Chapter 5: Development of dilute sulfuric acid pretreatment method for the
5.3. Results and discussion
5.3.1. Monomeric sugars yield during the pretreatment of biomass
5.2.3.2. Fermentation
The fermentation was performed in sterile 250 mL Erlenmeyer flasks containing 100 mL of fermentation medium which includes 2 mL of 50X concentrated nutrient solution (1.7 g of yeast nitrogen base, 1 g of urea and 6.56 g of peptone in 20 mL of water), 5 mL of inocula gives an initial cell concentration of 2 g/L and, added an appropriate quantity of hydrolysate to reach desired volume. Initial pH of the media was adjusted to 6 and incubated at 30 °C with 120 rpm. All fermentation samples were taken periodically for HPLC analysis.
5.2.4. Analytical methods
Each sample was filtered using a 0.2 μm filter and appropriate dilution was made with Milli Q water. Sugars, fermentative inhibitors, and ethanol quantitative analysis were performed using a Varian 210 HPLC system. The detailed HPLC specifications and operating conditions are described in section 2.2.4. Moreover, the total phenolic content present in the samples were also determined by the Folin–
Ciocalteu [166] method using gallic acid as a standard.
However, the present study was focused on xylose yield into pre-hydrolysate liquors with respect to pretreatment parameters. Sulfuric acid concentration shows a synergetic effect on hemicellulose hydrolysis. From Figure 5.2a, it can be seen that xylose content increased drastically from 96.02 mg to 150.2 mg due to increase in the sulfuric acid concentration and reaction time, i.e., 0.1 M to 0.2 M at 30 min to 120 min, respectively. Further increase in the sulfuric acid concentration (0.3‒0.4 M) showed a slight decrease in the xylose concentration at 60 to 120 min, which could be due to the cyclo-dehydration of xylose. Therefore, high acid concentration effectively hydrolysed the hemicellulose to release xylose units and concurrently influence its decomposition into furfural. From Figure 5.2b it is evident that 0.4 M sulfuric acid concentrations yield more amount of furfural compared with other acid concentration.
Figure 5.2: Effect of pretreatment parameter on a) xylose release and b) furfural formation
Blue, Orange, Grey and yellow bars indicates 0.1 M, 0.2 M, 0.3 M and 0.4 M sulfuric acid, respectively.
For instance, 150.2 mg of xylose yield was observed at 121 ºC, 0.2 M acid concentration and 120 min pre-treatment, whereas, 0.4 M H2SO4 yield 128.4 mg xylose under otherwise similar condition. Moreover, compositional analysis of pretreated biomass samples (by NREL procedure) derived at 0.2 M and 0.4 M H2SO4 revealed that the decomposition of pentose sugars increased with corresponding sulfuric acid
0 40 80 120 160
30 60 90 120
Xylose (mg/g)
Time (min)
a
0 1 2 3 4 5
30 60 90 120
Furfural (mg/g)
Time (min)
b
Table 5.2: Composition analysis of residual biomass according to NREL procedure
Components (mg)
Residual biomass 121 °C, 0.2M, 2h
(per 546 mg)
121 °C, 0.4M, 2h (per 528 mg)
Cellulose 319 315.5
Xylan 12.2 4.1
AIL* 175.4 171.8
* Acid insoluble lignin
(5.4)
(5.5)
Where, Ix is the initial amount of xylose present in the untreated biomass, Rx
stands for the amount of xylose present in the residual biomass derived from pretreatment and Lx, xb is the concentration of xylose and xylobiose present in the pre- hydrolysate liquor.
Theerarattananoon et al., (2010) in their study have used pressurized batch reactor for the pretreatment of different sorghum biomass varieties at 140 °C and 2%
(v/v) H2SO4. The results of their study revealed xylan conversion of about 82% in grain sorghum, 83% in BMR sorghum, 84% in photosensitive sorghum, 87% in forage sorghum and 90% in sweet sorghum along with 19.6 to 38% of xylose decomposition (Table 5.3). Further, increase in the temperature i.e., 165 °C leads to slightly increase the xylan conversion and concurrently higher xylose (51 to 69.8%) decomposition. The comparative analysis of the results obtained in the present study with that of the literature is shown in Table 5.3. It is very well known that decomposition of xylose
X X
X
I - R
conversion (%) 100
Xylan I
X X,Xb X
X
I -( + R )
(%) 100
I
Xylose decompostion L
generally initiated at a temperature higher than 120 °C [145]. Pre-treatment at 140 °C and 165 °C showed drastic degradation in the xylose which ultimately results in fermentative inhibitor formation.
Table 5.3: Summary of xylan conversion and xylose decomposition analysis results of the present study with literature
Type of Sorghum
Biomass
Pretreatment condition
Xylose (mg/g)*
%XC (Eq.
5.4)
Xylose (mg/g) (LX)
%XD (Eq. 5.5)
Grain
Untreated 140 °C, 30 min a 165 °C, 10 min a
246.0 (Ix) 43.4 (Rx) 25.8 (Rx)
– 82.5 89.8
– 152.9
99
– 20.3 49.4 BMR
Untreated 140 °C, 30 min a 165 °C, 10 min a
261.6 (Ix) 42.2 (Rx) 29.4 (Rx)
– 83.8 88.4
– 166.8
97
– 19.6
51 Photoperiod
sensitive
Untreated 140 °C, 30 min a 165 °C, 10 min a
274.0 (Ix) 42.6 (Rx) 24.2 (Rx)
– 84.6 91.2
– 154.4
81
– 28.4 61.6 Forage
Untreated 140 °C, 30 min a 165 °C, 10 min a
297.9 (Ix) 36.2 (Rx) 25.4 (Rx)
– 87.8 91.6
– 148.2
95
– 38.1 59.7 Sweet
Untreated 140 °C, 30 min a 165 °C, 10 min a
268.1 (Ix) 26.4 (Rx) 24.1 (Rx)
– 90.2
91
– 178.6
56.7
– 23.6 69.8 Present study
BMR
Untreated
121°C, 120 min b
197.4 (Ix) 21.7 (Rx)
– 93.8
– 150.2
– 16
* mg/g of biomass; XC, xylan conversion; XD, xylose decomposition; a 2% (v/v) H2SO4; b 0.2 M H2SO4
[or equivalent to 1.12% (v/v)]; - Not applicable; IX Initial xylan present in the untreated biomass; RX
residual xylan present pretreated biomass; LX xylose concentration in the liquid fraction or pre- hydrolysate.
Therefore, the present study suggests that, instead of using high temperature and high-pressure batch reactors, utilization of autoclave (121 °C) for pretreatment of sorghum biomass showed significant impact on xylan conversion (93.8%) with a lower
concentration of furfural. In addition to this, use of autoclave for pretreatment of lignocellulosic biomass has several advantages over high energy input batch reactors such as less capital investment, low power consumption, zero maintenance cost and easy to operate.
Apart from xylose and furfural, acid catalyzed pretreatment reaction can also produce different types of sugars and fermentative inhibitors. Sugars like glucose (Figure 5.3a) and arabinose (Figure 5.3b) are derived from cellulose and hemicellulose, respectively. Fermentative inhibitors such as 5-HMF (Figure 5.3c), formic acid (Figure 5.3d), acetic acid and phenolic compounds are derived from glucose, furfural, acetylated xylan and acid soluble lignin, respectively. Release and stability of sugars are depended on the pretreatment severity conditions. As pretreatment severity increases glucose dehydrates to form 5-HMF, furfural decomposes to form formic acid and, the accumulation of phenolic compounds may also increase in pre-the hydrolysate liquors. These are known to be potential toxic compounds which inhibit microbial growth during fermentation [34].
0 10 20 30 40 50 60
30 60 90 120
Glucose (mg/g)
Time (min)
a
0 2 4 6 8 10 12 14 16
30 60 90 120
Arabinose (mg/g)
Time (min)
b
Figure 5.3: Effect of pretreatment parameters on a) glucose, b) arabinose, c) 5-HMF and d) formic acid formation
Blue, Orange, Grey and yellow bars indicates 0.1 M, 0.2 M, 0.3 M and 0.4 M sulfuric acid, respectively.
However, in order to minimize the fermentative inhibitor effect on the microorganisms for efficient fermentation, it is essential to select an optimized condition which gives a comparatively high concentration of sugars with lower amounts of fermentative inhibitors. Therefore, from the results, the optimized sugars yield was obtained at 121 °C, 0.2 M H2SO4 and 120 min. At this condition, 209.9 mg/g of maximum sugars yield was obtained which includes xylose (150.2 mg/g) arabinose (14.3 mg/g), glucose (45.4mg/g) along with fermentative inhibitors like, 4.26 mg of furfural, 5 mg of 5-HMF, 2.64 mg of formic acid, 21.3 mg of acetic acid and 3.7 mg of phenolic compounds.