Bioaugmentation of Corn Stalk Saccharification with Aspergillusfumigatus Under Low/High Solid Loading Culture

In this study, pretreatment of corn stalks with the cellulosic fungus Aspergillusfumigatus CLL was performed to enhance its saccharification. The effect of feedstock loading (10 g/L and 20 g/L) on corn stalk pretreatment performance and the effects of Trichodermareesei cellulase and commercial cellulase on the saccharification of pretreated corn stalks were investigated. The results indicated that with a solid loading of 10 g/L for 2 days at 30 °C, 25.2% lignin was degraded, whereas the degradation was only 2.8% under solid loading of 20 g/L. Pretreated corn stalks (solid loading 10 g/L for 2 days at 30 °C) saccharified by 300 U/L T.reesei cellulose exhibited 3.4 times higher sugar yields (67.9%) compared to the raw feedstocks, and the value was comparable with the saccharification performance of commercial cellulase (60.8%). These results demonstrated that A.fumigatus CLL not only decomposed the structure of corn stalks but also complemented the incomplete cellulase system of T.reesei. A.fumigatus CLL can be used to pretreat lignocellulosic materials to enhance the saccharification performance.


Introduction
Lignocellulosic biomass is one of the most abundant resources on the Earth 1 which represents a large reservoir of glucose and an attractive renewable energy source which can be a potential feedstock for the biofuels and other high-value chemical products 2 . The utilization of lignocellulosic biomass to produce biofuels will bring signi cant opportunities for carbon-neutral economy 3 The hydrolysis of lignocellulosic feedstocks into monosaccharides is one of the key steps in the lignocellulosic biofuels production. Lignocellulosic biomass sacchari cation affected by the structure of lignocellulose feedstocks and the activity of cellulase 4 , cellulose and hemicellulose entangled together with lignin formed a rigid matrix in plant biomass which is recalcitrant to cellulase attacks 5 , in addition, the holocellulose hydrolysis performed with the synergism of multiple cellulase which increased the sacchari cation cost and reduced process operability of lignocellulosic biofuels 6 .
It has been generally agreed that effectiveness of pre-treatment and sacchari cation determines the yield of biofuels in the fermentation step. In previous studies, number of excellent physical/chemistry pretreatment methods such as acid or alkali treatment, hot water soak, steam explosion and ammonia ber expansion have been developed 7 . However, high energy consumption, high equipment requirements, heavy pollution and the production of downstream fermentation inhibitors during physical and chemical pretreatment should not be ignored 8 . On the other hand, the sacchari cation of lingnocellulose still remains as one of the critical bottle-necks. The enzymatic hydrolysis of cellulosic feedstocks achieved by cellulase cocktail which composed of endoglucanases, exoglucanases and β-glucosidases. Although many fungi have been reported for high-yield cellulase production, Trichoderma reesei cellulase is most extensively used in commercial processes, and frequently used in biomass sacchari cation 8, 9 . The amount of β-glucosidases secreted by T. reesei was insu cient for effective cellulose conversion and can be relieved by adding external β-glucosidases 10 , but the addition of β-glucosidase increases the cost of sacchari cation. As a ubiquitous lamentous fungal, Aspergillus fumigatus was reported to have a good ability to synthesize cellulase β-glucosidase 11,12 , the high βglucosidase activity of served it being a potential good supplement for T. reesei cellulase in lignocellulose sacchari cation. Meanwhile, the genus Aspergillus sp. has been reported to have the highest degradative capacity for aromatics 13 which suggest with ability of lignin degradation and decomposed the structure of lignocellulose feedstocks which facilitated the lignocellulose sacchari cation 14 .
In this study, A, fumigatus CLL was promoted to decompose the structure of corn stalks to enhance the sacchari cation performance of corn stalks, what's more, explored the feasibility of supplement β-glucosidase of to T. reesei cellulase system. The solid loading has a great in uence on the lignocellulose sacchari cation, Aspergillus fumigatus CLL was conducted to treat corn stalks under high solid loading (20 g/L) culture and low solid loading (10 g/L) culture. In addition, the sacchari cation results obtained from A, fumigatus CLL cellulase and T. reesei cellulase were compared with the sample sacchari cation by commercial cellulase sacchari cation in this study.

Raw materials and inoculum
Corn stalks obtained from the farm of Heilongjiang University, Harbin, Heilongjiang, China. The corn stalks were crushed and sieved through 60 sieves, then dried at 65 ℃ until weight kept constant for later use. The composition of lignocellulosic feedstocks was determined by ANKOM automatic ber analyzer (https://www.ankom.com/product-catalog/ankom-200-ber-analyzer) and shown in Table 1. Lignocellulosic fungi A. fumigatus CLL was obtained in the Microbiology Laboratory of Heilongjiang University of Science and Technology. A.fumigatus CLL was maintained on potato dextrose agar (PDA) plates at 4 ℃. The spores of A.fumigatus CLL that grew well on PDA were transferred to the modi ed Martin medium and cultured for 2 days. Spore suspension (10 7 spores per mL) was added (2% v w −1 , corresponding to 2 ×10 5 spores g −1 feedstock) for high solid loading culture and low solid loading culture. Trichoderma reesei(DSM 768)was obtained in the Microbiology Laboratory of Heilongjiang University of Science and Technology, and the cellulase of T. reesei cellulase was produced and separated according the method described by Zhao 15 . Brie y, T. reesei was cultured in the cellulase production medium ((NH 4 ) 2 SO 4  Diluted the commercial cellulase and T. reesei cellulase to 0.6-6.0 g/L with citrate buffer (0.05mmol/L, pH4.5) for future corn stalks sacchari cation. The sacchari cation of pretreated/untreated corn stalks was performed according the method described by Sheng 16 . Brie y,. mixing the commercial cellulase/T. reesei cellulase with different corn stalks samples (10/1, v/w) at 55°C, the sacchari cation was carried out for 24 hours and the samples were collected every 3 hours.

Analysis methods
Sugar was performed by HPLC described by Sheng 16 . The structure of corn stalks was determined by JSM6480 scanning electron microscope.
The changes in functional groups of corn stalks was determined by Perkin Elmer Spectrum 100 FT-IR Spectrometer. The cellulase activities determined according to the method described by Ghose 17 . The lignase activities were determined according to the method described by Morgan 18 . All tests were replicated three times, and the mean was described with standard deviation.
In this formula, W sugar released is sugar production during sacchari cation (mg), sugar conversion factor is hydrolysis sacchari cation correction factor (Glucose 0.9, Xylose 0.88) ,total cellulosic fraction is the fraction of cellulose and hemicellulose in corn stalks (mg) 19,20 . All tests were replicated three times, and the mean was described with standard deviation.

Effect of bioaugmentation on the composition of corn stalks
It is believed that lignin is the main obstacle of lignocellulosic biomass sacchari cation for cellulose and hemicellulose were bundled with lignin 20 , while the removal of lignin not only depolymerized the hash structure of the corn stalks but also facilitated the contact of cellulase with holocellulose 21 . It is reported that the high solid loading culture white rote fungi commonly used to decomposed the lignin of lignocellulosic feedstocks, which consumes low energy and environmental friendly 22 . Compared with high solid loading culture, low solid loading improved the biological reaction e ciency and shorten reaction time for more contact of ligninase with lignin 23 . In this study, as shown in Fig. 1(a), in the rst 30 days of high solid loading culture, the degradation ratio of cellulose and hemicellulose were similar with lignin. At this time, the lignin degradation ratio was 26.43%, the cellulose and hemicellulose degradation ratio were 28.78% and 25.45%, respectively. 30 days later, the lignin degradation ratio was higher than that of cellulose and hemicellulose. 60 days later, the lignin degradation ratio was 52.22%, while the degradation ratio of cellulose and hemicellulose were 44.78% and 33.86%, respectively. Compared with high solid loading culture, the corn stalks degradation ratio increased rapidly under low solid loading culture. As shown in Fig. 1(b), the lignin degradation ratio increased rapidly in 2 days and gradually stabilized after 16 days, 25.2% lignin was degraded in 2 days, and the degradation ratio of cellulose and hemicellulose was 7.48% and 6.93%, respectively. 20 days later, the lignin degradation ratio reached 50.5%. Meanwhile, hemicellulose and cellulose degradation ratio reached 27.95% and 29% respectively. While under high solid loading culture, the degradation ratio of lignin, cellulose, and hemicellulose were only 18.08%, 20.17%, and 21.41% in 20 days. The results indicate that A. fumigatus CLL able to degrade lignin under high/low loading culture effectively, compare with high solid loading culture, less holocellulose consumed under low solid loading culture which is more conducive to subsequent sacchari cation and utilization for A. fumigatus CLL. In previous studies, some lignocellulosic fungi, especially white rot fungi or brown rot fungi, were conducted to pretreat cellulosic feedstocks and some progress were obtained 24,25 . In contrast, Aspergillus sp. has been reported for high β-glucosidase production 26, 27 . More importantly, the product of degrading lignin is fatty acids rather than aromatic monomers 14 , was clearly brought about substantial demethoxylation and dehydroxylation, whereas white rot fungi degraded lignin closely resembled undegraded kraft lignin 28 , Compared with white rot fungi, enhanced the hydrophobicity typically enables stronger hydrophobic interactions between cellulase and lignin, reduced inhibitory effect of lignin and its derivatives on cellulase 29,30 .
The performance of lignin degradation is closely related to the ligninase. Therefore, the trend of ligninase activity in high/low solid loading culture should be clari ed. The major enzymes associated with lignin-degrading fungi are lignin peroxidase (EC 1.11.1.14), manganese peroxidase (EC 1.11.1.13) and laccase (EC 1.10.3.2). As shown in Fig. 1(c) and Fig. 1(d). During the low solid loading culture process, the laccase (Lac) activity and increased in the rst 10 days and the maximum Lac activity (15.6 U/mL) was obtained at 10 days, then decreased to 1.2 U/mL at 20 days. The trend of Lignin peroxidase (Lip) activity was similar to that of Lac activity, the maximum Lip activity of 14.6 U/mL was obtained at 10 days, 20 days later the activity of Lip was just 1.1 U/mL. Different from Lac and Lip, the peak of Manganese peroxidase (Mnp) activity obtained at 8 days, the maximum Mnp was 16.3 U/mL, 12 days later, the Mnp activity was only 10% of the peak.
Different from the low solid loading culture, the lignase activity of high solid loading culture peaked at 25 days and decreased quickly. Overall, the peak lignase activity of high solid loading culture similar with low solid loading culture, but the peak time for lignase has been doubled, which indicates that low solid loading culture is more conducive for A. fumigatus CLL to induce ligninase, and the better lignin degradation performance might be attributed to the increase of the contact of ligninase with corn stalks under low solid loading culture, and speed up the synthesis rate of ligninase. It is reported that the presence of proteins induced a high production of the ligninase 31 . In the low solid loading culture, peptone dissolved in the liquid medium, which increases the contact of the protein with A. fumigatus CLL which enhanced the activity of ligninase, what's more, a certain level of readily available carbon sources is necessary to induce and maintain the activities of ligninase 32 , it can be inferred that low solid loading culture provided soluble oligosaccharides for A. fumigatus CLL and the available carbohydrates enhanced the synthesis of ligninase. On the other hand, It has been suggested that the extracellular glucan plays a role in the degradation of lignin as an indirect source of hydrogen peroxide 33,34 . Another participation of the extracellular glucan in the fungus metabolism, and in particular in wood degradation is that they function as a supporting network on which some of the excreted ligninase adsorb 35 . They may also contribute in maintaining an optimal pH for ligninase 36 .

Effect of bioaugmentation on the structural features of corn stalks
The degradation of lignin not only released holocellulose from the lignin package, at the same time, loosen the structure of lignocellulose raw materials for subsequent use 37 . As shown in Fig. 2(a), the raw corn stalks show a dense layer of lignin structure, 1 day later, some breakage was obtained on the corn stalks surface(b), which suggest that the corn stalks start to degrade. 2 days later, the corn stalks surface has been destroyed more obviously, the cellulose and hemicellulose exposed from lignin while the structure is relatively complete(c).10 days later, as shown in Fig. 2(d), the surface structure of the corn stalks has been completely destroyed.
The FTIR result was shown in Fig. 2(e). The functional groups of corn stalks showed obvious changes during treated by A. fumigatus CLL. In the rst two days, compared with untreated raw materials, the 1512cm −1 band showed obvious absorption, which corresponds to the aromatic skeleton of lignin vibration C=C 16 . In addition, the characteristic peaks near the 1266cm −1 waveband appear to be signi cantly weakened, where it is the C-O bond 38 . Obvious absorption appeared at 2919-2922cm −1 and 3400cm −1 indicates that non-cellulose species such as lignin were degraded and the holocellulose were exposed and preserved to some extent 39 .

Effect of bioaugmentation on the sacchari cation of corn stalks
A key factor affecting the e ciency of cellulase hydrolysis was the availability of holocellulose. It is believed that the lignin content closely related to the lignocellulosic feedstocks sacchari cation performance 40 , removal the lignin and loosen the hard structure facilitated the lignocellulosic sacchari cation, therefore, removing lignin from lignocellulose and destroying the structure of lignin is a crucial step in the sacchari cation of lignocellulose feedstocks. To evaluate the bioaugmentation on the sacchari cation of pretreated/unpretreated corn stalks, commercial cellulase and T. reesei cellulase, which play a key role in the sacchari cation process was conducted to hydrolysis corn stalks which treated by for 1 day. As shown in Fig. 3(a), the sacchari cation ratio increased as the commercial cellulase concentration increased from 0.6 g/L to 4.8 g/L, increased the commercial cellulase to 6.0 g/L, the sacchari cation was barely increased. The maximum sacchari cation ratio (42.8%) was obtained as the 4.8g/L commercial cellulase.
The result of samples hydrolysis by T. reesei cellulase was shown in Fig. 3(b). The peak sacchari cation ratio also obtained at T. reesei cellulase 4.8g/L (45.6%). It is worth noting that both the peak sacchari cation ratio of T. reesei cellulase and commercial cellulase were both obtained at 21 hours then maintained stability. It is reported that cellulase is more susceptible to end-product inhibition caused by glucose, once glucose is accumulated in the medium in a higher amount, high concentration glucose can either block the active site for the substrate or prevent the hydrolyzed substrate from leaving 41 . Feedback inhibition exhibits inhibiting effect on the cellulase hydrolysis of lignocellulosic biomass.
Previous researchers found that the low β-glucosidase activity of T. reesei reduced the e ciency of lignocellulosic hydrolysis 42 , and the catalytic e ciency of T. reesei cellulase was lower than that of commercial cellulase composed of multiple fungi cellulase cocktail 43,44 . Most of the cellulase producer lamentous fungi are characterized by low secretion of β-glucosidase which advocates the activity to be insu cient to convert cellobiose (an intermediate product in cellulose hydrolysis) to glucose 45 . The less abundance of β-glucosidase even under conditions of cellulase induction and the product inhibition to which it is susceptible, limits the use of native cellulase preparations in lignocellulosic biomass sacchari cation 46 . It is worth noting that the sacchari cation performance of T. reesei cellulase was comparable with commercial cellulase in this study, which suggest that external β-glucosidase was added in the T. reesei cellulase system. In previous studies, some species of have been reported to have the ability of producing β-glucosidase with high activity 26, 47 . Therefore, it is can be inferred that the β-glucosidase produced by A. fumigatus CLL supplements the cellulase system of T. reesei and enhanced the sacchari cation performance of T. reesei cellulase.
To verify the inference that A. fumigatus CLL completed the cellulase system of T. reesei, the cellulase activities of untreated sample sacchari ed by T. reesei cellulase (group I), pretreated sample sacchari ed by commercial cellulase (group II), and pretreated sample without external cellulase(group III), pretreated sample sacchari ed by T. reesei cellulase (groupIV), were investigated at 5 g/L feedstocks, 55 ℃, 130 rpm for 24 hours, the activities of endo-glucanohydrolase (shorted for EG), exo-glucanohydrolase (shorted for CBH) and β-glucosidase (shorted for BG) were determined every 3 hours. As shown in Table 2, EG, CBH and EB were observed in the untreated sample sacchari ed by T. reesei cellulase, while the peak activity of EG (0.229IU/mL) and CNH (0.216IU/mL) were much higher than that of EG (0.087IU/mL). It is commonly believed that T. reesei has poor ability to produce β-glucosidase, the sacchari cation of lignocellulose feedstocks was accomplished by the synergy of EG, CBH and BG, the lack of BG reduced the hydrolysis e ciency of lignocellulose, since the external β-glucosidase was indispensable for the sacchari cation by T. reesei cellulase. Compared with the untreated sample sacchari ed by T. reesei cellulase, pretreated sample sacchari ed by T. reesei cellulase demonstrated a high BG activity (0.318IU/mL) which similar with the sample sacchari ed by commercial cellulase (0.242IU/mL for EG, 0.203IU/mL for CBH and 0.287IU/mL for EG), suggest the addition of strain CLL not only enhanced the BG activity, but also completed the cellulase system. Meanwhile, what's important is that the cellulase(EG, CBH and BG) activity obtained a obviously drop after 21 hours. For the untreated sample sacchari ed by T. reesei cellulase, the activities of EG, CBH and BG were about 54.1%, 50.4%, 28.7% of the peak, respectively. For the treated sample sacchari ed by T. reesei cellulase, the activities of EG, CBH and BG were about 61.9%, 60.9%, 58.8% of the peak, respectively, similar results were obtained from the sample saccharide by commercial cellulase which consistent with the results of sacchari cation ratio The pretreatment of lignocellulose was double-edged for the lignin removal companied with the loss of holocellulose, since appropriate pretreatment time is a key factor to improve the sacchari cation ratio of the lignocellulosic feedstocks. In this study, the corn stalks degrade by strain CLL under high/low solid loading culture were sacchari ed by T. reesei cellulase/commercial cellulase to investigate the effect of pretreatment duration on the feedstocks sacchari cation performance. As shown in Fig. 4(a), under low solid loading culture, the sacchari cation ratio of untreated sample was just 20.18%, with the extension of the pretreatment duration, the sacchari cation ratio gradually increased and the peaked at 2 d, the maximum sacchari cation was ratio up to 68.4%. 2 days later, extended the pretreatment time reduced the sacchari cation ratio of corn stalks, the sacchari cation ratio of A. fumigatus CLL treatment for 10 d and 16 d were just 41.3% and 32.4% respectively. Different from low solid loading culture, the maximum sacchari cation ratio (60.9%) under high solid loading culture was obtained at 10 d ( Fig. 4(b)), extend the duration to 30 d and 50d, the corn stalks sacchari cation ratio were 49.4% (cultured for 30 d under high solid loading) and 33.1%(cultured for 50 d under high solid loading) respectively. Compared with feed stocks sacchari cation by T. reesei cellulase, the feed stocks sacchari cation by commercial cellulase demonstrated a similar result suggest that the corn stalks treated by promoted the e ciency of T. reesei cellulase, and can be a potential client in the lignocellulose biomass energy re ning.

Conclusions
The A. fumigatus CLL degrade the lignin of corn stalks and decomposed the structure of corn stalks effectively. At the same time, strain CLL supplements β-glucosidase for the cellulase system of T. reesei to improve the sacchari cation e ciency of cellulase. A. fumigatus CLL promoted the corn stalks sacchari cation under low/high solid loading culture. Compared with high solid loading culture, low solid loading culture was more conducive to corn stalks sacchari cation. A. fumigatus CLL was a potential individual on the lignocellulosic biomass energy re ning and feasible to reduce the cost of the sacchari cation downstream process. Effect of cellulase concentration on the sacchari cation of pretreated corn stalks (a) samples sacchari ed by commercial cellulase (b) samples sacchari ed by Trichoderma reesei cellulase