**4. Production of microbial lipids from lignocellulose biomass**

The bioconversion of lignocellulose to the microbial lipids includes following steps: pretreatment of lignocellulose biomass, hydrolysis of structural carbohydrates to fermentable sugars, microbial production of lipids and isolation and purification of the product. Since most of the oleaginous microorganisms lack cellulase and hemicellulase activity, structural polysaccharides in lignocellulosic biomass has to be hydrolysed to fermentable sugars (mainly xylose and glucose) which microorganism can use as a carbon source. The structural polysaccharides are hydrolysed using cellulolytic enzymes or thermochemical process conduct at elevated temperature in the presence of concentrate acid catalyst. Enzymatic hydrolysis is preferred over thermochemical route since the reaction is carried out under mild conditions (pH and temperature) in non-corrosive environment. Furthermore, inhibitors that could potentially inhibit the microorganism are not formed [65–67]. The major drawbacks of enzymatic hydrolysis are longer hydrolysis time, higher price of enzyme and inhibition by end products [67–70]. Production of the oleaginous lipids from lignocellulosic biomass is carried out using three process configurations such as separate hydrolysis and lipid production (SHLP), simultaneous saccharification and lipid production (SSLP) and consolidate bioprocessing (CBP, **Figure 2**). The production of the lipids by SHLP involves two separate steps, enzymatic hydrolysis of lignocellulose followed by lipid production, while in SSLP these steps are integrated and carried out simultaneously in one vessel. In SHLP both steps are run under optimal conditions for microorganism (pH = 4.8–6.0, T = 25–30°C) and cellulases (pH = 4.5–6,0; T = 50–60°C) [25, 71, 72]. However, inhibition of cellulase by accumulated glucose and cellobiose decreases the yield of fermentable sugars. In SSLP, sugars released by hydrolysis are simultaneously assimilated by microorganism minimizing the inhibition effect by the end-product. Elimination of enzyme inhibition enhances the rate of carbohydrate hydrolysis and shortens the process time. Since the enzyme hydrolysis and microorganism growth are carried out in one vessel, the number of vessels needed for the process is reduced, decreasing the capital costs. The main disadvantage of SSLP in comparison to SHLP is the necessity of running the process at temperature favorable for the microbial growth (T = 30–32°C) which is usually suboptimal for the cellulase hydrolysis [67]. To compensate lower activity at the process temperature, enzyme loading is increased. Alternatively, lipids could be produced in a process known as 'Consolidate bioprocessing', which gain much attention in the production of lignocellulosic bioethanol [73]. CBP integrates cellulase production, carbohydrate hydrolysis and lipid production in one step. Besides high lipid productivity and titer, the industrially viable CBP-strain has to efficiently secrete cellulases for hydrolysis of carbohydrates. Suitable microorganism for the CBP could be isolated from nature or alternatively designed by genetic engineering using two strategies already used in development of CBP yeast strain for the lignocellulosic bioethanol production [74]. The first strategy includes a heterologous expression of the cellulose degrading genes in the oleaginous

microorganism and the second strategy includes a metabolic engineering of cellulolytic micro-

**Figure 2.** Production of microbial lipids from lignocellulosic biomass by separate hydrolysis and lipid production

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143

(SHLP), simultaneous saccharification and lipid production (SSLP) and consolidate bioprocessing (CBP).

Microbial production of oleaginous lipids from lignocellulosic biomass is carried out either by

**Tables 1** and **2** summarize processes of lipid production by SHLP and SSLP in submerged culture. Most of the researches have been done in shake-flask cultures at 30°C, pH between 5 and 6, using 10% (v/v) of inoculum and buffer to maintain constant pH [57, 75–77]. The most favorable feedstocks for lipid production are agriculture waste, corn stover (stalks, leaves and cob) and corn cobs. Other lignocellulosic feedstocks used for lipid production include energy crops (*Panicum virgatum* and *Jerusalem artichoke*), forest residue (*Douglas fir*) and agriculture waste (sweet sorghum bagasse). The performance of the process depends on the cultivation mode (batch and fed-batch), the pretreatment method, method for carbohydrate hydrolysis, the substrate loading and type of microorganism. Acid and alkali pretreatments are the most often used methods for improving the digestibly of lignocellulose by cellulase [57, 75, 78–84]. Hydrolysis of structural polysaccharides is commonly carried out using enzymatic hydrolysis [27, 57, 75–77, 84–86]. The efficiency of cellulase hydrolysis mostly depends on pretreatment method, but also on used commercial cellulase. For efficient hydrolysis, at least 10 different enzymes are needed including enzymes from the glycoside hydrolase families 7 (CBHI, EGI), 6 (CBHII), 5 (EGII), 10 and 11 (xylanases) and 3 (ß-glucosidases) as well as the acetyl xylan esterases. Commercial cellulase preparations are constantly improved and their prices are being reduced. Thus, Cellic CTech2 and Cellic CTech3 from Novozymes (www.novozymes.com)

organism for improved lipid accumulation.

submerged or by solid state cultivation.

**4.1. Submerged production of lipids**

**Figure 2.** Production of microbial lipids from lignocellulosic biomass by separate hydrolysis and lipid production (SHLP), simultaneous saccharification and lipid production (SSLP) and consolidate bioprocessing (CBP).

microorganism and the second strategy includes a metabolic engineering of cellulolytic microorganism for improved lipid accumulation.

Microbial production of oleaginous lipids from lignocellulosic biomass is carried out either by submerged or by solid state cultivation.

#### **4.1. Submerged production of lipids**

and extractive compounds present in the lignocellulosic biomass [45, 49–51]. The most common aromatic compounds in the lignocellulose acid hydrolysate are vanillin, syringaldehyde, 4-hydroxybenzoic acid, ferulic acid, etc. [45, 51]. Formation of degradation by-product strongly depends on the plant source and pretreatment process (temperature, pressure, reac-

The bioconversion of lignocellulose to the microbial lipids includes following steps: pretreatment of lignocellulose biomass, hydrolysis of structural carbohydrates to fermentable sugars, microbial production of lipids and isolation and purification of the product. Since most of the oleaginous microorganisms lack cellulase and hemicellulase activity, structural polysaccharides in lignocellulosic biomass has to be hydrolysed to fermentable sugars (mainly xylose and glucose) which microorganism can use as a carbon source. The structural polysaccharides are hydrolysed using cellulolytic enzymes or thermochemical process conduct at elevated temperature in the presence of concentrate acid catalyst. Enzymatic hydrolysis is preferred over thermochemical route since the reaction is carried out under mild conditions (pH and temperature) in non-corrosive environment. Furthermore, inhibitors that could potentially inhibit the microorganism are not formed [65–67]. The major drawbacks of enzymatic hydrolysis are longer hydrolysis time, higher price of enzyme and inhibition by end products [67–70]. Production of the oleaginous lipids from lignocellulosic biomass is carried out using three process configurations such as separate hydrolysis and lipid production (SHLP), simultaneous saccharification and lipid production (SSLP) and consolidate bioprocessing (CBP, **Figure 2**). The production of the lipids by SHLP involves two separate steps, enzymatic hydrolysis of lignocellulose followed by lipid production, while in SSLP these steps are integrated and carried out simultaneously in one vessel. In SHLP both steps are run under optimal conditions for microorganism (pH = 4.8–6.0, T = 25–30°C) and cellulases (pH = 4.5–6,0; T = 50–60°C) [25, 71, 72]. However, inhibition of cellulase by accumulated glucose and cellobiose decreases the yield of fermentable sugars. In SSLP, sugars released by hydrolysis are simultaneously assimilated by microorganism minimizing the inhibition effect by the end-product. Elimination of enzyme inhibition enhances the rate of carbohydrate hydrolysis and shortens the process time. Since the enzyme hydrolysis and microorganism growth are carried out in one vessel, the number of vessels needed for the process is reduced, decreasing the capital costs. The main disadvantage of SSLP in comparison to SHLP is the necessity of running the process at temperature favorable for the microbial growth (T = 30–32°C) which is usually suboptimal for the cellulase hydrolysis [67]. To compensate lower activity at the process temperature, enzyme loading is increased. Alternatively, lipids could be produced in a process known as 'Consolidate bioprocessing', which gain much attention in the production of lignocellulosic bioethanol [73]. CBP integrates cellulase production, carbohydrate hydrolysis and lipid production in one step. Besides high lipid productivity and titer, the industrially viable CBP-strain has to efficiently secrete cellulases for hydrolysis of carbohydrates. Suitable microorganism for the CBP could be isolated from nature or alternatively designed by genetic engineering using two strategies already used in development of CBP yeast strain for the lignocellulosic bioethanol production [74]. The first strategy includes a heterologous expression of the cellulose degrading genes in the oleaginous

**4. Production of microbial lipids from lignocellulose biomass**

tion time and presence of catalyst) [46–48, 51].

142 Advances in Biofuels and Bioenergy

**Tables 1** and **2** summarize processes of lipid production by SHLP and SSLP in submerged culture. Most of the researches have been done in shake-flask cultures at 30°C, pH between 5 and 6, using 10% (v/v) of inoculum and buffer to maintain constant pH [57, 75–77]. The most favorable feedstocks for lipid production are agriculture waste, corn stover (stalks, leaves and cob) and corn cobs. Other lignocellulosic feedstocks used for lipid production include energy crops (*Panicum virgatum* and *Jerusalem artichoke*), forest residue (*Douglas fir*) and agriculture waste (sweet sorghum bagasse). The performance of the process depends on the cultivation mode (batch and fed-batch), the pretreatment method, method for carbohydrate hydrolysis, the substrate loading and type of microorganism. Acid and alkali pretreatments are the most often used methods for improving the digestibly of lignocellulose by cellulase [57, 75, 78–84]. Hydrolysis of structural polysaccharides is commonly carried out using enzymatic hydrolysis [27, 57, 75–77, 84–86]. The efficiency of cellulase hydrolysis mostly depends on pretreatment method, but also on used commercial cellulase. For efficient hydrolysis, at least 10 different enzymes are needed including enzymes from the glycoside hydrolase families 7 (CBHI, EGI), 6 (CBHII), 5 (EGII), 10 and 11 (xylanases) and 3 (ß-glucosidases) as well as the acetyl xylan esterases. Commercial cellulase preparations are constantly improved and their prices are being reduced. Thus, Cellic CTech2 and Cellic CTech3 from Novozymes (www.novozymes.com)


**Table 1.** Overview of various pretreatment methods.

have improved cellobiohydrolases, endoglucanases, ß-glucosidases and additional oxidative activity (auxiliary activity family 9, formerly known as GH61) for enhanced sugar yield especially at the high substrate loading [87, 88]. Spent liquors from acid pretreatment of lignocellulosic biomass are also used as a carbon source [57, 75, 78–84]. Unlike the enzymatic hydrolysate, spent liquor obtained by acid pretreatment of a lignocellulosic biomass contains lignocellulosederived products that can inhibit microorganism growth and synthesis of product as well as the enzyme activity [47, 48].

Economically feasible process for industrial cellulosic lipid production requires high final lipid titer (**Table 2**). Most of the research has been done in batch SHLP using different oleaginous strains of yeasts. Concentration of lipid and productivity of batch SHLP process depends on lignocellulose feedstock, microorganism, pretreatment method, detoxification method and type of carbohydrate hydrolysis. As shown in **Table 2**, in most of the batch SHLP under optimized culture conditions, lipid concentration and lipid productivity was below 20 g/L and 0.15 g/L h, respectively.

Harde et al. [86] cultivated *Mortierella isabellina* on pretreated biomass and detoxified spent liquor obtained by SPORL pretreatment of *Douglas fir*. For lipid production, three strategies were investigated. First two strategies included separate processing of pretreated biomass and spent liquor. Lignocellulosic biomass was subjected to separate hydrolysis and lipid production and simultaneous saccharification and lipid production with prehydrolysis step. Third strategy included hydrolysis of whole lignocellulosic slurry, detoxification and lipid production (**Tables 2** and **3**). Lipid yield produced from whole lignocellulosic slurry was lower than those from other two strategies, where pretreated biomass and spent liquor were

**Feedstock** Corn stover

*Trichosporon* 

H2SO4 (0.1–1%,

Spent liquor from acid pretreatment

Batch

—

7.6

39

0.15

0.078

[78]

140–180°C, 5–10

 min)

*cutaneum*

AS 2.571

Sweet

*Cryptococcus* 

Microwave radiation

Enzymatic hydrolysate of

Batch

15.5

—

64

0.11 d\*

[26]

pretreated sweet sorghum bagasse

(endoglucanase 778–1022 CMC U/g

DM, ß-glucosidase126–186 pNG/g

DM, xylanase 625–950 ABXU/g DM)

Enzymatic hydrolysate of pretreated

Batch

10.9 14.1

4.78 34

—

0.05

[57]

2.48 29

—

0.027

[57]

corn stover (26 FPU/g DM, substrate

loading 5%)

H2SO4 (1%, 121°C,

As above

> 2 h)

> > Corn stover

*Trichosporon* 

Pre-soaking with

Enzymatic hydrolysate of pretreated

Batch

—

0.97

—

—

0.014

[75]

corn cobs (cellulase 7 FPU/g DM,

substrate loading 10%)

Enzymatic hydrolysate of pretreated

Batch

16.5 7.2

43

0.138d\* —

[79]

corn stover (cellulase10 FPU/g DM,

cellobiase 20 CBU/g DM, xylanase

mg/g DM, substrate loading 5%)

Spent liquor from acid pretreatment

Enzymatic hydrolysate of pretreated

Batch

38.4

12.3 32

0.131

0.047f [76]

Production of Microbial Lipids from Lignocellulosic Biomass

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corn cob residue (cellulase 15 FPU/g

Batch

6.1

—

28

—

0.072

[80]

H2SO4 (190°C, 3

 min)

*cutaneum*

Corn stover

*Cryptococcus* 

Ionic liquid (1-ethyl-3-methylimidazolium

acetate, 140°C, 1

 h) 10

*curvatus*

Jerusalem

*Cryptococcus* sp.

49 min)

HNO3 (0.57%, 117°C,

artichoke

Corn cobs

*Trichosporon* 


pretreatment

conditions

DM)

substrate loading 15%)

*cutaneum*

ACCC20271

Corn stover

*Cryptococcus* 

NaOH (0.5

M, 80°C,

Enzymatic hydrolysate of pretreated

Batch

27.7

—

44

0.155

0.156

[81]

145

corn stover (cellulase: 20 FPU/g DM,

ß-glucosidase: 40 CBU/g DM, xylanase

U/g DM, 10% substrate loading)

140

*curvatus* ATCC

75 min)

20509

residue

*curvatus* ATCC

(100°C, 4 min h)

sorghum

bagasse

Corn stover

*Mortierella* 

NaOH (1%, 121°C,

*isabellina* ATCC

2 h)

42613

20509

**Microbial strain**

**Pretreatment**

**Cultivation media**

**Fermentation** 

**Xa**

**Lb**

**wL**

**YL/S**

**d**

**Pre**

**Reference**

**c**

**(g/L)**

**(g/L)**

**(%)**

**(g/g)**

**(g/L/h)**

**mode**


have improved cellobiohydrolases, endoglucanases, ß-glucosidases and additional oxidative activity (auxiliary activity family 9, formerly known as GH61) for enhanced sugar yield especially at the high substrate loading [87, 88]. Spent liquors from acid pretreatment of lignocellulosic biomass are also used as a carbon source [57, 75, 78–84]. Unlike the enzymatic hydrolysate, spent liquor obtained by acid pretreatment of a lignocellulosic biomass contains lignocellulosederived products that can inhibit microorganism growth and synthesis of product as well as

**Pretreatment process Effect on lignocellulosic biomass Disadvantage**

porosity of biomass [52–54]

cellulose, increase of crystallinity, increase of

hemicellulose, increase the surface area, reduction of degree of polymerization and crystallinity of cellulose [52, 53, 57–59]

Complete hemicellulose and minimal lignin removal, cellulose depolymerization [60]

Depolymerization and deacetylation of hemicellulose, depolymerization and cleavage of lignin-carbohydrate bonds [63, 64]

redistribution of lignin in biomass, deacetylation of hemicellulose [56]

Toxic and corrosive process, formation of inhibitors [40, 52, 55, 56]

Less corrosive and expressive process than dilute acid hydrolysis, formation of inhibitors, less efficient for feedstock with high lignin

Is not effective for biomass with higher lignin content, formation of some inhibitors [46, 61, 62]

Does not use chemical catalyst, less corrosive, minimal formation of

content [44, 52, 53, 58]

inhibitor [56]

Dilute acid hydrolysis Hydrolysis of hemicellulose and amorphous

Mild alkaline hydrolysis Delignification, partial hydrolysis of

Hydrothermal process Partial hydrolysis of hemicellulose,

**Table 1.** Overview of various pretreatment methods.

Economically feasible process for industrial cellulosic lipid production requires high final lipid titer (**Table 2**). Most of the research has been done in batch SHLP using different oleaginous strains of yeasts. Concentration of lipid and productivity of batch SHLP process depends on lignocellulose feedstock, microorganism, pretreatment method, detoxification method and type of carbohydrate hydrolysis. As shown in **Table 2**, in most of the batch SHLP under optimized culture conditions, lipid concentration and lipid productivity was below 20 g/L and

Harde et al. [86] cultivated *Mortierella isabellina* on pretreated biomass and detoxified spent liquor obtained by SPORL pretreatment of *Douglas fir*. For lipid production, three strategies were investigated. First two strategies included separate processing of pretreated biomass and spent liquor. Lignocellulosic biomass was subjected to separate hydrolysis and lipid production and simultaneous saccharification and lipid production with prehydrolysis step. Third strategy included hydrolysis of whole lignocellulosic slurry, detoxification and lipid production (**Tables 2** and **3**). Lipid yield produced from whole lignocellulosic slurry was lower than those from other two strategies, where pretreated biomass and spent liquor were

the enzyme activity [47, 48].

Sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL)

144 Advances in Biofuels and Bioenergy

Ammonia fiber expansion

(AFEX)

0.15 g/L h, respectively.


**Feedstock**

**Microbial strain**

As above As above As above As above

> Corn stover

*Rhodosporidium* 

NaOH (0.4%, 80°C,

Enzymatic hydrolysate of pretreated

Batch

36.2

21.4 59

0.19

0.28

[84]

corn stover (cellulase 40

cellulose- substrate loading 20%)

mg protein/g

*toruloides*

As above As above As above aX: Biomass concentration, g cell/L.

bL: Lipid concentration, g lipids/L.

cwL: Lipid content, g lipid produced/g dry cell weight.

ePr: Lipid productivity, g lipid produced/h L

fLipid productivity was calculated based on time for prehydrolysis (3

gLipid productivity, lipid concentration (L)/time of cultivation (216

hLipid productivity, lipid concentration (L)/time of cultivation (120

iLipid productivity, lipid concentration (L)/time of cultivation (168

**Table 2.**

 h).

 h).

 h). Production of lipids by separate hydrolysis and lipid production (SHLP) from lignocellulosic hydrolysate.

days) and fermentation (8

days): lipid concentration/time.

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dYL/S: Lipid yield, g lipid/g of consumed carbon source; d\*YL/S: Lipid yield, g lipid/g pretreated lignocellulosic biomass.

As above

As above

As above

As above As above As above

DO-stat fed-batch

Pulse fed-batch

Online sugar

54

32

59

0.29

0.4

[84]

monitoring

fed-batch

43

26.7 62

0.24

0.35

[84]

42

25.2 60

0.23

0.33

[84]

2 160°C, 10 min)

h) and H2SO4 (0.8%,

SO2 (11 120 min)

g/L, 140°C,

As above

Batch

38.2

17.6 —

0.18

0.081i [86]

SO2 (11 60 min)

g/L, 140°C,

Detoxified enzymatic hydrolysate of

Batch

35.4

18.55 —

0.17

0.086i [86]

whole pretreated slurry (cellulase 14.6

FPU/g glucan, substrate loading 10%)

SO2 (11 120 min)

g/L, 140°C,

As above

SO2 (11 60 min)

g/L, 140°C,

Detoxified spent liquor

Batch Batch

11.6 7.7

—

0.16

0.046h [86]

**Pretreatment**

**Cultivation media**

**Fermentation** 

**Xa**

**Lb**

**wLc**

**YL/Sd**

**Pre**

**Reference**

**(g/L)**

16.05 8.4

—

0.18

0.050h [86]

**(g/L)**

**(%)**

**(g/g)**

**(g/L/h)**

**mode**


**Feedstock**

Sweet

*Lipomyces* 

No pretreatment

Enzymatic hydrolysate of sweet

sorghum stalks cellulase (8 FPU/g,

Celluclst 1.5

L: Novozyme 188

(ß-glucosidase) at a1:5 (vol/vol)

Undetoxified spent liquor form acid

Batch/65 h/

15.1 5.5

36

0.129

0.09

[82]

bioreactor

cultivation

Fed-batch with

75.4

30.6 39

0.146

0.15

[82]

constant C and N

feed

Fed-batch with

70.8

33.5 47

0.159

0.17

[82]

two stage feeding

strategy (1st

C +

C-source)

Batch

53.4

29.0 53

0.156

0.215

[83]

N-source, 2nd

pretreatment

sorghum

*starkeyi*

CBS 1807

stalks

Corn cobs

*Rhodotorula* 

Mixed acids (0.5%

*glutinis* CGMCC

H2SO4 + 1.5% H PO3 4,

123°C)

2.703

As above As above

> Switchgrass

*Lipomyces* 

H2SO4 (0.936%, 160°C,

Undetoxified spent liquor form acid

*tetrasporus*

15

min, 20% solids)

pretreatment

Y-11562

*Lipomyces* 

As above

As above

As above

47.7

28.1 59

0.161

0.179

[83]

*kononenkoae*

Y-7042

*Rhodosporidium* 

As above

As above

As above

42.6

26.2 61

0.159

0.128

[83]

*toruloides* Y-1091

Corn stover

*Mortierella* 

steam explosion

Enzymatic hydrolysate of pretreated

Batch

36.1

18.7 52

0.05f

0.039

[85]

corn stover (cellulase 30 FPU/g,

substrate loading 30%)

Enzymatic hydrolysate of pretreated

Batch

25.5

14.4 —

0.18

0.120g [86]

lignocellulose biomass (cellulase 14.6

FPU/g glucan, substrate loading 10%)

(200°C, 7 min)

*isabellina*

Douglas

*Mortierella* 

SO2 (11

g/L, 140°C,

*isabellina* NRRL

60 min)

fir forest

residue

1757

As above

SO2 (11 120 min)

g/L, 140°C,

As above

Batch

25.7

11.9 —

0.18

0.120g [86]

As above

As above

As above

As above

**Microbial strain**

**Pretreatment**

**Cultivation media**

**Fermentation** 

**Xa**

**Lb**

**wLc**

**YL/Sd**

**Pre**

**Reference**

**(g/L)**

**(g/L)**

**(%)**

**(g/g)**

**(g/L/h)**

**mode**

Batch

6.4

—

29

0.077

0.033

[77]

146 Advances in Biofuels and Bioenergy

processed separately. Despite the lower process efficiency, this approach is attractive from the economic point of view since it reduces the operational and capital costs for lipid production. Development of the strain with high tolerance toward inhibitors in spent liquor could reduce the number of steps in production and production cost. Harde et al. [86] developed sulfite tolerant strain of *M. isabellina* by gradual adaptation of the strain to inhibitors from spent liquor. The sulfite-adapted strain was able to grow in the presence of 2.0 g/L of sulfite in synthetic media and spent liquor [86].

lipid production by yeast *R. glutinis* using the undetoxified spent liquor from acid pretreatment of corn cobs as a carbon source [83]. In this study, the lipid productivity was remarkably improved using two feeding strategies regarding the dynamics of nitrogen supplementation. Since yeast *R. glutinis* showed high tolerance toward inhibitors, a corn cob acid hydrolysate was used without detoxification. First strategy included feeding with concentrated undetoxified spent liquor (790.2 g/L xylose and 40.5 g/L glucose) supplemented with the nitrogen source. The second strategy included feeding of the culture for the first 80 h of cultivation with concentrated undetoxified spent liquor supplemented with nitrogen source and afterwards only with the carbon source. The highest biomass concentration of 75.4 g/L was obtained using first feeding strategy, while second feeding strategy resulted with the highest lipid concentration of 33.5 g/L, which is the highest value of lipid concentration reported in literature

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Still most of the studies on lipid production have been done by SHLP using hydrolysate of lignocellulosic biomass as a carbon source. Research by Gong et al. [79, 81] showed that the efficiency can be improved by integrating the enzyme hydrolysis and microbial process applying SSLP (**Table 3**). Two SSLP processes were conducted in cultivation media with and without the addition of nitrogen source. In cultivation media-containing alkaline pretreated corn stover without nitrogen, cells did not grow due to the lack of nitrogen and carbon sources was used for lipid production. To obtain high lipid productivity, the culture media was inoculated at high inoculums size (7.2 g/L), while control culture supplemented with nitrogen was inoculated at average inoculums size (10% v/v). The highest lipid productivity of 0.195 g/L h was obtained in SSLP without nitrogen source and this is the highest value reported for the SSLP using the lignocellulose as a carbon source. In comparison to the SHLP using the same pretreated lignocellulosic biomass (**Table 2**), the productivity of SSLP was improved and loading of cellulase and xylanase was reduced for 50%, while ß-glucosidase was not used. The major disadvantage of this strategy is increased cost for cultivation of larger quantities of inoculum using enriched growth media [81]. Gong et al. [79] applied similar strategy using the corn stover pretreated with ionic liquids as a carbon source. However, lower lipid yields of 0.125 and 0.135 g/ g DM were obtained for SSLP and SHLP, respectively [79]. SSLP was also conducted with fungus *M. isabellina* using pretreated *Douglas fir* forest residue but without success. After prehydrolysis step, fungus was able to grow in semi-solid media obtaining 17.0 g/L of lipids. However, the productivity of lipid synthesis with fungus was half of those obtained in SHLP

with detoxified enzymatic hydrolysate of whole pretreated Douglas fir [86].

All cultivations were carried out at substrate loading of 10% (w/w) or lower suggesting possible problems with the enzyme hydrolysis and microorganism growth at higher substrate loadings. The product titer could be improved by increase of the substrate loading conducting so-called high-gravity fermentations which have been successfully applied in the bioethanol production from starch and lignocellulosic feedstock by simultaneous hydrolysis and fermentation. Significant savings in energy input, decrease of waste discharge, distillation costs and capital costs increased the competiveness of the process [91]. However, running the SSLP under high-gravity conditions imposes a number challenges with respect to the lignocellulosederived inhibitors and mixing and mass transfer in cultivation broth. Due to high substrate loading, the concentration of inhibitory by-products are increased and consequently lead to the

for culture grown on lignocellulose hydrolysate [82].

Slininger et al. [83] designed two step screening assay for the detection of the highly productive yeast strains with high tolerance to lignocellulose-derived inhibitors. Growth media contained undetoxified enzyme hydrolysate of corn stover pretreated by ammonia fiber expansion (AFEX) and acid pretreated switchgrass. Three yeast strains, *Lipomyces tetrasporus*, *Lipomyces kononenkoae* and *Rhodosporidium toruloides* were identified. Yeast strains were able to grow on the undetoxified switchgrass hydrolysate and accumulate 25–30 g/L lipids at the rate of 0.128–0.215 g/L h with lipid yield of 0.156–0.161 g/g of consumed substrate [83]. Those values are the highest values reported in literature for batch cultivation of oleaginous microorganisms using lignocellulosic hydrolysate. Contrary to expectation, performance of the isolated oleaginous yeasts was significantly better than other used yeasts in SHLP with detoxified spent liquor (**Table 2**). Some oleaginous microorganisms show high tolerance to most of lignocellulose-derived inhibitors. Indeed, yeast strain *R. toruloides* tolerates acetate, 5-hydroxymethylfurfural and syringaldehyde at concentrations below 70, 14.7 and 12 mM, respectively. Negligible effect on growth and lipid production showed the presence vanillin and *p*-hydroxybenzoate at concentrations below 10 mM. The strongest inhibitory effect on growth and lipid accumulation had furfural. At concentration of 1 mM, biomass and lipid concentrations dropped by 45.5 and 26.5% [89].

Fed-batch mode of cultivations in production of microbial lipids has already been proved to be superior to batch cultivation. High cell and lipid concentration of 106.5 and 71.9 g/L (67.5%), respectively, were obtained in pilot scale fed-batch in a 15 L stirred tank bioreactor cultivation by yeast *R. toruloides* using glucose as a carbon source with the productivity of 0.54 g/L h [90]. Fei et al. [84] applied fed-batch cultivation mode to improve the efficiency of lipid production by *R. toruloides* using lignocellulosic hydrolysate. Different feeding strategies of the culture were investigated including dissolved oxygen-stat (DO-stat) feeding mode, pulse feeding mode and online sugar control mode. All three fed-batch strategies improved processes performance in comparison to the batch cultivation in terms of cell concentration, lipid yield and process productivity. The highest lipid yield of 0.29 g/g and lipid productivity 0.4 g/(L h) was obtained using the online sugar control feeding mode. Those values are the highest reported in the literature obtained by using concentrated enzymatic hydrolysate of lignocellulose biomass. This study represents major breakthrough in the research of lipid production from lignocellulosic biomass that could improve feasibility of the bioprocess. However, production of concentrated lignocellulosic hydrolysate (~ 550 g/L) used in research relies on the cost-intensive evaporation [84].

Therefore, developing new methods for preparation of concentrated lignocellulosic hydrolysate could improve the process economics. Fed-batch cultivation was applied in process of the lipid production by yeast *R. glutinis* using the undetoxified spent liquor from acid pretreatment of corn cobs as a carbon source [83]. In this study, the lipid productivity was remarkably improved using two feeding strategies regarding the dynamics of nitrogen supplementation. Since yeast *R. glutinis* showed high tolerance toward inhibitors, a corn cob acid hydrolysate was used without detoxification. First strategy included feeding with concentrated undetoxified spent liquor (790.2 g/L xylose and 40.5 g/L glucose) supplemented with the nitrogen source. The second strategy included feeding of the culture for the first 80 h of cultivation with concentrated undetoxified spent liquor supplemented with nitrogen source and afterwards only with the carbon source. The highest biomass concentration of 75.4 g/L was obtained using first feeding strategy, while second feeding strategy resulted with the highest lipid concentration of 33.5 g/L, which is the highest value of lipid concentration reported in literature for culture grown on lignocellulose hydrolysate [82].

processed separately. Despite the lower process efficiency, this approach is attractive from the economic point of view since it reduces the operational and capital costs for lipid production. Development of the strain with high tolerance toward inhibitors in spent liquor could reduce the number of steps in production and production cost. Harde et al. [86] developed sulfite tolerant strain of *M. isabellina* by gradual adaptation of the strain to inhibitors from spent liquor. The sulfite-adapted strain was able to grow in the presence of 2.0 g/L of sulfite in synthetic

Slininger et al. [83] designed two step screening assay for the detection of the highly productive yeast strains with high tolerance to lignocellulose-derived inhibitors. Growth media contained undetoxified enzyme hydrolysate of corn stover pretreated by ammonia fiber expansion (AFEX) and acid pretreated switchgrass. Three yeast strains, *Lipomyces tetrasporus*, *Lipomyces kononenkoae* and *Rhodosporidium toruloides* were identified. Yeast strains were able to grow on the undetoxified switchgrass hydrolysate and accumulate 25–30 g/L lipids at the rate of 0.128–0.215 g/L h with lipid yield of 0.156–0.161 g/g of consumed substrate [83]. Those values are the highest values reported in literature for batch cultivation of oleaginous microorganisms using lignocellulosic hydrolysate. Contrary to expectation, performance of the isolated oleaginous yeasts was significantly better than other used yeasts in SHLP with detoxified spent liquor (**Table 2**). Some oleaginous microorganisms show high tolerance to most of lignocellulose-derived inhibitors. Indeed, yeast strain *R. toruloides* tolerates acetate, 5-hydroxymethylfurfural and syringaldehyde at concentrations below 70, 14.7 and 12 mM, respectively. Negligible effect on growth and lipid production showed the presence vanillin and *p*-hydroxybenzoate at concentrations below 10 mM. The strongest inhibitory effect on growth and lipid accumulation had furfural. At concentration of 1 mM, biomass and lipid

Fed-batch mode of cultivations in production of microbial lipids has already been proved to be superior to batch cultivation. High cell and lipid concentration of 106.5 and 71.9 g/L (67.5%), respectively, were obtained in pilot scale fed-batch in a 15 L stirred tank bioreactor cultivation by yeast *R. toruloides* using glucose as a carbon source with the productivity of 0.54 g/L h [90]. Fei et al. [84] applied fed-batch cultivation mode to improve the efficiency of lipid production by *R. toruloides* using lignocellulosic hydrolysate. Different feeding strategies of the culture were investigated including dissolved oxygen-stat (DO-stat) feeding mode, pulse feeding mode and online sugar control mode. All three fed-batch strategies improved processes performance in comparison to the batch cultivation in terms of cell concentration, lipid yield and process productivity. The highest lipid yield of 0.29 g/g and lipid productivity 0.4 g/(L h) was obtained using the online sugar control feeding mode. Those values are the highest reported in the literature obtained by using concentrated enzymatic hydrolysate of lignocellulose biomass. This study represents major breakthrough in the research of lipid production from lignocellulosic biomass that could improve feasibility of the bioprocess. However, production of concentrated lignocellulosic hydrolysate (~ 550 g/L) used in research

Therefore, developing new methods for preparation of concentrated lignocellulosic hydrolysate could improve the process economics. Fed-batch cultivation was applied in process of the

media and spent liquor [86].

148 Advances in Biofuels and Bioenergy

concentrations dropped by 45.5 and 26.5% [89].

relies on the cost-intensive evaporation [84].

Still most of the studies on lipid production have been done by SHLP using hydrolysate of lignocellulosic biomass as a carbon source. Research by Gong et al. [79, 81] showed that the efficiency can be improved by integrating the enzyme hydrolysis and microbial process applying SSLP (**Table 3**). Two SSLP processes were conducted in cultivation media with and without the addition of nitrogen source. In cultivation media-containing alkaline pretreated corn stover without nitrogen, cells did not grow due to the lack of nitrogen and carbon sources was used for lipid production. To obtain high lipid productivity, the culture media was inoculated at high inoculums size (7.2 g/L), while control culture supplemented with nitrogen was inoculated at average inoculums size (10% v/v). The highest lipid productivity of 0.195 g/L h was obtained in SSLP without nitrogen source and this is the highest value reported for the SSLP using the lignocellulose as a carbon source. In comparison to the SHLP using the same pretreated lignocellulosic biomass (**Table 2**), the productivity of SSLP was improved and loading of cellulase and xylanase was reduced for 50%, while ß-glucosidase was not used. The major disadvantage of this strategy is increased cost for cultivation of larger quantities of inoculum using enriched growth media [81]. Gong et al. [79] applied similar strategy using the corn stover pretreated with ionic liquids as a carbon source. However, lower lipid yields of 0.125 and 0.135 g/ g DM were obtained for SSLP and SHLP, respectively [79]. SSLP was also conducted with fungus *M. isabellina* using pretreated *Douglas fir* forest residue but without success. After prehydrolysis step, fungus was able to grow in semi-solid media obtaining 17.0 g/L of lipids. However, the productivity of lipid synthesis with fungus was half of those obtained in SHLP with detoxified enzymatic hydrolysate of whole pretreated Douglas fir [86].

All cultivations were carried out at substrate loading of 10% (w/w) or lower suggesting possible problems with the enzyme hydrolysis and microorganism growth at higher substrate loadings. The product titer could be improved by increase of the substrate loading conducting so-called high-gravity fermentations which have been successfully applied in the bioethanol production from starch and lignocellulosic feedstock by simultaneous hydrolysis and fermentation. Significant savings in energy input, decrease of waste discharge, distillation costs and capital costs increased the competiveness of the process [91]. However, running the SSLP under high-gravity conditions imposes a number challenges with respect to the lignocellulosederived inhibitors and mixing and mass transfer in cultivation broth. Due to high substrate loading, the concentration of inhibitory by-products are increased and consequently lead to the


**Table 3.** Production of lipids by simultaneous saccharification and lipid production (SSLP) from lignocellulosic hydrolysate. decrease or complete inhibition of growth and product accumulation along with their enzyme activity. Furthermore, increased viscosity of the lignocellulose slurry prohibits the efficient mixing, decreasing the heat and mass transfer (substrate, enzyme and oxygen) in bioreac

Production of Microbial Lipids from Lignocellulosic Biomass

http://dx.doi.org/10.5772/intechopen.74013

tor. Increasing the stirring rate in a conventional stirred tank bioreactor provides even mixing directly around the impeller, while the solid substrate settles down to the bottom and toward to the bioreactor's wall. To avoid the above motioned problems, cultivation should start with lower substrate loadings. The substrate should be gradually fed keeping the viscosity of cul

ture broth sufficiently low (fed-batch cultivation). Kinetics of substrate additions depends on the activity of cellulolytic enzymes and substrate consumption by working microorganism and it should be experimentally optimized. Gradual addition of substrate should enable the working microorganism to adapt to increasing inhibitors concentrations and convert some them to less toxic compounds (furfural and HMF into less toxic compounds such as furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively) [92]. Using this strategy Elliston et al. [93] produced 11.6% (vol/vol) ethanol from waste paper in a bioreactor with high shear mixing. Gradual addition of substrate resulted in cumulative substrate loading of 65% [93].

Solid state fermentation offers a number of advantages over submerged cultivation in the pro

mycopesticides and bioherbicides, biosurfactants, biofuel, aroma compounds, etc. [94

duction of microbial biomass and specific products of microbial metabolism. This technique of cultivation has been successfully used for the production of food (fermented sausages and sea food), products of microbial metabolism including antibiotics, giberellinic acid, aflatoxines, pigments, alkaloids, organic acids and plant growth factors, enzymes, biopesticides, including

major benefit of the solid state cultivation is higher bioprocess productivity, lack of catabolic repression, higher product concentration and low water and energy demands. In comparison to submerged culture, the risk of contamination is decreased due to lower water content in growth media. Furthermore, in comparison to the submerged culture, the product isolation is simpler and also less cost effective. The major drawback of solid state fermentation includes engineering problems with control of process parameters (temperature, water content, pH, substrate and oxygen concentration, etc.) and the scale-up of process to industrial size [96]. Several researches on lipid production by solid state fermentation using lignocellulose bio

mass have been described in literature (**Table 4**). Production of lipids by this type of cultivation depends on oleaginous microorganism's ability to hydrolyse the carbohydrates from lignocel

lulosic biomass to fermentable sugars. This bioprocess of lipid production is also called con

solidate process (CBP). Desirable characteristics of CBP-strain are efficient lipid accumulation, high lipid productivity, high cellulase and hemicellulase activity and the ability to grow on insoluble substrate in the absence of free water. The oleaginous microorganisms used in the submerged production of lipid are not able to grow on the solid substrate or secrete cellulase and hemicellulase. Several fungi strains were isolated with 20–35% (w/w) of accumulated lip

ids in cell dry weight. Low lipid yield in solid state fermentation is a consequence of insufficient cellulolytic activity of isolated CBP-strains and low efficiency of lipid accumulation [97]. The cellulolytic activity in submerged cultivation was between 10 and 20 FPU/g and 4–15 FPU/g

of dry matter of lignocellulosic biomass in SHLP and SSLP, respectively (**Tables 2** and

**4.2. Production of lipids by solid state fermentation**


151







**3**). The

–96]. The

decrease or complete inhibition of growth and product accumulation along with their enzyme activity. Furthermore, increased viscosity of the lignocellulose slurry prohibits the efficient mixing, decreasing the heat and mass transfer (substrate, enzyme and oxygen) in bioreactor. Increasing the stirring rate in a conventional stirred tank bioreactor provides even mixing directly around the impeller, while the solid substrate settles down to the bottom and toward to the bioreactor's wall. To avoid the above motioned problems, cultivation should start with lower substrate loadings. The substrate should be gradually fed keeping the viscosity of culture broth sufficiently low (fed-batch cultivation). Kinetics of substrate additions depends on the activity of cellulolytic enzymes and substrate consumption by working microorganism and it should be experimentally optimized. Gradual addition of substrate should enable the working microorganism to adapt to increasing inhibitors concentrations and convert some them to less toxic compounds (furfural and HMF into less toxic compounds such as furfuryl alcohol and 2,5-bis-hydroxymethylfuran, respectively) [92]. Using this strategy Elliston et al. [93] produced 11.6% (vol/vol) ethanol from waste paper in a bioreactor with high shear mixing. Gradual addition of substrate resulted in cumulative substrate loading of 65% [93].

#### **4.2. Production of lipids by solid state fermentation**

**Substrate** Corn stover

*Trichosporon* 

Pre-soaking with H2SO4

Prehydrolysis for 6

7 FPU/g DM, substrate loading

10%

h, cellulase

Batch/80

h/bioreactor

3.03

—

0.042

[75]

150 Advances in Biofuels and Bioenergy

*cutaneum*

Corn stover

*Cryptococcus* 

Ionic liquid (1-ethyl-3-

Cellulase4 FPU/g DM,

Batch/2 days/

6.0

0.112

0.125d [79]

no nitrogen source

cellobiase 8 CBU/g DM,

xylanase 5 loading 5%

mg /g DM, substrate

methylimidazolium acetate,

140°C, 1 h)

*curvatus*

Corn stover

*Cryptococcus* 

NaOH (0.5

M, 80°C, 75 min)

Cellulase 10 FPU/g DM,

Batch/3 days/

11.9

0.129

0.168

[81]

xylanase 80 loading 10%

As above

Batch/3 concentration of 7.2

 g/L, media without nitrogen

source

days/ high inoculums

15.9

0.159

0.195

[81]

U/g DM, substrate

*curvatus* ATCC

20509

*Cryptococcus* 

As above

*curvatus* ATCC

20509

Douglas

*Mortierella* 

SO2 (11

g/L, 140°C, 60 min)

Prehydrolysis for 24

 h,

Batch/168 h

17.0

0.21b\*

0.101e [86]

cellulase 14.6 FPU/g glucan,

substrate loading 10%

*isabellina* NRRL

fir forest

residue

1757

*Mortierella* 

SO2 (11

g/L, 140°C, 120

 min)

As above

Batch/ 168 h

11.7

0.18 b\* 0.070e [86]

*isabellina* NRRL

1757

aL: Lipid concentration, g lipids/L.

cPr: Lipid productivity, g lipid produced/h L.

dLipid productivity, lipid concentration (L)/time of cultivation (48

eLipid productivity, lipid concentration (L)/time of cultivation (168

**Table 3.**

 h).

 h). Production of lipids by simultaneous saccharification and lipid production (SSLP) from lignocellulosic hydrolysate.

YL/S: Lipid yield, g lipid/g pretreated lignocellulosic biomass; b\* YL/S: Lipid yield, g lipid/g theoretical sugar yield from pretreated biomass.

b

(190°C, 3 min)

**Microbial strain**

**Pretreatment**

**Enzyme hydrolysis**

**Fermentation mode/time/**

**La**

**YL/S**

**b**

**Prc**

**Reference**

**(g/L)**

**(g/g)**

**(g/L/h)**

**note**

Solid state fermentation offers a number of advantages over submerged cultivation in the production of microbial biomass and specific products of microbial metabolism. This technique of cultivation has been successfully used for the production of food (fermented sausages and sea food), products of microbial metabolism including antibiotics, giberellinic acid, aflatoxines, pigments, alkaloids, organic acids and plant growth factors, enzymes, biopesticides, including mycopesticides and bioherbicides, biosurfactants, biofuel, aroma compounds, etc. [94–96]. The major benefit of the solid state cultivation is higher bioprocess productivity, lack of catabolic repression, higher product concentration and low water and energy demands. In comparison to submerged culture, the risk of contamination is decreased due to lower water content in growth media. Furthermore, in comparison to the submerged culture, the product isolation is simpler and also less cost effective. The major drawback of solid state fermentation includes engineering problems with control of process parameters (temperature, water content, pH, substrate and oxygen concentration, etc.) and the scale-up of process to industrial size [96].

Several researches on lipid production by solid state fermentation using lignocellulose biomass have been described in literature (**Table 4**). Production of lipids by this type of cultivation depends on oleaginous microorganism's ability to hydrolyse the carbohydrates from lignocellulosic biomass to fermentable sugars. This bioprocess of lipid production is also called consolidate process (CBP). Desirable characteristics of CBP-strain are efficient lipid accumulation, high lipid productivity, high cellulase and hemicellulase activity and the ability to grow on insoluble substrate in the absence of free water. The oleaginous microorganisms used in the submerged production of lipid are not able to grow on the solid substrate or secrete cellulase and hemicellulase. Several fungi strains were isolated with 20–35% (w/w) of accumulated lipids in cell dry weight. Low lipid yield in solid state fermentation is a consequence of insufficient cellulolytic activity of isolated CBP-strains and low efficiency of lipid accumulation [97]. The cellulolytic activity in submerged cultivation was between 10 and 20 FPU/g and 4–15 FPU/g of dry matter of lignocellulosic biomass in SHLP and SSLP, respectively (**Tables 2** and **3**). The unrestricted carbon source supply is required for the efficient growth and lipid accumulation. Therefore, enhancement of the cellulase activity in cultivation media was recognized as crucial for the improvement of bioprocess performance. Enhancement of cellulase activity was obtained by the optimization of moisture content of solid substrate, cultivation temperature, addition of complex substrates (e.g. wheat bran) and addition exogenous cellulase [97, 98]. The most promising CBP-strain for solid state cultivation is fungus *A. tubingensis* TSIP9 with high cellulase activity and moderate lipid content of 20.5% [99, 100]. Different modes of the solid state fermentation were applied to improve the lipid yield including batch, fed-batch and batch with repeated substrate replacement. Simple strategy of substrate addition in fed-batch cultivation (0.0719 g/g DM) did not improve the lipid yield in comparison to the batch cultivation (0.0799 g/g of substrate dry matter). The batch cultivation with repeated substrate replacement was the most efficient strategy for the production of lipids on the solid substrate. Repeated cycles of the batch cultivations with replacement of 90% fermented substrate with fresh one shortened the process time in comparison to the batch cultivation. Furthermore, cleaning and sterilization of the bioreactor between the batches and inoculum preparation was avoided that additionally saved the time, energy as well as labor [99]. Regardless the fermentation mode, the bioprocess efficiency of solid state fermentations was lower than in the submerged culture (**Tables 2** and **3**). Lipid yields in solid state fermentations were at least two times lower than the submerged cultures. In addition to strain characteristic, significant impact on process efficiency have concentration gradients of hydrogen ions, oxygen, fermentable sugars, products of metabolism formed in the layer of solid substrate during cultivation that inhibited growth of microorganism and cellulase activity.

**Substrate Microbial strain Pretreatment Fermentation mode/**

exploded (121°C, 1 h)

exploded (15% water, 1.5 MPa, 10 min)

*Microsphaeropsis* sp. As above Batch (75% moisture,

*Sclerocystis* sp. As above As above Cellulase

*Phomopsis* sp. As above As above Cellulase

*Cephalosporium* sp. As above As above Cellulase

*Nigrospora* sp. As above As above Cellulase 0.069

4:1 g/g)

30°C, 10 days, 27°C, ratio of wheat straw to wheat bran

Batch (50–80% moisture, 6 days, 30°C, weight ration of wheat straw to wheat bran 2:8 g/g)

(65% moisture, 28°C

Batch,

5 days)

9:1 g/g)

*Colletotrichum* sp. — Batch Cellulase 1.84

*Colletotrichum* sp. — Batch + Exogenous

*Alternaria* sp. — Batch Cellulase 1.21

*Alternaria* sp. — Batch + exogenous

Acid (0.7% H2 SO4 , 121°C,

Acid (0.5% H2 SO4 , 121°C,

1 h)

1 h)

*Microsphaeropsis* sp. As above As above Addition of

*Microsphaeropsis* sp. Steam

*Microsphaeropsis* sp. Steam

Wheat straw and wheat bran mixture

Wheat straw and wheat bran mixture

Rice straw and wheat bran

Wheat straw and wheat bran mixture

Palm pressed fiber and palm empty fruit bunches

*Aspergillus oryzae*

*Aspergillus tubingensis* TSIP9

A-4

**time/note**

Batch (75% moisture, 10 days, 27°C ratio of wheat straw to wheat bran 4:1 g/g)

Batch (75% moisture, 30°C, 10 days, 27°C, ratio of wheat straw to wheat bran

**Enzyme activity**

Production of Microbial Lipids from Lignocellulosic Biomass

http://dx.doi.org/10.5772/intechopen.74013

Cellulase 0.31–0.54 FPU/g DM<sup>b</sup>

0.34–0.52 FPU/g DM<sup>b</sup>

0.32–0.56 FPU/g DM<sup>b</sup>

0.39–0.58 FPU/g DM<sup>b</sup>

FPU/g DM<sup>b</sup>

exogenous cellulase 10 FPU/g DM<sup>c</sup>

Addition of exogenous cellulase 10 FPU/g DM<sup>c</sup>

FPU/g DM

cellulase 10 FPU/g DM

FPU/g DM

cellulase 10 FPU/g DM

Cellulase: 1.69 FPU/g DM<sup>d</sup>

Cellulase: 26.1 U/g DM<sup>b</sup> xylanase 59.3

Cellulase: 0.32 FPU/g DM<sup>c</sup>

**YL/S (g/g)a Reference**

153

0.024–0.042e [97]

0.019–0.028b [97]

0.021–0.027b [97]

0.026–0.034b [97]

0.023e [97]

0.042c [98]

0.074 c [98]

0.08 c [98]

0.0682 [101]

0.0843 [101]

0.0603 [101]

0.0817 [101]

0.06287 [102]

0.0885 [100]
