Research Article pubs.acs.org/journal/ascecg
Field-Grown Transgenic Hybrid Poplar with Modified Lignin Biosynthesis to Improve Enzymatic Saccharification Efficiency Zhouyang Xiang,† Suman Kumar Sen,‡ Douyong Min,‡ Dhanalekshmi Savithri,‡ Fachuang Lu,† Hasan Jameel,*,‡ Vincent Chiang,§ and Hou-min Chang‡ †
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, 381 Wushan Road, Guangzhou 510640, China ‡ Department of Forest Biomaterials and §Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, 2820 Faucette Drive, Raleigh, North Carolina 27695, United States S Supporting Information *
ABSTRACT: Hybrid poplars (Populus nigra L. × Populus maximowiczii A.) were genetically modified through antisense insertion of the 4-coumarate:coenzyme A ligase (4CL) gene. Compositional changes in response to this genetic change were measured in the field after 2 and 3 years of growth. The stem samples were treated with either green liquor or dilute acid pretreatments, representing alkaline and acid pretreatments. The enzymatic saccharification of the untreated and pretreated transgenic poplars were evaluated. After transgenic species were transplanted into the environment, they showed reduced recalcitrance to chemicals (i.e., pretreatments) and enzymes despite their lignin content and S/V ratio being comparable to those of the wild types. Compared to the field-grown poplars, the sugar yield increased up to 103% for untreated transgenic samples and increased 22% for acid- and green liquor-pretreated transgenic samples. This shows that field-grown transgenic hybrid poplars with modified lignin biosynthesis have improved enzymatic saccharification efficiency (sugar recovery and yield). KEYWORDS: Transgenic, 4CL gene, Enzymatic saccharification, Hybrid poplar, Field-grown, Lignin
■
break during pretreatments.7,8 Trees with a high syringyl to guaiacyl lignin ratio (S/G ratio) have also been produced through genetic modification. By overexpressing coniferaldehyde-5-hydroxylase (CAld5H)9 or ferulate 5-hydroxylase (F5H)10 genes, poplar trees cultivated in the greenhouse produced wood with an increased S/G ratio. Regulating the 4CL and CAld5H genes at the same time could produce poplar wood with both reduced lignin content and an increased S/G ratio.11 Some recent studies demonstrated that the perturbation of some 4CL mutants could also result in a high S/G ratio in the lignin of the transgenic plants.12−15 Regulating the PtrGT8D gene, Li et al.16 produced transgenic Populus trichocarpa with reduced xylan content. Transgenic woody or nonwoody plants grown in the greenhouse demonstrated lower recalcitrance to chemical reactions and enzymatic treatments. Transgenic poplars grown in the greenhouse with reduced lignin and xylan content had higher enzymatic saccharification efficiency compared to that of the wild types.17−19 Arabidopsis thaliana13 and switchgrass14 with 4CL gene perturbations
INTRODUCTION Lignin and hemicelluloses are regarded as inhibitory factors during the processing of lignocellulosic resources into chemicals, biofuels, and biomaterials.1 Pretreatment methods have been extensively studied to modify the lignocellulosic recalcitrance to chemical and biological reactions. In recent years, modern biotechnology has aimed at alternating the chemical compositions of lignocellulosic materials and is becoming another method to assist the biorefinery industry in meeting processing specifications. Lignin or xylan-modified transgenic trees have the potential to benefit paper making, fiber, biofuel, and biochemical industries. All of them use a significant amount of energy when disposing of unwanted lignin and hemicelluloses, which results in environmental issues.2−4 By downregulating the 4coumarate:coenzyme A ligase (4CL) gene, poplar5 and pine6 trees cultivated in a greenhouse produced wood with reduced lignin content. In addition to the lignin content, the lignin structure can cause the tree to be recalcitrant during lignocellulosic bioprocessing. Syringyl (S) lignin is preferred over guaiacyl (G) lignin in the wood because guaiacyl lignin has a free C-5 carbon on its aromatic ring and can easily form 5−5 linkages with other G lignin units, which are relatively hard to © 2017 American Chemical Society
Received: November 14, 2016 Revised: February 1, 2017 Published: February 13, 2017 2407
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
Research Article
ACS Sustainable Chemistry & Engineering
Table 1. Lignin Content and S/V Ratio (Mean ± SE, n = 2) of Transgenic Hybrid Poplar Tree Stems Grown in a Greenhouse (GH) and in Mountain and Coastal Plain Regions mountain
coastal plain
year 2 transgenic lines/trees as4CL-11.6 as4CL-15.7 as4CL-20.3 WT-22.1 a
GH lignin content % 11.6 15.7 20.3 22.0
± ± ± ±
0.3a 0.2a 0.3a 0.0
GH S/V ratio 2.1 2.6 1.6 1.7
± ± ± ±
0.0a 0.1a 0.0 0.1
lignin content % 21.1 22.1 20.6 22.5
± ± ± ±
0.2a 0.5 0.3a 0.4
year 3 S/V ratio
2.0 2.2 2.0 1.9
± ± ± ±
0.0a 0.0a 0.0a 0.0
lignin content % 23.7 22.5 23.6 23.6
± ± ± ±
0.2 0.2 0.0 1.1
year 2
S/V ratio 0.6 1.9 1.7 1.8
± ± ± ±
0.0a 0.0a 0.0a 0.0
lignin content % 23.2 24.8 22.9 24.6
± ± ± ±
0.3a 0.3 0.1a 0.0
year 3 S/V ratio
2.0 2.1 1.9 2.0
± ± ± ±
0.0 0.0a 0.0a 0.0
lignin content % 22.9 22.4 24.1 25.1
± ± ± ±
0.0a 0.2a 0.6 0.3
S/V ratio 2.0 1.9 2.0 1.8
± ± ± ±
0.0a 0.0a 0.0a 0.0
Value is significantly different from that of the corresponding wild type sample (by paired-samples t test with confidence interval = 95%). Structural Analysis. For carbohydrate content to be determined, the solid sample was hydrolyzed, and an ICS-3000 ion chromatography (IC) system (Dionex, Sunnyvale, CA, USA) was used to quantify the neutral sugar contents following the procedure described by Xiang et al.22 Lignin content and the lignin syringaldehyde to vanillin ratio (S/V ratio) were determined by nitrobenzene oxidation according to the procedure described by Min et al. 17 The measurement was conducted in duplicate for each tree stem sample. Pretreatments. For the acid hydrolysis (AH) pretreatment, approximately 1.0 g of the extractive-free sample was reacted with 4.0 mL of 0.1 wt % sulfuric acid at 185 °C for 30 min in a sealed stainless steel reactor. For the green liquor pretreatment (GL), approximately 1.0 g of the extractive-free sample was reacted with 4.0 mL of green liquor at 185 °C for 15 min in a sealed stainless steel reactor. The green liquor was prepared by dissolving 0.375 g of Na2S and 1.125 g of Na2CO3 in 0.1 L of deionized (DI) water resulting in a 6% total titratable alkali (TTA). After the reaction was completed, the mixture was filtered and washed with 500 mL of DI water. The pretreatment was conducted in duplicate for each tree stem sample. Hydrolysis was conducted on the liquid filtrates from the acid pretreatments to analyze the sugar content. For this step, 9.74 mL of each acid-pretreated filtrate was mixed with 0.26 mL of 72% sulfuric acid and autoclaved for 1.5 h. After cooling, the solution was analyzed by the IC system for neutral sugar contents. This sugar content was then added to the sugar content from enzymatic hydrolysis to determine the total sugar yield for the pretreated samples. Enzymatic Saccharification. Cellulase CTec 2 (VCP10007, 139 FPU, Novozyme North America, Inc.) was used for the enzymatic saccharification. Acetate buffer (0.02 M, pH 4.8) supplemented with 40 μg mL−1 of tetracycline was prepared. Approximately 0.5 g of each of the native or pretreated stem samples was mixed with 10 mL of the prepared acetate buffer in a 40 mL sample flask. Cellulase with an enzyme charge of 5 FPU of cellulase per gram of cellulose was added. The slurry was incubated at 50 °C in a shaking air bath maintained at 150 rpm for 48 h. Then, the slurry was filtrated, and the residue was washed with 40 mL of DI water several times. Approximately 50 mL of the filtrate was collected and boiled for 10 min to denature enzymes. The saccharified sugars in the filtrate were quantified by the IC system according to the procedure described in Structural Analysis section. The saccharification was conducted in duplicate for each tree stem sample. Sugar Yield and Carbohydrate Saccharification Efficiency. Saccharification yield is used to evaluate the amount of mono sugars that can be produced from the original untreated woody materials through pretreatments and enzymes. Saccharification sugar yield is calculated by the following equation, where 0.9 is the correction coefficient of hydration
grown in a greenhouse also demonstrated improved enzymatic saccharification efficiency. Transgenic poplars grown in a greenhouse with a high S/G ratio can improve delignification19 and increase pulping efficiency.20 Even though the transgenic trees showed expected chemical compositional changes in the greenhouse, some studies found that 4CL-downregulated transgenic poplar’s lignin content reversed back to the level of the wild types when they were planted in the field.15,21 Voelker et al.21 also found that the saccharification efficiency of the field-grown 4CL-downregulated poplar showed no significant difference compared to that of the wild types. However, in Xiang’s study,15 field-grown poplar trees with a similar gene modification showed improved saccharification efficiency, indicating that the lignin may be less recalcitrant than the lignin found in wild-type trees. Hybrid poplars grow much faster than their regular counterparts and are an important raw material for bioethanol production. For the possibility of using field-grown transgenic energy plants to be evaluated further, the responses of field-planted 4CLdownregulated hybrid poplar trees regarding cell wall composition and enzymatic saccharification were analyzed.
■
MATERIALS AND METHODS
Raw Materials. The wood stems in this study were harvested from hybrid poplars (Populus nigra L. × Populus maximowiczii A.) and abbreviated as NM6. NM6 samples were grown inside a greenhouse and a field for either two or three years. Transgenic lines labeled “as4CL” were constructed by fusing the Pt4CL1 cDNA encoding the 4-coumarate:coenzyme A ligase (4CL) gene from P. trichocarpa in an antisense orientation with respect to a duplicated-enhancer cauliflower mosaic virus 35S promoter.5 The gene expressions of 4CL were successfully reduced resulting in low lignin contents for transgenic NM6 trees grown in a greenhouse. The NM6 trees evaluated included one line of wild type with greenhouse lignin content of 22.1% [WT (22.1)] as control and three as4CL transgenic lines with low, medium, and high greenhouse lignin content as shown in the following labeling scheme [as4CL (11.6), as4CL (15.7), and as4CL (20.3)]. The Forest Biotechnology Group at North Carolina State University cultivated the samples in a greenhouse for 4 months before planting them in one of two terrains: either the coastal plains located in Wallace, NC or the mountains located in Fletcher, NC. The remainder of the NM6 trees continued to grow in the greenhouse for another 6 months as compared to the trees planted in the field. Only one tree was analyzed for each wild type or each genetic line from each growing site. The trees selected for the analysis were considered healthy without any visible signs of disease. A second tree from each line of 2 year-growth was selected for another lignin content measurement to ensure that the tree variation of lignin content is negligible (Table S1). Before analysis, the collected tree stems were air-dried, debarked, and milled using a Wiley mill to a size between 40 and 60 mesh. Extractives were removed from the ground stem samples by extraction using a 2:1 (volume ratio) benzene-to-ethanol solution.
sugar yield =
total mono sugar obtained × 0.9 × 100% mass of the original untreated woody materials
(1) Sugar recovery or saccharification efficiency is used to evaluate the percent of carbohydrates in the woody material converted to mono sugars through pretreatments and enzymes, which is calculated by 2408
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
Research Article
ACS Sustainable Chemistry & Engineering
Table 2. Carbohydrate Content (Mean ± SE, n = 2) of Transgenic Hybrid Poplar Tree Stems Grown in Mountain and Coastal Plain Regions mountain year 2 transgenic lines/trees
glucan %
as4CL-11.6 as4CL-15.7 as4CL-20.3 WT-22.1
year 3
xylan %
total sugar %
glucan %
43.3 ± 0.6 40.4 ± 0.0a
16.5 ± 0.3 18.2 ± 0.0a
62.8 ± 1.0 61.1 ± 0.0
41.6 ± 0.7
17.0 ± 0.4
± ± ± ±
a
39.7 36.1 38.6 62.1 ± 1.1 37.5 coastal plain
xylan % a
0.0 0.1a 0.3 1.3
year 2
a
14.5 14.9 13.8 14.1
± ± ± ±
0.0 0.1a 0.2 0.6
total sugar % 58.1 54.7 56.5 55.5
± ± ± ±
0.1a 0.6 0.6 2.3
year 3
transgenic lines/trees
glucan %
xylan %
total sugar %
as4CL-11.6 as4CL-15.7 as4CL-20.3 WT-22.1
41.9 ± 0.0a 39.9 ± 0.1a
16.8 ± 0.0a 17.3 ± 0.1a
62.6 ± 0.0a 60.4 ± 0.1a
41.2 ± 0.0
16.9 ± 0.1
61.9 ± 0.1
glucan % 39.6 36.7 38.2 35.1
± ± ± ±
0.1a 0.6a 0.1a 0.2
xylan % 13.4 15.4 12.6 14.4
± ± ± ±
0.1a 0.1a 0.3a 0.0
total sugar % 56.7 55.7 54.6 53.8
± ± ± ±
0.4a 0.8a 0.4a 0.3
Value is significantly different from that of the corresponding wild type sample (by paired-samples t test with confidence interval = 95%). sugar recovery =
total mono sugar obtained × 0.9 × 100% carbohydrate content of woody materials
However, the differences were much smaller compared to those of the greenhouse data. The hybrid poplars with lignin biosynthesis perturbation growing lignin contents like the wild type in the real environment might be further evidence for the control of lignin biosynthesis by environmental stresses.24 The S/V ratios of the transgenic NM6 tree stems were also brought back to a level close to that of the wild type with a few exceptions. Transgenic line as4CL-11.6 showed a much lower S/V ratio (0.6) compared to that of the wild type (1.8) after 3 years of growth in the mountain site. Interestingly, it was observed that, in both the mountain and coastal plain sites, the wild type (22.1%) and transgenic line as4CL-20.3 initially having higher lignin content showed an increase in their S/V ratios, whereas the other two transgenic lines, as4CL-15.7 and as4CL-11.6, that initially had moderate and low lignin contents, respectively, showed a decrease in their S/V ratios. The changes in S/V ratio in NM6 transgenic tree stems while grown in the field could be explained by the increase or decrease in the production of S or G lignin. For lines with a very low initial lignin content in the greenhouse, more condensed lignin units, e.g., the G unit, were produced. The reason could be that condensed lignin units take less biosynthesis steps to be produced,25 and the net structure of condensed lignin units brings higher mechanical strength to the tree stems, helping the tree with low initial lignin content survive in a severe environment. Uncondensed lignin units, e.g., the S unit, are mostly linear, whereas condensed lignin units have network structures and strong 5−5 linkages that strengthen the lignocellulosic matrix.7,8,26 For lines with high lignin content, it is possible that more S lignin units were produced because their initial lignin content was already enough to maintain the normal functionality of plants as the tree grew. Despite starting with three significantly different lignin contents within the greenhouse, all the transgenic lines that grew in the field, irrespective of the growing region, showed an increase in lignin content, making them comparable to the corresponding wild types in terms of both lignin content and S/V ratio. Line as4CL-11.6 grown in the mountains for 2 or 3 years and most of the lines grown in the coastal plains had significantly higher glucan content, 5−12% higher, compared to the wild type, indicating the higher cellulose content highlighted in Table 2. In the earlier investigation, it was observed that
(2) Statistical Analysis. The paired sample t test and linear regression analysis was conducted using PASW Statistics 18.0 (SPSS) with a confidence interval level of 95%.
■
RESULTS AND DISCUSSION Compositional Analysis of Field-Grown Transgenic Hybrid Poplars. In the greenhouse, all the trees from the as4CL genetic lines had reduced lignin content compared to that of the wild type (Table 1) due to the expression of the antisense 4CL gene. Greenhouse grown as4CL-15.7 and -11.6 samples had higher syringyl to vanillin ratios (S/V ratio) than that of the wild type. The lignin S/V ratio is an indication of the lignin S/G ratio when using the nitrobenzene oxidation method to determine lignin structure.15,23 Some recent studies have shown that the perturbation of some 4CL mutants could also result in a high S/G ratio in the plant stems.13−15 The reason for this could be that S lignin is mainly deposited in the interfascicular tissues whereas G lignin is primarily deposited in the xylem. The antisense insertion of the 4CL gene may have different effects on different tissues causing the changes in the S/G ratio.12 The lignin content and S/V ratios of the field-grown transgenic poplars grown in both the mountain and coastal plain regions for 2 and 3 years are also shown in Table 1. In the mountain region, after growth in the real environment, all the transgenic as4CL lines had lignin contents approaching that of the wild type, which had a value of approximately 22%. A few samples with lignin content significantly lower than that of the wild types were observed. Lines as4CL-11.6 and as4CL-20.3 grown for 2 years showed 21.1% and 20.6% lignin content, respectively, which were significantly lower than that of the corresponding wild type at 22.5%. In the coastal plains, a similar observation was made. Several samples had lignin content significantly lower than that of the wild type. Lines as4CL-11.6 and as4CL-20.3 grown for 2 years showed 23.2% and 22.9% lignin content, respectively, and lines as4CL-11.6 and as4CL15.7 grown for 3 years showed 22.9% and 22.4% lignin content, respectively. The values were significantly lower than that of the corresponding wild types at 24.6% and 25.1%, respectively. 2409
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
Research Article
ACS Sustainable Chemistry & Engineering
Table 3. Sugar Yields and Percentages of Xylan and Lignin Removal from Different Pretreatment Methods for Selected Transgenic and Wild Type Trees with Lignin Content of ∼22%a transgenic lines
tree age and grow site
as4CL-11.6 (21.1)
2 years mountain
as4CL-15.7 (22.1)
2 years mountain
as4CL-15.7 (22.5)
3 years mountain
as4CL-15.7 (22.4)
3 years coastal plain
WT-22.1 (22.5)
2 years mountain
pretreatment method
glucan removal %
xylan removal %
lignin removal %
sugar recovery %
none acid GL none acid GL none acid GL none acid GL none acid GL
0 7.2 9.9 0 5.2 8.2 0 4.2 13.9 0 9.5 12.0 0 0.96 8.4
0 63.0 27.9 0 72.5 30.2 0 53.7 30.2 0 63.6 33.1 0 59.4 21.2
0 7.1 32.7 0 11.3 26.7 0 5.3 38.2 0 10.7 36.6 0 13.8 25.3
18.8 78.6 10.2 58.3 71.8 46.5 71.4 64.4 47.4 72.8 85.6 12.1 48.8 64.0
a
(1) as4CL-11.6 (21.1) had a greenhouse lignin content of 11.6% and a current lignin content of 21.1%; (2) glucan, xylan, and lignin percentage removals are based on the xylan and lignin contents of the original untreated wood.
Table 4. Sugar Yields and Percentages of Xylan and Lignin Removal from Different Pretreatment Methods for Selected Trees with Lignin Content of ∼24%a transgenic lines
tree age and grow site
as4CL-11.6 (23.7)
3 years mountain
as4CL-15.7 (24.8)
2 years coastal plain
as4CL-20.3 (23.6)
3 years mountain
as4CL-20.3 (24.1)
3 years coastal plain
WT-22.1 (24.4)
average of wild types with lignin content of ∼24%
pretreatment method
glucan removal %
xylan removal %
lignin removal %
sugar recovery %
none acid GL none acid GL none acid GL none acid GL none acid GL
0 4.0 12.6 0 3.8 6.8 0 3.6 10.6 0 4.7 8.6 0 4.0 8.9
0 58.6 29.7 0 65.3 31.8 0 61.6 30.2 0 57.9 23.8 0 64.7 27.2
0 5.5 27.0 0 12.5 39.1 0 12.7 40.7 0 16.6 36.5 0 14.1 30.5
22.9 49.8 61.1 13.0 41.3 68.6 26.9 61.1 73.0 33.7 61.6 80.1 21.3 59.4 75.3
a
(1)as4CL-11.6 (23.7) had a greenhouse lignin content of 11.6% and a current lignin content of 23.7%; (2) glucan, xylan, and lignin percentage removals are based on the xylan and lignin contents of the original untreated wood.
inantly to remove the xylan content, whereas the purpose of green liquor pretreatment is to remove lignin for cellulose to be more accessible for subsequent enzymatic hydrolysis. The compositional changes of the transgenic tree stem samples after pretreatment were recorded (Tables S2−S5). For the effect of lignin on pretreatment to be eliminated, the transgenic and wild type trees were divided into two groups. One group had lignin content of 22%, and the other group had a lignin content of 24%. This classification may give us a better understanding on how lignin biosynthesis perturbation affects biomass pretreatments in addition to lignin content. When the lignin content was 22%, during the acid pretreatment, most of the transgenic hybrid poplar had higher removal of xylan than the wild type, which is shown in Table 3. The highest xylan removal of 72.5% was observed with as4CL-15.7 (22.1) compared to the 59.4% removed in the corresponding wild type. For green liquor pretreatment, most of the transgenic hybrid poplar had higher lignin removal than the wild type. The
cellulose biosynthesis was enhanced for the compensation of low lignin contents.5,11,18 This study shows that compensation for transgenic hybrid poplars may have prolonged effects after either 2 or 3 years of growth in field-environments if the samples contain less lignin than their wild-type counterparts, as highlighted in Tables 1 and 2. The high glucan content in the transgenic hybrid poplar may result in a higher enzymatic saccharification yield compared to that of the wild type. Pretreatments before Enzymatic Saccharification. Pretreatments on the extractive free sawdust from both the transgenic and wild type samples from both the mountain and coastal plain were conducted to enhance the enzymatic saccharification efficiency. Two types of pretreatments were applied: dilute acid pretreatment with 0.1% (mass concentration) sulfuric acid and green liquor pretreatment with 6% TTA (see Pretreatments section). These pretreatments were chosen to represent an acidic and an alkaline pretreatment, respectively, because they remove different components during pretreatment. The purpose of acid pretreatment is predom2410
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
Research Article
ACS Sustainable Chemistry & Engineering highest lignin removal was 38.2% with as4CL-15.7 (22.5) compared to the 25.3% removed from the wild type. When the lignin content was greater than 24% during acid pretreatment, the opposite results were observed. Most of the transgenic hybrid poplars had lower xylan removal than that of the wild type, as shown in Table 4. Only as4CL-15.7 (24.8) had comparable xylan removal to that of the wild type at 65.3% versus 64.7%. However, for green liquor pretreatment, most of the transgenic hybrid poplar still had more lignin removed than their wild type counterparts, except for as4CL-11.6 (23.7). The highest lignin removal was with as4CL-20.3 (23.6), which removed 40.7% of the lignin as compared to 30.5% for the wild type. As the exception, line as4CL-11.6 (23.7) had very low lignin removal of 27.0%, which is probably due to its very low S/V ratio of 0.6 (Table 1). This indicates that S/V ratio may have some effects on delignification for the field-grown transgenic hybrid poplars. In general, the S/V ratios of the transgenic hybrids were very close and thus other reasons may be dominant in controlling the delignification of green liquor pretreatment for transgenics. In summary, most of the transgenic hybrid poplar samples had more xylan or lignin removed after pretreatments compared to those of the wild types. The results may indicate that the xylan or lignin in transgenic tree stems is less recalcitrant. The reduced recalcitrance could be due to the possible alteration of lignin-carbohydrate linkages, which has already been proven to be closely related to the enzymatic saccharification efficiency of the NM6 tree stems grown in the greenhouse.27 Enzymatic Saccharification. Enzymatic hydrolysis of all of the field-grown transgenic samples was conducted with 5% solids using a buffer of pH 4.8 with 5 FPU of cellulase per gram cellulose at 50 °C for 48 h. Transgenic samples without any additional pretreatment and after either the dilute acid or green liquor pretreatments were used to evaluate the effects of pretreatment on enzymatic saccharification. The enzyme loading was chosen because a higher dosage is not economically viable. After enzymatic hydrolysis, the sugar content in the hydrolysate was quantified, allowing sugar yield and recovery for the samples to be calculated. The calculations are defined in the Sugar Yield and Carbohydrate Saccharification Efficiency section. Sugar recovery is more related to the recalcitrance of the transgenic hybrid poplar wood stems, whereas sugar yield is more about the ability of the biomass to produce sugars. In general, the enzymatic sugar yield for the pretreated and untreated samples had an order of (1) green liquor pretreatment, (2) acid pretreatment, and (3) no pretreatment, as illustrated in Figures 1 and 2. These results again confirm that removing lignin or xylan is an effective way to increase enzyme accessibility to cellulose.26,27 Overall, lignin may still play a more vital role than xylan during enzymatic saccharification for these transgenic tree stem samples because green liquor pretreatment, which removes more lignin than xylan, led to the highest enzymatic sugar yield as shown in Tables 3 and 4. These results are consistent with our previous studies on enzymatic hydrolysis on acid- and green liquor-pretreated wood samples.17,18 Sugar recovery from the samples from the mountain site after 2 and 3 years of growth are shown in Figure 1. Most of the transgenic lines without any pretreatment exhibited significantly higher sugar recovery compared to that of their corresponding wild types. The increase in sugar recovery for the unpretreated samples relative to that of the wild type for the transgenic line
Figure 1. (a) Sugar recovery and (b) sugar yield (with standard error bar) after enzymatic hydrolysis (EH) of transgenic sawdust with or without pretreatments after 2 and 3 years of growth in the mountain region (based on the original extractive-free sawdust) (Y2, year 2; Y3, year 3), e.g., as4CL-11.6 (23.7) had a greenhouse lignin content of 11.6% and a current lignin content of 23.7%; acid pretreatment for Y2as4CL-21.1 was not conducted due to limited sample; asterisks (*) indicate that the value is significantly different from that of the corresponding wild-type sample (by paired-samples t test with confidence interval = 95%).
as4CL-11.6 from the two-year growth period was 55.4%, whereas the samples from the three-year growth period, as4CL20.3 and as4CL-15.7, were 18.0 and 103.2%, respectively. This may indicate that the transgenic NM6 tree stems are less recalcitrant to enzymatic hydrolysis. After dilute acid pretreatment, all the transgenic lines, except as4CL-11.6 from the three-year growth period, showed a significant increase in sugar recovery compared to that of the corresponding wild types. Line as4CL-15.7 from the two-year growth period showed an increase in sugar recovery of 19.5%. The three-year growth period transgenics, as4CL-20.3 and as4CL-15.7, had an increase in sugar recovery after dilute acid pretreatment of 3.7 and 21.2%, respectively. Line as4CL-15.7 without any pretreatment and after dilute acid pretreatment demonstrated the highest sugar recovery among the transgenics. After green liquor pretreatment, only the samples from the two-year growth period showed significant improvement in sugar recovery, whereas none of the transgenics from the three-year growth period showed an increase in sugar recovery comparable to that of the corresponding wild type. The increases in sugar recovery compared to the wild type for the two-year growth period transgenic lines as4CL-15.7 and as4CL-11.6 were 12.2 and 22.8%, respectively. Transgenic line as4CL-11.6 from the two2411
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
Research Article
ACS Sustainable Chemistry & Engineering
sugar yield numbers had similar patterns compared to the sugar recovery numbers. Sugar recovery for all of the samples grown in the coastal plains region for two or three years is presented in Figure 2. All of the untreated transgenics from the two-year growth period did not show any improvement in sugar recovery, whereas all of the untreated transgenics from the three-year growth period showed a significant increase in sugar recovery relative to that of the wild type. The transgenic lines as4CL-20.3, as4CL-15.7, and as4CL-11.6 from the three-year growth period exhibited an increase in sugar recovery relative to the wildtype by 35.3, 90.4, and 58.2%, respectively. Except for as4CL-15.7 from the threeyear growth period, none of the transgenic lines showed any improvement in sugar recovery after dilute acid treatment in comparison to that of the wild type. The increase in sugar recovery for as4CL-15.7 from the three-year growth period in comparison to the corresponding wild type was 6.4%. After green liquor pretreatment, again none of the transgenics grown for two years showed any improvement in sugar recovery; however, all of the transgenics grown for three years showed a significant improvement in sugar recovery after green liquor pretreatment in comparison to that of the wild type. The increases in sugar recovery for the transgenic samples as4CL20.3, as4CL-15.7, and as4CL-11.6 as compared to the wild type were 2.2, 9.2, and 16.2%, respectively. The highest sugar recovery was seen with transgenic line as4CL-15.7 grown for three years without additional pretreatment after dilute acid pretreatment and as4CL-11.6 grown for three years with green liquor pretreatment. In addition, the sugar yield numbers had similar trends compared to the sugar recovery numbers for the wild type and transgenics grown in the coastal plains region. Overall, it was observed that lignin still plays a vital role in the saccharification efficiency for the wild types and transgenics because green liquor pretreatment removed more lignin and resulted in higher sugar recovery and sugar yield than those of acid pretreatment (Tables 3 and 4). However, even though the field-grown transgenics increased their lignin content close to the level of the wild types, they still expressed higher enzymatic sugar recovery and sugar yield than the wild type for some transgenic lines. Transgenic samples without pretreatment had the highest increase in sugar recovery and sugar yield, and samples with acid pretreatment had the smallest increase compared to the wild type. Comparing the samples between mountain and coastal plain regions, transgenic trees grown in the coastal plains region had a higher increase in sugar recovery and sugar yield, which could be due to environmental differences between the two regions causing changes to the lignin or hemicellulose biosynthesis. Here, we can state that field-grown transgenic hybrid poplar (NM6) with modified
Figure 2. (a) Sugar recovery and b) sugar yield (with standard error bar) after enzymatic hydrolysis (EH) of transgenic sawdust with or without pretreatments after 2 and 3 years of growth in the coastal plain region (based on the original extractive-free sawdust) (Y2, year 2; Y3, year 3), e.g., as4CL-11.6 (22.9) had a greenhouse lignin content of 11.6% and a current lignin content of 22.9%; asterisks (*) indicate that the value is significantly different from that of the corresponding wildtype sample (by paired-samples t test with confidence interval = 95%).
year growth period showed the highest sugar recovery after green liquor pretreatment. If the transgenics and wild type had the same cellulose content, their sugar yield should have the same pattern or trend in the sugar recovery data. However, some transgenic lines had higher cellulose content than the wild types, such as as4CL-11.6 grown in the mountains for both growth periods and in the coastal plains for three years (Table 2). Consequently, it may also be interesting to evaluate the sugar yields. From Figure 2, it can be observed that untreated as4CL-11.6 samples from the three-year growth period with the same sugar recovery value had ∼5% higher sugar yield than the corresponding wild type. However, for other samples grown in the mountain region, the
Table 5. Results of Linear Regression Analysis Conducted by PASW Statistics 18.0 (SPSS) Using Saccharification Efficiency as Dependent Variable and Using Lignin Content and Xylan Content as Independent Variables for Wild Type and Transgenics (Confidence Interval = 95%) sugar recovery (z) dependent variable sugar recovery (z)
independent variables
R2
samples
n
lignin content (x)
wild type
12
z = −5.5x − 2.2y + 185.9
0.848
xylan content (y)
transgenics
29
z = −4.5x − 2.2y + 164.9
0.809
models
2412
correlation coefficients
Pearson correlation
partial correlation
significance
lignin content (x) xylan content (y) lignin content (x) xylan content (y)
−0.841 −0.612 −0.804 −0.582
−0.870 −0.694 −0.843 −0.679
0.000 0.018 0.000 0.000
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
Research Article
ACS Sustainable Chemistry & Engineering
The statistical analysis indicated that, by analyzing the wild type and transgenic samples separately, lignin or xylan contents had similar correlations with the sugar recovery. However, by comparing the wild type and transgenic samples together, at similar lignin content, the transgenics overall showed higher sugar recovery. It can be postulated that even though the lignin biosynthesis for the transgenic hybrid poplar seemed to become normal after they were planted in the real environment, the transgenic tree stems became less recalcitrant to chemical reactions (AH and GL pretreatments) and enzymatic saccharification. It is hypothesized that other chemical or physical structural features of transgenic tree stems may be alternated during their growth in the real environment. Finally, we can state that field-grown transgenic hybrid poplar (NM6) with modified lignin biosynthesis has improved enzymatic sugar recovery and sugar yield. These findings are very important to biotechnology research aimed at producing biofuels with less cost.
lignin biosynthesis has improved enzymatic sugar recovery and sugar yield. The Roles of Xylan and Lignin in Enzymatic Saccharification of Transgenics. For correlations between lignin content, xylan content, and saccharification efficiency to be evaluated, linear regression analysis was conducted using PASW Statistics 18.0 (SPSS) with a confidence interval level of 95%. The dependent variable was sugar recovery, and the independent variables were lignin and xylan content (of the pulp or original wood). The R-squared values of the generated linear models for the wild types and transgenics were 0.848 and 0.809, respectively, indicating that the lignin content and xylan content were linearly related to enzymatic saccharification efficiency (Table 5). The individual Pearson correlation and partial correlation coefficients between lignin or xylan content and sugar recovery were also calculated. It was found that wild type and transgenic trees had similar correlation coefficients. The Pearson and partial correlations between lignin content and saccharification efficiency were approximately −0.8 and between xylan content and saccharification efficiency were approximately −0.6 for both wild type and transgenics (Table 5). The results were consistent with the previous theory that removing lignin or xylan is an effective way to increase enzyme accessibility to cellulose.28,29 Nevertheless, with higher Pearson and partial correlation coefficients, it can be postulated that lignin content had stronger effects on sugar recovery than the xylan content. By evaluating the wild type and transgenics separately, lignin and xylan contents had very similar effects on the enzymatic saccharification efficiency due to the very close R-squared and correlation coefficient numbers. However, at a similar lignin level, some transgenic samples demonstrated higher sugar yield with or without pretreatments than the wild-type samples (Tables 3 and 4). The reason could be the higher lignin and xylan removed after pretreatment, but this could not explain the higher sugar recovery for some untreated transgenic samples. In other words, the transgenic hybrid poplar tree stems were less recalcitrant to chemical reactions (AH and GL pretreatments) and enzymatic saccharification than that of the wild types. It is possible that other chemical or physical structural features of transgenic tree stems such as lignin-carbohydrate linkage27 or cellulose crystallinity30,31 may be alternated during their growth in the real environment.
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02740. (Table S1) Lignin contents of a second tree selected from each line of 2 year growth, (Tables S2−S5) detailed weight loss and chemical composition data before and after pretreatments of transgenic trees grown in the mountain and coastal plain regions after 2 or 3 years (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]; Tel: 1-919-522-1923. ORCID
Zhouyang Xiang: 0000-0003-1840-9237 Hasan Jameel: 0000-0002-9947-7313 Notes
The authors declare no competing financial interest.
■
■
ACKNOWLEDGMENTS This study was supported by United States Department of Agriculture CSREES Grant (2009-10001-05113). The authors would also like to acknowledge support from National Natural Science Foundation of China (31600470) and China Postdoctoral Science Foundation (2016M590783). The authors thank Dr. John King, Aletta Davis, and Anna Stout for cultivating and harvesting the transgenic trees and valuable discussions. The authors are also grateful to Novozymes North America, Inc. for providing the enzymes used in this study.
CONCLUSIONS In this study, hybrid poplars (Populus nigra L. × Populus maximowiczii A.), abbreviated as NM6, were genetically modified through antisense of the 4-coumarate:coenzyme A ligase (4CL) gene. Their compositional changes to these transgenics in the real environment were recorded using field trials in different regions of North Carolina. The results of enzymatic saccharification on those transgenic poplars were evaluated. In the greenhouse, the transgenic hybrid poplar expressed successful lignin biosynthesis suppression. However, when they were transplanted in the field, most of the transgenics had their lignin content and S/V ratios brought back to levels closer to those of the wild type. Even though they have the same lignin content level, most of the transgenics demonstrated higher lignin and xylan removal rates after green liquor and dilute acid pretreatments. Many of the transgenics showed significantly higher enzymatic sugar recovery and sugar yield than those of the wild type for untreated and pretreated samples.
■
REFERENCES
(1) Zhu, J. Y.; Pan, X. J. Woody biomass pretreatment for cellulosic ethanol production: Technology and energy consumption evaluation. Bioresour. Technol. 2010, 101, 4992−5002. (2) Akhtar, M.; Attridge, M. C.; Myers, G. C.; Blanchette, R. A. Biomechanical pulping of loblolly pine chips with selected white-rot fungi. Holzforschung 1993, 47, 36−40. (3) Pilate, G.; Guiney, E.; Holt, K.; Petit-Conil, M.; Lapierre, C.; Leple, J. C.; Pollet, B.; Mila, I.; Webster, E. A.; Marstorp, H. G.; Hopkins, D. W.; Jouanin, L.; Boerjan, W.; Schuch, W.; Cornu, D.; 2413
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
Research Article
ACS Sustainable Chemistry & Engineering Halpin, C. Field and pulping performances of transgenic trees with altered lignification. Nat. Biotechnol. 2002, 20, 607−612. (4) Xing, R.; Qi, W.; Huber, G. W. Production of furfural and carboxylic acids from waste aqueous hemicellulose solutions from the pulp and paper and cellulosic ethanol industries. Energy Environ. Sci. 2011, 4, 2193−205. (5) Hu, W. J.; Harding, S. A.; Lung, J.; Popko, J. L.; Ralph, J.; Stokke, D. D.; Tsai, C. J.; Chiang, V. L. Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees. Nat. Biotechnol. 1999, 17, 808−812. (6) Wagner, A.; Donaldson, L.; Kim, H.; Phillips, L.; Flint, H.; Steward, D.; Torr, K.; Koch, G.; Schmitt, U.; Ralph, J. Suppression of 4-Coumarate-CoA Ligase in the Coniferous Gymnosperm Pinus radiata. Plant Physiol. 2009, 149, 370−383. (7) Besombes, S.; Mazeau, K. The cellulose/lignin assembly assessed by molecular modeling. Part 1: adsorption of a threo guaiacyl β-O-4 dimer onto a Iβ cellulose whisker. Plant Physiol. Biochem. 2005, 43, 299−308. (8) Besombes, S.; Mazeau, K. The cellulose/lignin assembly assessed by molecular modeling. Part 2: seeking for evidence of organization of lignin molecules at the interface with cellulose. Plant Physiol. Biochem. 2005, 43, 277−286. (9) Li, L. G.; Cheng, X. F.; Leshkevich, J.; Umezawa, T.; Harding, S. A.; Chiang, V. L. The last step of syringyl monolignol biosynthesis in angiosperms is regulated by a novel gene encoding sinapyl alcohol dehydrogenase. Plant Cell 2001, 13, 1567−1585. (10) Stewart, J. J.; Akiyama, T.; Chapple, C.; Ralph, J.; Mansfield, S. D. The effects on lignin structure of overexpression of ferulate 5hydroxylase in hybrid poplar. Plant Physiol. 2009, 150, 621−635. (11) Li, L. G.; Zhou, Y. H.; Cheng, X. F.; Sun, J. Y.; Marita, J. M.; Ralph, J.; Chiang, V. L. Combinatorial modification of multiple lignin traits in trees through multigene cotransformation. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 4939−4944. (12) Li, Y.; Kim, J. I.; Pysh, L.; Chapple, C. Four isoforms of Arabidopsis 4-coumarate: CoA ligase have overlapping yet distinct roles in phenylpropanoid metabolism. Plant Physiol. 2015, 169, 2409− 2421. (13) Van Acker, R.; Vanholme, R.; Storme, V.; Mortimer, J. C.; Dupree, P.; Boerjan, W. Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana. Biotechnol. Biofuels 2013, 6, 46. (14) Xu, B.; Escamilla-Treviño, L. L.; Sathitsuksanoh, N.; Shen, Z.; Shen, H.; Zhang, Y. H. P.; Dixon, R. A.; Zhao, B. Silencing of 4coumarate:coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production. New Phytol. 2011, 192, 611−625. (15) Xiang, Z.; Sen, S. K.; Roy, A.; Min, D.; Savithri, D.; Jameel, H.; Chiang, V.; Chang, H. M. Wood characteristics and enzymatic saccharification efficiency of field-grown transgenic black cottonwood with altered lignin content and structure. Cellulose 2015, 22, 683−693. (16) Li, Q. Z.; Min, D. Y.; Wang, J. P. Y.; Peszlen, I.; Horvath, L.; Horvath, B.; Nishimura, Y.; Jameel, H.; Chang, H. M.; Chiang, V. L. Down-regulation of glycosyltransferase 8D genes in Populus trichocarpa caused reduced mechanical strength and xylan content in wood. Tree Physiol. 2011, 31, 226−236. (17) Min, D. Y.; Li, Q. Z.; Jameel, H.; Chiang, V.; Chang, H. M. Comparison of pretreatment protocols for cellulase-mediated saccharification of wood derived from transgenic low-xylan lines of cottonwood (P. trichocarpa). Biomass Bioenergy 2011, 35, 3514−3521. (18) Min, D. Y.; Li, Q. Z.; Jameel, H.; Chiang, V.; Chang, H. M. The cellulase-mediated saccharification on wood derived from transgenic low-lignin lines of black cottonwood (Populus trichocarpa). Appl. Biochem. Biotechnol. 2012, 168, 947−955. (19) Min, D. Y.; Yang, C.; Shi, R.; Jameel, H.; Chiang, V.; Chang, H. M. The elucidation of the lignin structure effect on the cellulasemediated saccharification by genetic engineering poplars (Populus nigra L.× Populus maximowiczii A.). Biomass Bioenergy 2013, 58, 52− 57.
(20) Huntley, S. K.; Ellis, D.; Gilbert, M.; Chapple, C.; Mansfield, S. D. Significant increases in pulping efficiency in C4H-F5H-transformed poplars: Improved chemical savings and reduced environmental toxins. J. Agric. Food Chem. 2003, 51, 6178−6183. (21) Voelker, S. L.; Lachenbruch, B.; Meinzer, F. C.; Jourdes, M.; Ki, C. Y.; Patten, A. M.; Davin, L. B.; Lewis, N. G.; Tuskan, G. A.; Gunter, L.; Decker, S. R.; Selig, M. J.; Sykes, R.; Himmel, M. E.; Kitin, P.; Shevchenko, O.; Strauss, S. H. Antisense down-regulation of 4CL expression alters lignification, tree growth, and saccharification potential of field-grown poplar. Plant Physiol. 2010, 154, 874−886. (22) Xiang, Z.; Anthony, R.; Tobimatsu, Y.; Runge, T. Emulsifying properties of an arabinoxylan-protein gum from distillers’ grains and the co-production of animal feed. Cellulose 2014, 21, 3623−3635. (23) Min, D. Y.; Xiang, Z.; Liu, J.; Jameel, H.; Chiang, V.; Jin, Y.; Chang, H. M. Improved protocol for alkaline nitrobenzene oxidation of woody and non-woody biomass. J. Wood Chem. Technol. 2014, 35, 52−61. (24) Kaur, H.; Shaker, K.; Heinzel, N.; Ralph, J.; Galis, I.; Baldwin, I. T. Environmental stresses of field growth allow cinnamyl alcohol dehydrogenase-deficient Nicotiana attenuata plants to compensate for their structural deficiencies. Plant Physiol. 2012, 159, 1545−1570. (25) Weng, J. K.; Li, X.; Stout, J.; Chapple, C. Independent origins of syringyl lignin in vascular plants. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 7887−7892. (26) Chen, C. L. Nitrobenzene and cupric oxide oxidation. In Method in Lignin Chemistry; Lin, S. Y., Dence, C. W., Eds.; Springer-Verlag: Berlin, 1992; pp 300−323. (27) Min, D. Y.; Yang, C.; Chiang, V.; Jameel, H.; Chang, H. M. The influence of lignin−carbohydrate complexes on the cellulase-mediated saccharification II: Transgenic hybrid poplars (Populus nigra L. and Populus maximowiczii A.). Fuel 2014, 116, 56−62. (28) Kumar, P.; Barrett, D. M.; Delwiche, M. J.; Stroeve, P. Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind. Eng. Chem. Res. 2009, 48, 3713−3729. (29) Taherzadeh, M. J.; Karimi, K. Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int. J. Mol. Sci. 2008, 9, 1621−1651. (30) Berlin, A.; Balakshin, M.; Gilkes, N.; Kadla, J.; Maximenko, V.; Kubo, S.; Saddler, J. Inhibition of cellulase, xylanase and betaglucosidase activities by softwood lignin preparations. J. Biotechnol. 2006, 125, 198−209. (31) Hendriks, A.; Zeeman, G. Pretreatments to enhance the digestibility of lignocellulosic biomass. Bioresour. Technol. 2009, 100, 10−18.
2414
DOI: 10.1021/acssuschemeng.6b02740 ACS Sustainable Chem. Eng. 2017, 5, 2407−2414
