3.1 Changes of TDS content in recycled alkali black liquor
Alkali treatment can remove the linkage bonds between lignin, cellulose and hemicellulose, destroy the internal structure of lignin, reduce the crystallinity of cellulose and increase the internal lignocellulose porosity [3]. Changes of TDS content in alkali black liquor circulation at different cycles and the enzymatic hydrolysis rate of pretreated corn straw by recycled alkali black liquor combined with ozone are shown in Fig. 1. It could be seen that the TDS contents increased with circulation times of alkali black liquor. The content of TDS at the sixth cycle was 186.80 mg/mL, which was 2.63 times more than that of the primary cycle. From the overall trend of the TDS content change, it can be observed that the solid content increased sharply from the zeroth to the third cycle, with the increase of 35.33 mg/mL, 38.07 mg/mL and 27.33 mg/mL respectively, whereas the increase was reduced to 8.33 mg/mL at fourth cycle, 69.52% lower than previous cycle.
With the increase of the TDS content, the cellulase hydrolysis rate showed a downward trend, which was most significantly decreased in the fourth cycle. It is indicated that when circulated to the fourth time, the accumulation of degradation compounds has almost reached a threshold, the alkali black liquor was unable to break down the linkages between cellulose, hemicellulose and lignin and internal structure effectively, resulting in the decrease of delignification abilities and limited contact of cellulase with substrate. Or a certain amount of organic substances which inhibit the hydrolysis of cellulase were gradually accumulated during alkali black liquor circulation. The inhibitors might be attached to corn straw by recycled alkali black liquor pretreatment and could not be completely removed, which led to decreased enzymatic hydrolysis efficiency.
3.2 Changes of the content of OM and IM in recycled alkali black liquor
Alkali black liquor is a mixture of organic and inorganic materials. The contents and proportions of OM and IM in different cycles of alkali black liquor are listed in Table 1. The organic and inorganic compound concentration underwent an upward trend with the increasing cycles of alkali black liquor, which was in consistent with the trend of TDS content. However, the OM and IM content increased slowly at the fourth cycle, indicating that the ability of recycled alkali black liquor to dissolve/remove OM and IM was reduced which was not as effective as fresh NaOH solution.
Table 1 Changes in content of organic matter (OM) and inorganic matter (IM) in different cycles of alkali black liquor
Cycle times
|
OM
|
IM
|
Content
(g/15mL)
|
increment
|
proportion (%)
|
Content
(g/15mL)
|
increment
|
proportion (%)
|
0
|
|
-
|
63.22%
|
0.392±0.05c
|
-
|
36.78%
|
1
|
0.959±0.09d
|
0.285
|
60.09%
|
0.637±0.07b
|
0.245
|
39.91%
|
2
|
1.292±0.10c
|
0.333
|
59.64%
|
0.875±0.08a
|
0.238
|
40.36%
|
3
|
1.637±0.10b
|
0.345
|
63.52%
|
0.940±0.10a
|
0.065
|
36.47%
|
4
|
1.739±0.13ab
|
0.102
|
64.38%
|
0.963±0.10a
|
0.023
|
35.62%
|
5
|
1.822±0.13ab
|
0.082
|
65.34%
|
0.966±0.10a
|
0.004
|
34.65%
|
6
|
1.834±0.12a
|
0.012
|
65.73%
|
0.956±0.11a
|
-0.010
|
34.26%
|
Note: The OM and IM contents were determined in 15 mL recycled alkali black liquor. The proportion of OM was the ratio of the content of OM to the total content of OM and IM, and so on. The increment was the difference between the content of the current cycle and that of the previous cycle.
There were slight changes in the organic matter proportion (OMP) and inorganic matter proportion (IMP) during the alkali black liquor recycling from zeroth to sixth cycle. The OMP tended to fall then rise, which was contrary to the IMP. And at the second cycle, the OMP reached a minimum value of 59.64%, whereas the maximum value of 40.36% of the inorganic. It can be found that the ability of IM removal in 0-2 cycles was stronger in comparison with that in 3-6 cycles, in contrast, the alkali black liquor recycling with 3-6 cycles had better effect on OM removal than 0-2 cycles. There was no obvious increase in the concentration of OM and IM after the fourth treatment, but the OMP kept rising from the fourth cycle to sixth cycle, indicating that the accumulation of substances in the recycled alkali black liquor had gradually reached saturation, and the OM was related to the decrease of cellulase hydrolysis rate.
3.3 Changes of alkali and acid precipitation in recycled alkali black liquor
3.3.1 Changes in contents of alkali and acid precipitation
The effects of different cycles of alkali black liquor circulation treatment on alkali and acid precipitation contents are presented in Fig. 2. The alkali precipitation content rose first, then descended slightly and reached a maximum of 0.707g at the fourth cycle, an increase by 0.546g was observed compared with the zeroth cycle. Nevertheless, the change of acid precipitation was different from the alkali precipitation, which generally showed an upward trend. The acid precipitation content was 1.626g at the sixth treatment, about an eleven-fold increase from the zeroth treatment. The total precipitation contents was 0.295 g, 1.041 g, 1.411 g, 1.708 g, 1.955 g, 2.175 g and 2.19 g from zeroth to sixth cycle, respectively, showing an overall upward (rising) trend but gradually declining in the increment which was consistent with the trend of TDS content but in contrast to the trend of cellulase hydrolysis yield in 3.1. The decrease of alkali precipitation content after the fourth cycle might be attributed to its deposition on the substrate or reaction and degradation into soluble components in the complex black liquor. In general, compared with alkali precipitation, acid precipitation content was higher and kept increasing indicating that the accumulation of acid insoluble component has effect on the decrease of cellulase hydrolysis.
3.3.2 Changes of alkali precipitation composition in recycled alkali black liquor
The effect of alkali black liquor circulations on the content of OM and IM in alkali precipitation is shown in Fig. 3. The inorganic content gradually rose during 0-4 cycles and then reached stabilized. The organic content showed tendency of first increasing and then descending, which was same as the alkali precipitation and peaked at the fourth cycle. It was indicated that the OM was the major factor to effect the change of alkali precipitation content.
3.3.3 Changes of acid precipitation composition in recycled alkali black liquor
Fig. 4 shows that the content of OM and IM in acid precipitation with different alkali black liquor recycling cycles. The organic content showed the same trend as the acid precipitation, rapid increase at 0-2 cycles and 5-6 cycles and steady growth at 3-4 cycles. Differ from the organics, the inorganic content no longer increased after the fourth time. The results of 3.1 showed that the enzymatic hydrolysis rate of cellulose decreased in the fourth cycle. Combined with the changes in the cellulase hydrolysis rate, it can be seen that with the increase of circulation times of alkali black liquor, the organics of acid precipitation was the main factor inhibiting the enzymatic hydrolysis of cellulose.
3.4 Changes of lignin content in recycled black liquor
The content of lignin in recycled alkali black liquor is presented in Fig. 5. A gradual increase in the lignin content from 2.050 mg/mL to 6.144 mg/mL with increasing alkali black liquor recycling times was observed. At the zeroth cycle, the alkali black liquor showed a good liquidity and light color, which had a tend to present dark color, high viscosity and poor fluidity after several cycle treatment. For all the six cycles, the increment in lignin concentration decreased gradually from 1.049 mg/mL to 0.299 mg/mL. For the 0-3 cycles, the increase in lignin content was between 35% and 50% of the zeroth cycle, showing that the delignification efficiency maintained the similar levels as the zeroth treatment with fresh NaOH solution [39]. After 4-6 cycles, the growth was 15%-30% compared with the zeroth cycle, indicating that the solubilization power of recycled alkali black liquor reduced and the lignin dissolution reached saturation gradually. The lignin content of circulating black liquor could indirectly reflect the lignin removal ability of straw. The delignification efficiency declined after the six treatment of recycled black liquor, speculating that the accumulation of degradation products reduced the utilization of alkali or high viscosity of black liquor hindered alkali liquor diffusion in lignocellulose [23,27,39].
However, recycling of alkali black liquor for the zeroth time resulted in a maximum cellulase hydrolysis yield of 87.67%, which underwent a downward trend by further recycling. The lower enzymatic hydrolysis rate of corn straw that pretreated with the recycled black liquor can be explained that the residual lignin in recycled black liquor caused reduced delignification efficiency, resulting high lignin content in pretreated straw. And the undegraded lignin was a physical barrier to prevent direct contact of the enzyme with cellulose [15], which reduced the cellulase accessibility.
3.5 GC-MS analysis of recycled alkali black liquor
The identities and the relative abundances of the organic components from recycled alkali black liquor were recognized by using GC-MS, the main identified compounds are listed in Table 2.
53 types of organic compounds were isolated and identified which can be classified into six categories: phenols, benzene ring and heterocyclic, furans, acids, alcohol ketones, and esters. As seen from the table, the total relative content of compounds in alkali black liquor exhibited an overall upward trend with the increasing cycles. And the phenols, benzene ring and heterocyclic, and furans were the three major products with high relative yields, accounting for more than 80% of the total. Among them, 2, 3-dihydrobenzofuran showed the highest relative yield, which was in the range of 31.359% - 34.510%. Then other substances with high relative abundances were 4-vinyl-guaiacol, 2,6-di-tert-butyl-4-methylphenol and 2,4-di-tert-butylphenol, which are typical lignin degradation products.
Lignin is a complex polymer in which guaiacyl, syringyl and p-hydroxyphenyl units are interconnected [40]. Guaiacyl and syringyl units are main composition of corn straw lignin which contain small amounts of p-hydroxyphenyl units. The compounds obtained from degradation of lignin were mainly monomeric aromatic products of which the phenols compounds were the predominant components [41]. Alkali pretreatment can cause scission of linkages between lignin and carbohydrates, expose the cellulose inside the lignocellulose and convert lignin macromolecules into small molecules of aromatic compounds that dissolved or deposited in alkali black liquor. The alkali black liquor mainly contained lignin degradation compounds. It can be speculated that continuous accumulation of small molecular compounds with the increasing cycle numbers resulted in increased concentration of alkali black liquor and reduced the amount of effective alkali, and the infiltration of the refractory organic compounds with alkali liquor to straw limited the swelling effect of alkali and hindered the breaking of the ether and ester bonds between lignin and carbohydrates, thereby reducing the enzymatic hydrolysis rate.
Table 2 Compounds and its relative intensity detected by GC-MS in recycled alkali black liquor from different cycles
Type
|
Components
|
Relative content/ area (%)
|
0
|
1
|
2
|
3
|
4
|
5
|
6
|
Phenols
|
2,6-Di-tert-butyl-4-methylphenol
|
4.283
|
4.596
|
4.370
|
4.372
|
4.355
|
4.881
|
4.715
|
4-Vinyl-guaiacol
|
13.922
|
13.253
|
12.175
|
12.236
|
12.445
|
11.740
|
12.495
|
2,4-Di-ter-butyl phenol
|
3.717
|
4.242
|
4.765
|
4.769
|
4.873
|
4.580
|
4.928
|
∑
|
21.922
|
22.091
|
21.310
|
21.377
|
21.673
|
21.201
|
22.138
|
|
|
|
|
|
|
|
|
|
Benzene ring and heterocyclic
|
1,2,3,6-Tetrahydrophthalic anhydride
|
-
|
2.071
|
-
|
0.783
|
-
|
0.316
|
1.099
|
1,4-Diisopropylnaphthalene
|
-
|
-
|
1.046
|
-
|
1.795
|
0.804
|
-
|
2,3,4-4a, 8,8a Hexahydropyran [3,2-b] pyran
|
1.231
|
-
|
-
|
4.958
|
-
|
-
|
-
|
4- (2-Methyloctadecyl-4-phenoxymethyl) -2,2-dimethyl-1,3-dioxolane
|
1.200
|
3.330
|
3.481
|
3.992
|
4.471
|
5.450
|
4.777
|
2-Methoxypyrimidin-4-amine
|
17.080
|
16.652
|
15.538
|
13.517
|
18.069
|
17.895
|
17.441
|
Pyridine
|
2.839
|
0.919
|
0.685
|
1.164
|
-
|
-
|
-
|
3-Methyl pyridine
|
0.572
|
-
|
0.634
|
-
|
0.570
|
-
|
-
|
2,5-Dimethylbenzaldehyde
|
0.472
|
0.338
|
0.562
|
0.551
|
0.411
|
0.931
|
-
|
Others
|
1.841
|
2.572
|
2.863
|
1.082
|
1.520
|
1.309
|
3.317
|
∑
|
25.235
|
25.882
|
24.809
|
26.047
|
26.836
|
26.705
|
26.634
|
|
|
|
|
|
|
|
|
|
Furans
|
2,3-Dihydrobenzofuran
|
31.383
|
31.359
|
34.105
|
34.276
|
33.760
|
33.945
|
34.510
|
|
5-Fluoro-1-α-ribofuranosyl-imidazole-4-carboxylic acid amide
|
0.209
|
0.512
|
0.628
|
0.697
|
1.132
|
1.174
|
0.976
|
|
∑
|
31.592
|
31.871
|
34.733
|
34.973
|
34.892
|
35.119
|
35.486
|
|
|
|
|
|
|
|
|
|
Acids
|
2-(2- Methoxyethoxy) acetic acid
|
-
|
-
|
-
|
-
|
0.200
|
0.692
|
0.357
|
3,6,9-Trioxaundecanedioic acid
|
1.263
|
0.245
|
0.811
|
1.360
|
1.227
|
-
|
1.187
|
3-(Trimethylsilyl)propionic acid
|
-
|
0.406
|
0.865
|
-
|
0.772
|
0.847
|
-
|
DL-Mandelic acid
|
-
|
1.066
|
0.402
|
0.582
|
-
|
-
|
-
|
Acetic Acid
|
-
|
0.534
|
0.352
|
0.118
|
0.404
|
1.095
|
0.906
|
Others
|
-
|
0.182
|
0.935
|
1.16
|
0.396
|
1.153
|
0.600
|
∑
|
1.263
|
2.433
|
3.365
|
3.22
|
2.999
|
3.787
|
3.050
|
|
|
|
|
|
|
|
|
|
Esters
|
N-allyl-N- [2- (tert-butyldimethylsilyloxy) propyl] methyl carbonate
|
-
|
-
|
-
|
0.419
|
0.610
|
0.345
|
0.424
|
Methyl 2- (2-methoxyethoxy) acetate
|
-
|
0.476
|
0.421
|
-
|
0.428
|
-
|
-
|
5- (2,3,4,5-Tetrahydro-3-hydroxy-4-ureidothiophen-2-yl) valerate
|
-
|
-
|
-
|
-
|
0.300
|
0.310
|
0.350
|
Vanillone lactone
|
1.227
|
1.081
|
0.995
|
1.048
|
0.860
|
0.975
|
1.080
|
1-O-butyl 2-O-octyl benzene-1,2- dicarboxylate
|
0.439
|
0.384
|
0.320
|
0.534
|
-
|
0.355
|
-
|
3,2,4-Tridecyl methoxy acetate
|
-
|
0.389
|
0.580
|
0.583
|
1.335
|
0.825
|
0.795
|
1,3-Diacetoxypropane-2-yldodecanoate
|
-
|
-
|
-
|
0.397
|
-
|
0.557
|
0.440
|
3- Tetradecyl methoxy acetate
|
-
|
0.124
|
0.227
|
-
|
-
|
0.355
|
0.376
|
others
|
0.398
|
0.524
|
0.848
|
0.906
|
0.455
|
-
|
-
|
∑
|
2.064
|
2.978
|
3.391
|
3.887
|
3.988
|
3.722
|
3.465
|
Alcohol ketones
|
|
|
|
|
|
|
|
|
3-Amino-4, 6-dimethyl-1h-pyridine-2-ketone
|
-
|
0.194
|
0.297
|
-
|
0.560
|
-
|
-
|
3-Methyl-1, 4-oxythio-heterocyclohexane 2-ketone
|
0.450
|
1.788
|
1.900
|
1.864
|
1.501
|
1.759
|
1.566
|
Hexahydro-1,6-pentanedione
|
0.584
|
-
|
-
|
-
|
0.242
|
0.133
|
-
|
others
|
0.365
|
0.531
|
-
|
0.353
|
-
|
0.332
|
0.650
|
∑
|
1.399
|
2.513
|
2.197
|
2.217
|
2.303
|
2.224
|
2.216
|
Note: others in each category means some compounds that not listed in Table 2. Other compounds: (acids type):Propyl thioacetic acid, 2- (4-Fluoro-6-oxy-1,6-dihy dropyrimidine-2-imine) -propionic acid, O-acetyl-L-serine; (benzene ring and heterocyclic type):1-(4- Nitrophenyl) ethyl thiosemicarbazide, 5-Isopropyl-2-methyl-1, 3-oxythio-heterocyclic hexane, 1-Nitroso 3-pyrroline, 2-Cyanoquinoline, 3-Ethyltetrahydrothiophene, 4-Hydroxymethyl-5-methylimidazole, C-(3-methyl-isoquinolin-1-yl) -methylamine, Octahydro-2, 6-cyclothianan [3,2-b] pyran, 1, 4-Diol -2, 3-dimethyl-5-trifluoromethyl benzene, 4,5-Dihydro-2- (4-chlorophenyl) thiazole; (esters type): 2,3,4,6-Tetraacetoxy-5,5-bis (ethylsulfanyl) hexyl acetate, 2- (2-Chloroacetyl) oxypropyl-2-chloroacetate, 2,2,2-Trifluoroethyl methanesulfonate, 7-Dimethoxymethylbic yclo [2.2,1] heptane-1-carboxylic acid methyl ester, Dimethylsilanediol; (alcohol ketones): Mercaptoethanol, 4, 4-Dimethyl-2, 5-cyclohexadiene -1-ketone, 9-Thiabicyclo [3.3.1] non-7-en-2-ol, Aminoacetaldehyde diethanol; 3,6-Dioxa-8-mercaptooctan-1-ol.