is
caused by the reduction in hemicellulose and lignin content,
decrease in the crystallinity of cellulose and increase in the
accessible surface area. At high process severity, the sugars
are converted to F and HMF which are known fermentation
Page 5 of 15 134
inhibitors. This pretreatment necessitates expensive reactor
construction materials due to the corrosive nature of acids.
In bioethanol industry, cost of material of construction for
pretreatment reactors is estimated to be 16–19% of the total
material cost (Aden et al. 2002) and this cost needs to be
reduced substantially. This is also expected to be true for
fermentative biogas production if acid pretreatments are
employed.
CO2 explosion
This process is like SE and AFEX in which wet biomass
is exposed to CO2 at high pressure and moderate tempera-
ture. The gas penetrates the small pores in the biomass and
the sudden release of pressure causes explosive disruption
of the biomass, increasing the accessible surface area. The
CO2 forms carbonic acid when dissolved in water, which
assists in the hydrolysis of hemicellulose without creating
sugar degradation products. The high cost of equipment to
withstand high pressure conditions is a major limitation for
scale-up of this process to industrial scale.
Organosolv
This is a very selective method yielding three separate frac-
tions: (1) lignin—pure, sulphur free, low molecular weight
solid (2) hemicellulose—an aqueous semi-pure fraction (3)
cellulose—a relatively pure solid fraction. The mechanical
size reduction of wood is not necessary and a variety of
organic or aqueous-organic solvent mixtures, with or with-
out a catalyst such as HCl or H2SO4, have been used in this
pretreatment. The process is conducted at high temperatures
(100–250 °C) using low boiling solvents (methanol and etha-
nol) or high boiling alcohols (ethylene glycol, glycerol, tet-
rahydrofurfuryl alcohol). The alcohol removes lignin and
dissolves hemicellulose by—(1) hydrolysing internal lignin
bonds and ether and 4-O-methylglucuronic acids ester bonds
between lignin and hemicellulose and (2) hydrolysing gly-
cosidic bonds in hemicellulose and partially in cellulose,
depending on the process severity.
The addition of acid catalyst is not necessary at high tem-
peratures (> 185 °C) because organic acids such as acetic
acid, uronic acid etc. produced in situ from the biomass will
act as a catalyst to rupture the lignin-carbohydrate complex.
However, acid catalysed degradation of monosaccharides
can generate the inhibitors F and 5-HMF. Delignification
kinetics vary with the solvent used during pretreatment. At
the end of the process, solvents can be recovered via evapo-
condensation and recycled to reduce costs. A commercial
example of organosolv pretreatment is the Lignol process
(Zhao et al. 2009). Residual organic solvents left in the pre-
treated substrate may be inhibitory to enzymatic hydrolysis
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134 Page 6 of 15
and/or fermentation. This pretreatment is expensive due to
solvent and catalyst costs and recyclability issues.
Ionic liquids (IL)
These are solvents with low melting points (< 100  °C),
high polarities, high thermal stabilities and negligible
vapour pressure consisting entirely of ions (cations and
anions). Most ILs used in biomass pretreatment are imida-
zonium salts such as 1-allyl-3-methylimidazonium chloride
(AMIMCl) and 1-butyl-3-methylimidazonium chloride
(BMIMCl). ILs can dissolve cellulose from wood which
can be reprecipitated in water, ethanol or acetone. In the
Lyocell process, N-methylmorpholine N-oxide (NMMO), is
used to generate cellulose fibres (Perepelkin 2007). NMMO
increases wood porosity and water swelling capacity and
considerably reduces cellulose crystallinity as well as reduc-
ing lignin on the wood surface (Shafiei et al. 2014). The
use of IL as a wood pretreatment appears to be attractive
but the solvents are expensive and need to be recovered and
reused by pervaporation, reverse osmosis, salting out and
ion exchange, which can add considerably to process costs.
Biological
Industrial enzymes such as hemicellulases and/or lignin
hydrolysing enzymes (laccase, lignin and manganese per-
oxidase) are used to break down hemicellulose and lignin.
The high cost of industrial enzymes is the main concern;
this can be overcome by producing in-house extracellular
enzymes from brown-, white-, and soft rot-fungi. White rot
degrades lignin by secreting various enzymes such as per-
oxidases or laccases. The optimum pH for growth of ligni-
nolytic white rot fungus is between 4 and 5. Uncontrolled
pH deviations from the optimum pH reduce the production
of laccase enzymes and efficiency of microbial pretreatment.
Biological pretreatment is considered environmentally safe
and energy-saving process as it is performed at ambient
temperature and pressure without requiring any chemicals
or sophisticated apparatus. However, due to low reaction
rates, biological pretreatments must be combined with other
pretreatment methods. Another major concern is the loss of
carbohydrates used in the growth and metabolism of enzyme
producing microbes. Microbial consortia can be an effec-
tive method to improve biomass degradation performance.
A microbial consortium contains various fungal and bacte-
rial species with different biomass degradation efficiencies,
functioning under a range of environmental conditions using
a variety of biomass degradation mechanisms. It possesses
an enhanced ability to colonize wood compared to individual
microbial species.
Biological pretreatment strategies are compatible and
synergistic with AD, as no potential inhibitors of anaerobic
13
World Journal of Microbiology and Biotechnology (2021) 37:134
fermentation are formed. In addition to hydrolysis, biologi-
cal pretreatments can also improve the quality of the sub-
strate by eliminating compounds that reduce the efficiency
of the AD process. In Table 2 the effect of different pretreat-
ments on biogas production from various types of wood is
given.
Fermentative gaseous biofuel production
Biogas from anaerobic digestion of wood
Anaerobic digestion is well described in the scientific lit-
erature and the technology is established at scale as a waste
management option and for renewable energy production
(Matsakas et al. 2016). The process comprises four key
steps: hydrolysis, acidogenesis, acetogenesis, and methano-
genesis. A mixed culture of microorganisms acts symbioti-
cally to convert complex organic substrates into biogas and
other reduced products. The cellulose and hemicellulose
fractions of wood are hydrolysed into soluble sugars. These
sugars are then transformed into volatile fatty acids (VFA)
in the acidogenesis stage. During the subsequent stage of
acetogenesis, the VFAs are converted into acetate, CO2 and
H2. Finally, these compounds are fully reduced to biogas by
methanogenesis. The biogas from AD consists mainly of
CH4 (55–75%) and CO2 (25–45%) along with other trace
gases depending on the substrates and co-substrates used.
The advantage of using a woody substrate is that contami-
nant gases such as hydrogen sulphide and ammonia will not
compromise the quality of the biogas.
Process conditions
Anaerobic digestion occurs over a wide range of tempera-
tures; from 10 to 20 °C (psychrophilic), through the more
common 30–40 °C (mesophilic) to 50–60 °C (thermophilic)
range. The active microbial community structure at each
temperature range is different (Goswami et al. 2016). There
is a rate advantage at thermophilic temperatures arising from
the higher hydrolysis coefficients and faster reaction kinetics
(Ferrer et al. 2010; Bolzonella et al. 2012). The acidogenesis
step of AD is favoured at acidic pH values of 4.5–6 due to
VFA formation. The optimum pH range for methanogenesis
is between 7.0 and 8.0. Another key process parameter par-
ticularly relevant for woody substrates is the C:N ratio of the
feedstock. The optimum range of C:N for AD is between 20
and 35; this requires N rich co-substrates or ammonia-based
alkaline pretreatment of the woody biomass to achieve this
ratio. Many other trace elements such as Na, Ni, Co, Fe,
Zn, Mg, Ca and K as cations and molybdate, tungstate and
phosphate as anions are required for synthesis of enzymes,
prosthetic groups, and coenzymes (Goswami et al. 2016).
World Journal of Microbiology and Biotechnology (2021) 37:134
Table 2 Effect of various pretreatments on conversion of wood to gaseous biofuels
Pretreatment Feedstock Pretreatment condition
Physico-chemical
Ionic liquid (a) spruce (a) NMMO at 130 °C for
(b) pine 15 h
(c) mixed forest residue (b) 85% NMMO at 120 °C
for 30 min
(c) 75% NMMO at 120 °C
for 15 h
Alkaline (a) pine (a) 8% NaOH for 60 min at
(b) birch 0 °C or 10 min at 100 °C
(c) poplar (b) 7% NaOH for 2 h at
(d) Eucalyptus globules 100 °C
(c) 5% NaOH for 96 h at
ambient temp
(d) 1% NaOH for 20 min at
125 °C
Organosolv (a) forest residue (a) 50% C2H5OH or CH3OH
(b) pine with 1% H2SO4 at 190 °C
for 1 h
(b) 75% aqueous C2H5OH
with 1% H2SO4 at a solid-
to-liquid ratio of 1:8 at
180 °C for 60 min
Aq. Ammonia willow
Acid (a) pine (a) 32% w/w at 25 °C for
(b) poplar 72 h (b) and C) H3PO4
(c) berry (85%) at 60 °C for 45 min
followed by C2H5OH and
water wash. Neutralisation
with 2 M Na2CO3
Hydrothermal with SO2 (a) birch, spruce and pine (a) SO2 at 212 °C for 4 to
(b) spruce 8 min at pH 1.6 to 1.8
(b) steam refining with SO2
Thermal
 Hot water (a) Norway spruce (a) water extraction at
(b) Eucalyptus globules 140 °C for 5 h
(b) hot water extraction at
125 °C, 20 min
Steam explosion (a) Japanese cedar (a) 258 °C for 5 min
(b) birch (b) 220 °C for 10 min
(c) birch (c) 210 °C for 10 min
(d) bamboo followed by Caldicellulo-
(e) Eucalyptus globules siruptor bescii treatment
at 62 °C
(d) 0.8% H2SO4 at 230 °C
for 3 min
(e) at 25 atm, 3 min
Biological
Milling + enzyme Cedar 10% slurry + 200 mL
enzyme cocktail at 50 °C
for 4 h
Page 7 of 15 134
Increase in yield compared References
to control
(a) 66 to 245 mL CH4/g VS (a) (Teghammar et al. 2012)
(b) 66 to 224 mL CH4/g VS (b) (Shafiei et al. 2014)
(c) 53 to 92 mL CH4/g VS (c) (Aslanzadeh et al. 2014)
(a) 33 to 107 mL CH4/g VS (a) (Salehian and Karimi
(b) 250 to 460 mL CH4/g VS 2013; Salehian et al. 2013)
(c) 127 to 272 mL CH4/g VS (b) (Mirahmadi et al. 2010)
(d) 14 to 134 mL CH4/g TS (c) (Yao et al. 2013)
(d) (Nakamura and Mtui
2003)
(a) 50 to 340 mL CH4/g VS (a) (Kabir et al. 2015)
(b) 38.7 to 71.4 mL CH4/g (b) (Mirmohamadsadeghi
carbohydrate et al. 2014)
80 to 155 mL CH4/g VS (Jurado et al. 2013)
(a) 25 to 35 mL CH4/g VS (Mirmohamadsadeghi et al.
(b) reduces from 32 to 2016)
22 mL CH4/g VS
(c) reduces from 80 to
44 mL CH4/g VS
(a) 40 to 276 mL CH4/g VS (a) (Matsakas et al. 2015)
(b) 304 mL CH4/g VS (b) (Janzon et al. 2014)
(a) 210 mL CH4/g COD (a) (Ghimire et al. 2021)
(b) 14 to 124 mL CH4/g TS (b) (Nakamura and Mtui
2003)
(a) 45 to 180 mL CH4/g VS (a) (Take et al. 2006)
(b) 250 to 369 mL CH4/g VS (b) (Vivekanand et al. 2013)
(c) maximum production (c) (Mulat et al. 2018)
rate increased from 4 to 9 (d) (Dai et al. 2021)
–14 mL CH4/g VS/day (e) (Nakamura and Mtui
(d) 204.4 mL H2/L culture 2003)
for SE to 1055.8 mL H2/L
culture for SE + acid
(e) 14 to 194 mL CH4/g TS
253 mL CH4/g wood (Navarro et al. 2020)
13
134 Page 8 of 15 World Journal of Microbiology and Biotechnology (2021) 37:134

Table 2 (continued)
Pretreatment Feedstock Pretreatment condition Increase in yield compared References
to control

Microbial (a) sawdust (a) 12 mg dry microbial (a) 232 to 312 mL CH4/g VS (a) (Ali et al. 2017)
(b) albizia consortium/g sawdust at (b) 4.4 to 10.8 mL CH4/g VS (b) (Ge et al. 2015)
(c) Japanese cedar 30 °C for 10 days (c) 0 to 43 mL CH4/g TS (c) (Take et al. 2006)
(b) 5.18 g of chips/mL of
Ceriporiopsis subvermis-
pora
(c) different Basidiomycete
fungi incubated stationary
for 20 days at 37 °C
Chemo- Enzymatic oil palm 0.3 g lime/g palm trunk at 749 to 2249 mL H2/L sub- (Sitthikitpanya et al. 2017)
121 °C for 120 min fol- strate (2.04 mol/mol sugar
lowed by CTec2 treatment consumed)
at 30 FPU/g substrate at
50 °C for 72 h

VS volatile solids, COD chemical oxygen demand, NMMO N-methylmorpholine-N-oxide, SE steam explosion, TS total solids

Microbiology Biomethane yield

Key bacterial groups are responsible for the conversion of
substrates at each stage. Homoacetogens produce acetate
from CO2 and H2. CO2 and H2 are also transformed into
CH4 by hydrogenophilic methanogens. The acetoclastic
methanogens are Archaebacteria which transform acetate
into methane (Goswami et al. 2016). Firmicutes are the
dominant bacterial phyla in the thermophilic digestion
of both pretreated and untreated birch wood (Mulat et al.
2018). Members of Firmicutes, found in the human gut as
one of the dominant bacterial phyla (Magne et al. 2020),
are also dominant in anaerobic digesters along with
Bacteroidetes (Solli et al. 2014; Abendroth et al. 2015;
Stolze et al. 2016). The hydrogenotrophic Methanobacte-
riaceae is the most dominant methanogen in thermophilic
anaerobic digesters with birch wood as a substrate, rather
than the common acetoclastic methanogens (Mulat et al.
2018). Another study, investigating the natural micro-
bial flora anaerobically decomposing poplar wood chips,
found Firmicutes (46%) and Proteobacteria (32%) to be
the most abundant microbial phyla, followed by the Bac-
teroidetes (9.9%) (van der Lelie et al. 2012). Clostridia
was the most abundant class of Firmicutes in the bacterial
community. The dominant Proteobacteria was Alphapro-
teobacteria; of this, the family Rhodospirillaceae made
up 21.1% and the genus Magnetospirillum 19.9%, making
this the most abundant genus in the community. The Bac-
teroidetes were the third most abundant group of microor-
ganisms, representing nearly 10% of the total community.
No methane formation was reported in this process and no
Archaea community analysis was presented.
Under appropriate conditions anaerobic digestion can con-
vert cellulose, starch and hemicellulose (nearly 70% of
the woody material) into CH4, a greater energy yield than
bioethanol fermentation where only cellulose is used as
a substrate. Even inhibitory compounds such as F, HMF
and soluble lignin generated during pretreatment can be
transformed into CH4, if not highly concentrated (Fox et al.
2003). The theoretical methane yield based on the elemental
composition of the substrate (CaHbOcNd), assuming 100%
conversion, can be estimated by the following equation
(Lübken et al. 2010).
YTheoretical CH4 (L∕g VS) = 22.4 (4a + b − 2c − 3d)÷
(1)
8 (12a + b + 16c + 14d)
Based on Eq. (1), the theoretical CH4 yields from ligno-
cellulosic biomass (C5H9O2.5NS0.025) and woody biomass
(molecular weight 217.8 g/mol) are estimated as 480 (Frigon
and Guiot 2010) and 470 mL/g VS respectively. Table 2
shows CH4 yields obtained from untreated and pretreated
woody biomass at laboratory scale. Methane yields from
untreated wood are very low due to the recalcitrance of wood
to anaerobic microorganisms. Significant improvements to
CH4 yields after various pretreatments have been reported
(Table 2). CH4 yields can be as low as 30% of theoretical
for wood due to non-biodegradable lignin, peptidoglycan
and insoluble cellulose, hemicellulose and protein polymers
(Frigon and Guiot 2010). Unless lignin is separated from
biomass by pretreatment, it can limit holocellulose access to
enzymes through enmeshment and inhibit the holocellulose

13
World Journal of Microbiology and Biotechnology (2021) 37:134
degradation (Monlau et al. 2011). However, there is evi-
dence that parts of lignin or pseudo-lignin are used as sub-
strates for biogas production when birch is pretreated by SE
at a specific severity (Vivekanand et al. 2013).
Significant differences exist between softwood and hard-
wood CH4 yields. Softwood (pine), semi softwood (poplar)
and hardwood (berry) have been compared for CH4 produc-
tion potential in solid state AD (Mirmohamadsadeghi et al.
2016). Untreated hardwood produced consistently higher
CH4 yields to that of softwood. The effect of hydrothermal
and alkaline pretreatments on CH4 yield is also different
depending on the wood type. Pretreated hardwood (birch)
had higher CH4 yields than softwood (pine and spruce)
(Mirahmadi et al. 2010; Matsakas et al. 2015). This has
been attributed to the higher lignin content of softwood and
the different types of lignin present in softwoods and hard-
woods. Hardwood lignin contains a mixture of guaiacyl and
syringyl units and softwood lignin contains mainly guaia-
cyl units. Alkaline pretreated birch wood produced 460 mL
CH4/g volatile solids (Mirahmadi et  al. 2010), which is
comparable to CH4 yields from many other lignocellulosic
feedstocks (Martínez-Gutiérrez 2018).
Besides pretreatment, co-digestion can be an option for
improving methane yields from wood. Co-digestion is the
simultaneous digestion of a homogenous mixture of two
or more substrates. The use of co-substrates can improve
biogas yield due to the synergism established in the diges-
tion process, providing a better nutritional balance and
moisture content of the digester feed. The co-digestion of
manure and woody biomass is an example of utilization of
two substrates in an AD process (Li et al. 2019). Together
the materials complement each other and the risk of inhi-
bition from an uncontrolled C:N ratio and high ammonia
concentration decreases. The manure fraction helps balance
the C:N ratio of the feedstock and provides a wide range of
nutrients (Ingersoll 2020). The combination of wood pre-
treatment and co-digestion with animal manure results in a
remarkable increase (76%) in methane yield (Li et al. 2019).
Biohydrogen from dark fermentation of wood
Dark fermentation to produce biohydrogen is the most
widely studied fermentation based H2 production process,
with a Technology Readiness Level (TRL) of 7 (Islam
et al. 2021). Dark fermentation to produce H2 is like the
acidogenic fermentation phase of AD but carried out in
the absence of light. Dark fermentation requires the least
amount of energy input of the H2 production pathways of
electrochemical (electrolysis), thermochemical (gasification)
and biological (biophotolysis, photo and dark fermentation,
microbial electrolysis) (Wang et al. 2008; Islam et al. 2021).
The production of biohydrogen from cellulose, hemicel-
lulose and starch without the need for photoenergy and a
Page 9 of 15 134
reduced space requirement makes dark fermentation 340
times cheaper than photosynthetic production (Atif et al.
2005).
Feedstocks and conversion chemistry
A variety of organic wastes such as agricultural, food,
sewage sludge and municipal wastewaters have been used
for biohydrogen production, but only a few studies have
explored the utilization of woody biomass (Islam et  al.
2021). Carbohydrate rich feedstocks produce better H2 yields
than protein and lipid rich feedstocks (Osman et al. 2020).
The carbohydrate rich holocellulose in lignocellulosic bio-
mass is proven as an effective substrate for biohydrogen pro-
duction, making woody biomass a potential future candidate
(Demirel et al. 2010; Guo et al. 2010).
The cellulose and hemicellulose portion of wood can be
hydrolysed to produce C5 and C6 sugars for fermentation.
These sugars, when dark fermented, produce H2 and CO2
and organic acids and alcohols in the liquid phase. Theo-
retically, 12 mol of H2 can be produced from a mole of
glucose (Eq. 2). However, glycolysis under dark fermen-
tation produces acetic, propionic and butyric acids as by-
products, limiting H2 production from glucose to 1—4 mols
(Eqs. 3–5). Equations 3–5 show that more H2 is produced
from the acetic acid pathway than from the butyric and pro-
pionic acid pathways. Similarly, more H2 is produced from
glucose than from xylose (Eqs. 3–7) (Antonopoulou et al.
2008; Kongjan et al. 2009; Osman et al. 2020). Therefore,
efficient pretreatments and enzymatic hydrolysis that break
down complex woody biomass to simple sugars are critical
in improving H2 yield from wood.
C6 H12 O6 + 6H2 O → 6CO2 + 12H2 (2)
C6 H12 O6 + H2 O → 2CH3 COOH + 2CO2 + 4H2 (3)
C6 H12 O6 → CH3 CH2 CH2 COOH + 2CO2 + 2H2 (4)
C6 H12 O6 → CH3 COOH + CH3 CH2 COOH + CO2 + H2
(5)
C5 H10 O5 + 1.67H2 O → 1.67CH3 COOH + 1.67CO2 + 3.33H2
(6)
C5 H10 O5 → 0.83CH3 CH2 CH2 COOH + 1.67CO2 + 1.67H2
(7)
Process microbiology and yields
Biohydrogen production from biomass via dark fermen-
tation is carried out by facultative and anaerobic micro-
organisms (Rittmann and Herwig 2012). Enterobacter,
Bacillus (facultative) and Clostridium (anaerobic) are
13
134 Page 10 of 15
the common heterotrophic microorganisms responsi-
ble for dark fermentation. Among them Clostridium sp,
Enterobacter sp and Thermoanaerobacterales (Family
III) sp are the most widely studied bacterial species in
pure and mixed culture dark fermentation studies (Monlau
et al. 2011; Rittmann and Herwig 2012). H2 yields from
Clostridium sp and Enterobacter sp are found to be 2 and
1 mol H2/mol glucose respectively (Monlau et al. 2011). A
H2 yield of 3.6 mmol/g cellulose (~ 0.6 mol H2/mol cellu-
lose) was obtained from microcrystalline cellulose using a
pure culture of Clostridium acetobutylicum as the biocata-
lyst (Ntaikou et al. 2010). Using a co-culture of Clostrid-
ium acetobutylicum and Ethanoigenens harbinense, an
improved yield of 8.1 mmol H2/g cellulose (1.3 mol H2/
mol cellulose) was obtained in the same study. Levin et al.
(2006) investigated H2 production from wood fibres using
the pure culture strain Clostridium thermocellum. Under
controlled laboratory conditions, a H2 yield of 1.6 mol/
mol glucose was obtained, which equates to 40 and 80%
of the theoretical H2 production from acetate and butyrate
pathways, respectively. However, monoculture processes
require aseptic conditions and pure substrates that make
them expensive to operate and limit the types of feedstocks
that can be used (Li and Chen 2007; Ntaikou et al. 2010).
Mixed cultures involve multiple H2 producing organ-
isms that can collectively improve H2 yields beyond the
theoretical limit (Thauer limit) of 4 mol H2/mol glucose
(or 497.8 mL H2/g-glucose) obtained for single cultures
(Ergal et al. 2020; Wang et al. 2020). By precision design
of artificial microbial consortia, biohydrogen yield has
been increased beyond the “Thauer limit” indicating that
dark fermentation is a promising pathway along with AD
(Ergal et al. 2020). Similarly, mixed culture organisms
can symbiotically break down complex organic substrates,
allowing increased H2 production together with other co-
products. Hydrogen yields of 6.25 mmol H2/g glucose and
3.43 mmol H2/g protein are reported at an initial neutral
pH using mixed cultures of microorganisms (Xiao et al.
2010). The limitation, however, was the proliferation of
non-hydrogen-producing organisms such as methano-
gens, homoacetogens and lactic acid bacteria within the
mixed culture, which produced unwanted by-products and
consumed the H2 produced. The homoacetogenic bacte-
ria present in mixed culture glucose fermentations can
consume H2 and CO2 to form acetic acid and water. Both
H2 production and consumption pathways are thermody-
namically favourable, but H2 accumulation improves the
thermodynamic favourability of H2 utilization pathways,
requiring the produced H2 to be removed to reduce its par-
tial pressure (Bhatia et al. 2021). High H2 partial pressures
tend to decrease H 2 production by reducing ferredoxin,
affecting hydrogenase activity. If the process is not care-
fully controlled, proliferation of non-hydrogen-producing
13
World Journal of Microbiology and Biotechnology (2021) 37:134
bacteria can lead to no H2 production (Guo et al. 2010;
Ntaikou et al. 2010).
Microbial cultures extracted from rotted wood crumbs
have been used to produce biohydrogen from wheat straw
(Cao et al. 2013). The enriched culture produced 3.5 mmol
H2/g substrate. Recently (Dai et al. 2021) used bamboo
hydrolysate to produce biohydrogen. The integration of
acid and steam explosion pretreatments enhanced H2 fer-
mentation efficiency, resulting in a yield of 1056 mL H2/L
culture. It is potentially a desirable pretreatment option for
large-scale applications. Dilute acid pretreated pine wood
produced 1629 mL H2/L/d at a yield of 0.99 mL H2/mol
sugar (Gonzales et al. 2016). The rates and yields of H2
production from wood were comparable to H2 production
from empty palm fruit bunch and rice husks. These results
also suggest that inhibitors produced by pretreatment have a
significant effect on H2 yield. These studies demonstrate that
woody biomass can be an alternative to other lignocellulosic
biomass feedstocks.
Biohythane from dark fermentation
and methanogenesis
Biohythane is a H2-CH4 blend containing up to 30% H2
by volume. Biohythane can be produced from a two-stage
fermentation process like a two-stage AD process that has
separate dark fermentation and methanogenic stages (Fig. 3).
The soluble metabolites produced by dark fermentation are
utilized by methanogenic bacteria to produce CH4 and CO2.
The separation of the dark fermentation and methanogenesis
stages reduces fermentation time and allows better process
control (Monlau et al. 2011; Liu et al. 2013; Si et al. 2016).
Although a sequential two-stage process can be used to pro-
duce biohythane, the two stages require different environ-
mental, nutritional and physiological conditions (Kongjan
et al. 2011). The first stage of dark fermentation requires a
pH of 5–7 and a hydraulic retention time (HRT) of 1–3 days
typical of the acidogenic stage of AD. The methanogen-
esis stage requires a neutral pH of 7–8 and a longer HRT
of 15–20 days to sustain the slow growing methanogens
(Mamimin et al. 2015).
The production of biohythane from a single feedstock
using a staged fermentation process has been experimentally
demonstrated. Staged production of biohydrogen and biom-
ethane from oil palm trunk yielded 2 mol H2/mol sugar but
the contribution of biohydrogen to the overall energy yield
was less than 1% (Sitthikitpanya et al. 2017). A two-stage
biohydrogen and biomethane production system using pop-
lar wood waste increased overall energy yield by 33% and
18% for liquid and solid samples over a single-stage biom-
ethane production system (Akobi 2016). The H2 in hythane
gas is reported to improve the combustion characteristics
of CH4. Several countries have initiated testing to blend H2
World Journal of Microbiology and Biotechnology (2021) 37:134
Fig. 3 Schematic representation of biohythane production steps
into existing natural gas network with early results indicat-
ing up to 20% H2 could be safely added to gas networks
without affecting the existing gas distribution infrastructure
(Quarton and Samsatli 2018). This further strengthens the
case for future production and use of commercial scale bio-
hythane gas.
Future perspectives
Although, wood to biogas production is not at the TRL
of 9, biogas production from other feedstocks has been
commercialised in many countries including Germany,
United Kingdom and Sweden, creating significant biogas
supplies for domestic, industrial and transportation sec-
tors. Wood is identified as a viable feedstock for pellets
and briquettes (solid biofuels) and bio oil (liquid biofuels)
with commercial operations in many jurisdictions. How-
ever, wood to biogas (gaseous biofuel) production is still
in its infancy, mainly due to techno-economic challenges
associated with pretreatment. Part of the pretreatment
costs can be offset by valorising the side streams contain-
ing lignin, digestate and CO2. Pretreatment technologies
have evolved to support commercial bioethanol produc-
tion from lignocellulosic biomass and these technologies
can be adapted and combined to further improve the rate
and extent of enzymatic hydrolysis to produce metabo-
lizable sugars and precursor molecules from wood. New
Page 11 of 15 134
discoveries of fungal species able to hydrolyse a greater
fraction of recalcitrant softwood offer prospects for further
increasing sugar yields (O’Callahan et al. 2020). Hyper-
thermophilic fungal hydrolysis is a recent development
with the potential to reduce costs associated with physico-
chemical pretreatments (Ingersoll 2020). Furthermore, dry
fermentations (high solids fermentations) can reduce the
capital cost of fermentation, making AD more affordable.
Current biohydrogen yields from lignocellulosic bio-
mass are a major hindrance to wider adoption and scale up
of this process. Radical approaches to increasing rates and
yields are needed to make low-energy intensive fermenta-
tion pathways commercially viable. Process integration to
improve overall yields by sequential dark and photo fer-
mentation, and dark fermentation with microbial electroly-
sis have been proposed, but technical and scale up issues
need to be resolved to realise their full potential. Further
options for improving productivity are through microbial
inoculum selection, design of artificial microbial consor-
tia, improved substrate pretreatment and hydrolysis, opti-
mization of process operating parameters and metabolic
engineering of biocatalysts. Consolidated bioprocessing
with surface display technology can directly convert cellu-
lose and hemicellulose to target products without an inter-
mediate enzymatic hydrolysis step (Mbaneme and Chinn,
2015). Engineering microorganisms to simultaneously
metabolise cellulose and hemicellulose can significantly
increase H2 production (Xiong et al. 2018).
13
134 Page 12 of 15
Conclusions
Woody biomass as a feedstock for fermentative gaseous
fuel production has attracted research interest in the last
two decades. Woody material is a particularly challenging
biomass to ferment but offers benefits such as scale, exist-
ing logistics and high density. The rigid wood structure
necessitates a combination of two or more pretreatments
as a first step to enhance production of gaseous biofuels.
These pretreatments reduce cellulose crystallinity, sepa-
rate the recalcitrant lignin fraction and provide access for
enzymes to hydrolyse the cellulose and hemicellulose frac-
tions of wood. Different pretreatment methods of varying
severity have been researched for different wood types
with mixed success. A common finding is that thermal pre-
treatments and organosolv are appropriate for softwoods
whereas alkaline, hydrothermal and SE are advantageous
for hardwoods. Acid pretreatment is advantageous for H2
production whereas a combination of thermal and chemi-
cal (SE, wet oxidation and alkaline) pretreatments is better
for CH4 production. Biogas yield from woody biomass is
dependent on wood type and pretreatment; optimizing and
controlling both these factors can result in biogas yields
comparable to other lignocellulosic feedstocks. Improved
biogas yields can be achieved through co-digestion with
nutrient rich substrates such as manure. Some studies
investigating biohydrogen production have demonstrated
the potential to reach and pass the Thauer Limit. Bio-
hythane production could be an effective route to improve
energy yields from wood, with the advantage that it can be
integrated into existing natural gas distribution networks.
While there are technological barriers to overcome before
there is wider adoption of wood derived gaseous biofuels
at scale, current research shows that woody biomass is a
promising feedstock for gaseous biofuel production, and
there is plenty of potential to further improve gas yields.
Acknowledgements The authors would like to acknowledge Kim
McGrouther, Dr Mark West and Dr Paul Bennett for providing critical
comments and Dale Corbett and Milla Baker for graphics and bibliog-
raphy support respectively.
Author contributions Both SW and AV have formulated the idea, per-
formed the literature search, data analysis and drafted this manuscript.
Funding This work was financially supported by the New Zealand
Government’s Ministry of Business, Innovation and Employment via
Scion’s Strategic Science Investment Fund.
Declarations
Conflict of interest The authors declare that there are no conflicts of
interest with this work.
13
World Journal of Microbiology and Biotechnology (2021) 37:134
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