The Effects of Myrothecium verrucaria (MV) on Corn Stover During Co-Composting Process Under Room Temperature Conditions

In China, the production of crop straw has been estimated to be approximately 800 Million tons yearly of which about 40% was burned. Corn stover is one of the main agricultural wastes in China. It has shown that lignin in corn stover could be effectively removed byMyrothecium verrucaria. The effects of the pretreatment of corn strover by Myrothecium verrucaria on compost were studied. The results showed that corn stover pretreatment by Myrothecium verrucaria, the Cellulose, Hemicellulose, and lignin were degraded and the results were 33.43%, 11.53% and 18.70% respectively. Scanning electron microscope (SEM) analysis showed that the surface structure of corn stover was changed. Fourier transform infrared spectroscopy (FTIR) analysis showed that the degradation products of lignin were increased. The exposed area of cellulose and hemicellulose was increased. Compared with the control group, the pH value was stable and the temperature was higher. The content of nitrogen in the material decreased, while the contents of total phosphorus and total potassium increased.The C/N ratio of materials decreased after composting.The results showed that the pretreatment of Myrothecium verrucaria improve the degradation of lignocelluloses, a great contribution was made to reduce the causes loss of plant nutrient and to fight against environmental pollution.

In this study, corn stover was pretreated by Myrothecium verrucaria before being cocomposed with cow manure. We propose the hypothesis that the pretreatment of corn stover by Myrothecium verrucaria can directly enhance corn stover lignocellulose degradation before co-composting, and then help to improve the subsequent co-composting process, as this avoids both the competition and suppressive effects during the co-composting. The novel aspect of this treatment is that the direct effects of Myrothecium verrucaria on corn stover lignocellulose degradation were conducted before co-composting. The compost stability and maturity index were measured and compared with the treatments without MI and also without Myrothecium verrucaria addition or the treatments with MI inoculation during the cocomposting process. The main objective of this research was to evaluate the feasibility of corn stover pretreated by Myrothecium verrucaria before the co-composting of cow manure with corn stover, to optimize the composting procedures, and to improve the utilization of agricultural byproducts. It specifically aims to (1) this study constitutes a pioneer base of knowledge and the Effect of Myrothecium verrucaria on corn stover co-composting process of straw comprehensive utilization, finally to allow a better understanding by scientists and farmers the process of co-composting with cow manure; (2) Pretreatment of corn Stover and enhancement of the lignocelluloses subsequently before the composting process.

2.1.Experimental site
The experiment was conducted at the key Laboratory of Straw Biology and Utilization, the Ministry of Education-Jilin Agricultural University.

2.2.Experimental apparatus
The type of in-vesel made compost was chosen, because it has an advantage, can process large amounts of waste without taking up as much space as the windrow method and it can accommodate virtually any type of organic waste (e.g, meat, animal manure, biosolids, food scraps). This allows good control of the environmental conditions such as temperature, moisture, and airflow. The material is mechanically turned or mixed to make sure the material is aerated. The size of the vessel can vary in size and capacity.
This method produces compost in just a few weeks. It takes a few more weeks or months until it is ready to use because the microbial activity needs to balance and the pile needs to cool.
We used rectangular vessels. We therefore installed 3 vessels (boxes) in one line of each, and each vessel had the length of 72cm, 52cm of width and a height of 43cm. All vessels were numbered and named from 1-3 in the following manner ( control group or CK, microbial innoculant or MI and Myrothecium verrucaria or MV).
It signed to report the first vessel (CK) is considered as control will not be malled and microorganism will not be added or inoculated.
Corn straw was collected from the experimental field of Jilin Agricultural University, and dried under natural conditions after harvest. Corn stover was smashed to pass through 0.45mm screen. The composition of the corn stoverand Cow manure are shown in Table 1.

Biological material
The biological materials that we used during our research were in particular constituted of different samples of corn stover pretreated with microorganisms (commercial microbial inoculants:CMI and Myrothecium verrucaria ) on the one hand and on the other hand the samples of co-composting of the corn stover, the cow manure and the microorganisms (commercial microbial inoculants:CMI and Myrothecium verrucaria) were collected at room temperature, manually in the composting room at our key laboratory ofstraw biology and utilization, the ministry of education.
Myrothecium verrucaria Strain 3H6 (Preservation No: CCTCC M 2018800) was stored in a -80 ৹ C freezer at the (Key Laboratory of Straw Biology and Utilization, the Ministry of Education). This strain was recovered by growing aerobically on Potato medium agar (PDA: potato 200 g/ L, glucose 20 g/ L, and agar 18 g/L), at 30 ৹ C, pH 7.0 for 48 h.

2.3.Methods a) Pretreatment of Corn Stover before co-composting with cow manure
Boxes of polystyrene (100L)) were used as composting reactors with the length of 72cm, 52cm of width, and the height of 43cm. untreated corn stover (CK: control group), corn stover pretreated with commercial microbial inoculants (MI: group), and corn stover pretreated with Myrothecium verrucaria (MV: treatment group). Each treatment had 3 replicates A total of 3kg of corn stover was mixed with 1% of Myrothecium verrucaria as the amount of inoculant. The corn stover was adjusted to 65% moisture and kept for 20 days under room temperature conditions. After pretreatment, the corn stover was used as the substrate for the subsequent co-composting with cow manure. The corn stover after co-pretreatment was then mixed with the cow manure according to the gotten results of C/N ratio (30:1) to perform the co-composting, the core temperature and pH of the co-composting pile was measured daily using Digital thermometer, which was plunged into the compost at a depth of 15 cm. For the pH, asample of 20g was mixed with 100ml of H2O, then shaken for few minutes then left for 1h-3h, the measure was done using a pH meter 818 (SMART SENSOR), respectively.
Pile-turning was conducted every 2-3days during the high temperature stage and every 4-6 days during the moderate temperature stage.
The moisture content was then maintained in the range of 60%-65% by adding distilled water during the composting pile.

c) Measurement of physio-chemical parameters
❖ Effects of Co-pretreatment on the structure of corn stover before/after ❖ Scanning electron microscope (SEM) analysis The untreated corn stover and the pretreated corn stover were dehydrated using a freeze dryer. The different surface morphologies of untreated and pretreated corn stover were characterized by scanning electron microscopy (S-3400 N, Hitachi, Japan).

❖ Fourier transform infrared spectroscopy (FTIR) analysis
The chemical changes of corn stover before and after different pretreatment methods were investigated by Fourier transform infrared spectroscopy (FTIR) spectrometer (Nicolet 6700, Thermo Fisher Scientific Inc.). Samples were prepared by KBr pelleting. The resolution of the spectra was 2 cm −1 in the range of 500-4000 cm −1 , and 32 scans were carried out for each sample.

❖ Compositionanalysis of corn stover before/after
The content of cellulose, hemicellulose, and lignin was analyzed according to the method of the National Renewable Energy Laboratory, Golden, CO, USA [9 ] to evaluate the effect of pretreatment for corn stover. Untreated corn stover was selected as the control. Each analysis was performed in triplicate.
❖ Determination of total nitrogen: was determinedusing the Kjeldahl method [ 10] ❖ Determination of total carbon: was determined by using the potassium dichromate external bath heating method [11] ❖ Determination of potassium: was determined by usingammonium acetate flame photometer method [12] ❖ Determination of phosphorus: was determined by using ammonium vanadate colorimetry method [13].
❖ The temperature and pH: were determined by using measured daily using Digital thermometer which was plunged into the compost at a depth of 15 cm and pH meter, a sample of 20g was mixed with 100ml of H2O, then shaken for few minutes then left for 1h-3h, the measure was done using a pH meter 818 (SMART SENSOR), respectively. analysis, we notice that the surface and the physicochemical structure or even its composition has not changed at all, a has not undergone a modification from the physicochemical point of view and especially structural by keeping its shape intact with a strong, rigid and solid layer.  Several of these studies show that Myrothecium verrucaria (MV) or WRF and microbial innoculant (MI) degrade by secreting the enzymes which facilitate the reduction of the surface and the physico-chemical structure of the biomas (lignocelluloses) and to allow these latter (biomas) to partially lose their rigidity. It should therefore be noted that the degradation of the complete biomass remains a challenge nowadays because it takes more time a little longer, means cost but above all make use or intervene several microorganisms finally to facilitate a good degradation of the biomass (lignocelluloses).

Effect of co-pretreatment on Tempreature and pH
Examining the result obtained on the temperature taken daily in the morning at the  Myrothecium verrucaria (MV), in even reaching up to 40.8 °C and this phenomenon will be observed once more throughout the process for more than two weeks. And as when taking the temperature in the morning during the last 10 days, we will observe almost the same temperature on all three CK, MI and MV treatments with an average of 29 °C.
By comparing the two results on the temperature sampling, in the morning and in the evening on all 3 treatments CK, MI and MV, we noted that the temperature was high in the evening than in the morning in general but more particularly, high on MI that at CK and MV is with an average of 33.3 °C MI in the morning against an average of 35.3 in the evening.

Discussion
After the Scanning electron microscope (SEM) analysis of these three treatments it was although clear that the surface and the physicochemical structure underwent a modification, a change, a degradation because we can see the presence of lesions, holes, with a relative reduction of the biomas on the two treatments microbial innoculant (MI) and Myrothecium verrucaria (MV), while CK did not undergo any modification, degradation while keeping its surface and its physicochemical structure almost intact. But it should be noted that the degradation, the reduction of the rigidity of the lessions, of the very clear holes was observed more on Myrothecium verrucaria (MV) than on microbial innoculant (MI) where the degradation of the surface and of the physico-chemical structure was not really as on Myrothecium verrucaria (MV). Similar results were obtained or observed in [14 ] , [15 ] , [