Suitability of Korshinsk peashrub branches as substrate ingredient for P. tuoliensis production
Korpb are shrub wastes that accumulate continuously throughout the year and contain high amounts of cellulose, hemicellulose, and lignin, as well as crude protein and crude fat [21]. As shown in Table 1S, Korpb had a total carbon content of 32.2% and a total nitrogen content of 2.24%. The C/N ratio (14.391:1) was comparable to the C/N ratio in wheat bran (11.956:1), but significantly lower than the C/N ratio in corncob (52.821:1), cotton seed hull (28.088:1), and maize powder (31.725:1), which are all conventional substrate materials for edible fungus production [22, 23]. The above result showed that Korpb are a substrate material with high nitrogen content and can be an ideal raw material for edible fungus production.
To determine if the lignocellulose byproduct, Korpb, are suitable as a substrate ingredient for P. tuoliensis cultivation, eight substrate formulas containing various proportions of Korpb were designed.
Effect of Korshinsk peashrub branches on the growth of P. tuoliensis mycelium
The mycelial growth rate in different substrate formulas varied significantly (Fig. 1). The mycelial growth rate in T7 was the highest, followed by that in T3 and T4, all of which were significantly higher than that in CK. While the mycelial growth rate in T2, T5, and T6 was comparable to that in CK. However, the mycelial growth rate in T1, which contained 65% Korpb, was the lowest.
Previous studies have shown that the mycelial growth rate depends mainly on the C/N ratio in the cultivation substrate [24]. The optimum C/N ratio of P. tuoliensis mycelium was in the range of 20:1 - 30:1. Too much nitrogen will inhibit mycelial growth and delay the formation of fruiting bodies [25]. Formula T1 had a C/N ratio of 19:1 and a very high nitrogen content, which can well explain why the growth rate of mycelium in T1 was the lowest.
Effects of Korshinsk peashrub branches on the yield and biological conversion rate of P. tuoliensis
As shown in Table 1, the yield and biological efficiency (BE) of formula T2, which contained 45% Korpb, 20% cottonseed hull, and 15% wheat bran, were the highest, at 380 ± 65 g per bag and 52.6%, respectively, significantly higher than those of CK by 5.26% and 9.02%, respectively (p<0.05). The BE and yield of T3 and T6 were similar to those of CK, while those of T7 without cotton seed hull were lowest. The yield and BE of T2 were the highest, demonstrating that the formula composition of T2 can provide more nutrients for mushroom growth and well promote the bioconversion of the substrate to mushroom products.
Table 1 Agriculture traits of P. tuoliensis cultivated on substrates with various proportions of Korpb
Formulas
|
Average yield
(g/bag)
|
Biological efficiency
(%)
|
Pileus length
(mm)
|
Pileus width
(mm)
|
CK
|
361±66bc
|
48.0±3.56bc
|
161±36a
|
119±27a
|
|
T1
|
344±69bcd
|
46.3±2.54bc
|
153±25a
|
122±22a
|
|
T2
|
380±65a
|
52.6±4.28a
|
157±24a
|
119±25a
|
|
T3
|
359±55bc
|
50.0±3.79bc
|
160±31a
|
120±23a
|
|
T4
|
357±49bc
|
48.0±4.21bc
|
159±28a
|
118±15a
|
|
T5
|
330±60cd
|
46.0±3.12cd
|
157±19a
|
123±22a
|
|
T6
|
363±69bc
|
50.1±5.42bc
|
160±24a
|
120±20a
|
|
T7
|
311±47d
|
43.5±5.11d
|
153±22a
|
116±23a
|
|
Values followed by the same letter are not significantly different (ANOVA; Tukey’s test, p < 0.05).
The biological conversion of substrate to mushroom product can also be reflected on the utilization rate of lignocellulose biomass in the substrate. The growth and development of edible fungus rely on the degradation of lignocellulose by lignocellulosic degrading enzymes secreted from mycelia [40]. In our study, due to the addition of various proportions of Korpb, the different substrate formulas exhibited varied cellulose (15-28%), hemicellulose (7-14%), and lignin (13-21%) contents. After 110 days of P. tuoliensis cultivation, the degradation degree of cellulose and hemicellulose in the cultivation substrates were in the range of 13.52-56% and 8.5-55%, respectively, while lignin contents were only slightly changed (Table 2S), which corresponds to the results of [26], who demonstrated that Pleurotus spp. were the best laccase producers, but the weakest lignin degraders. These results also demonstrated that the lignocellulose biomass in cultivation substrates has been successfully converted into P. tuoliensis food.
Effects of Korshinsk peashrub branches on agriculture traits and nutritional composition of P. tuoliensis
The ideal commodity traits of P. tuoliensis include a big fan pileus and a short stipe [27]. As shown in Table 1 and Fig. 2, the pileus length and width of T1 - T7 were not significantly different from the regular formula (CK). This data demonstrates that the addition of Korpb in the cultivation substrate did not affect the agricultural traits of P. tuoliensis fruiting bodies.
The analysis index, such as crude protein, fat, ash, sugar, and fiber, are conventional indicator to evaluate the nutritional quality of edible fungi [27]. As shown in Table 2, no significant differences in crude protein content were detected in fruiting bodies grown in formulas CK, T1, T2, T3, T4, and T6, whereas crude protein content in T5 and T7 was significantly lower compared to CK. Similarly, the crude fiber content of fruiting bodies in CK, T1, T2, T3, and T6 were high, whereas the crude fiber contents in other formulas were slightly lower, with that for T7 being the lowest. The highest fat content was detected in T2 fruiting bodies, which was twice that of CK. In the CK formula, which did not include Korpb, the ash content was higher than that in other formulas. Crude sugars, including reducing and non-reducing sugars, were higher in T1, T5, and T7 than in CK; whereas that of other formulas were slightly lower. Nutritional index analysis showed that the addition of Korpb to substrate did not influence the nutritional value of P. tuoliensis fruiting body.
Table 2 Nutritional composition of P. tuoliensis cultivated on substrates with various proportions of Korpb
Formula
|
Crude protein
g/100g
|
Crude fiber
g/100g
|
Fat
g/100g
|
Ash
g/100g
|
Crude sugar
g/100g
|
CK
|
22.25±0.25a
|
6.28±0.02d
|
2.91±0.01c
|
6.35±0.01a
|
8.38±0.06e
|
T1
|
22.05±0.25a
|
6.67±0.05b
|
2.92±0.01c
|
6.00±0.01d
|
9.98±0.03c
|
T2
|
22.50±0.20a
|
6.14±0.07e
|
5.21±0.01a
|
5.94±0.01e
|
7.24±0.00g
|
T3
|
21.80±0.10b
|
6.48±0.01c
|
2.76±0.01d
|
6.02±0.01c
|
7.44±0.09g
|
T4
|
21.50±0.80c
|
5.29±0.03g
|
2.71±0.01f
|
5.78±0.01f
|
7.85±0.02f
|
T5
|
19.85±0.25d
|
5.70±0.04f
|
2.68±0.01g
|
5.70±0.01h
|
11.71±0.03a
|
T6
|
22.75±0.15a
|
6.90±0.10a
|
3.62±0.01b
|
6.33±0.02b
|
8.76±0.07d
|
T7
|
18.95±0.15e
|
4.48±0.01h
|
2.51±0.01h
|
4.92±0.01i
|
10.95±0.04b
|
Values followed by the same letter are not significantly different (ANOVA; Tukey’s test, p< 0.05).
Based on the comparison of growth and development indices of P. tuoliensis, such as mycelium growth rate, yield, biological convention rate, agriculture traits, and nutritional analysis, the formula T2 including 45% Korpb, 20% cotton seed hull, 15% corncobs, 10% wheat bran, 5% maize powder, 4% lime and 1% gypsum was considered as an optimized formula for the cultivation of P. tuoliensis. The main component of this formula is Korpb waste, therefore, the promotion and using of Korpb for P. tuoliensis production could consume a large number of Korpb waste to solve the problem which is availability low of Korpb. In addition, this formula can replace 40% cotton seed hull in a conventional substrate, vastly alleviating the shortage of traditional raw material and saving raw material costs. Using Korpb as the main subatrate ingredient for edible fungi cultivation would have excellent economic and ecological value.
Biogas production by anaerobic digestion of spent mushroom substrates containing various proportions of Korshinsk peashrub branches
To further promote the conversion of Korpb to circular economy and solve the SMS acumulation, the gas potential of SMSs containing Korpb via AD were evaluated. The parameters of eight kinds of SMS and inoculum for AD are shown in Table 3. After drying at 105 oC for 4 h, the TS contents of SMS ranged from 56.13 - 64.53%. The VS contents of these feedstocks were in the range of 27.51 - 33.88%. Their total carbon and total nitrogen contents ranged from 29.10 - 36.28%, and 1.57-1.88%, respectively, which resulted in C/N ratios of 15.88-22.75, indicating rich nitrogen content in SMS. In addition, cellulose, hemicellulose, and lignin contents varied among these SMSs, which may cause significant differences in their degradability.
Table 3 Characterization of SMS and inoculum
Material
|
CK
|
T1
|
T2
|
T3
|
T4
|
T5
|
T6
|
T7
|
Inoculum
|
Total solids (%)
|
63.63
|
57.37
|
56.13
|
59.03
|
59.23
|
63.3
|
63.1
|
64.53
|
5.46
|
Volatile solids (%)
|
27.91
|
31.17
|
33.33
|
31.58
|
33.88
|
28.72
|
29.68
|
27.51
|
3.1
|
Total carbon (%)
|
35.52±0.04
|
29.80±0.17
|
30.21±0.58
|
32.22±0.23
|
36.28±0.24
|
33.29±0.25
|
29.34±0.77
|
29.10±0.19
|
35.87±0.10
|
Total nitrogen (%)
|
1.57±0.13
|
1.88±0.02
|
1.88±0.01
|
1.71±0.07
|
1.7±0.03
|
1.83±0.02
|
1.81±0.05
|
1.62±0.02
|
2.92±0.02
|
C/N ratio
|
22.75±0.82
|
15.88±0.17
|
16.08±0.27
|
18.82±0.58
|
21.34±0.48
|
18.16±0.25
|
16.19±0.05
|
18.00±0.20
|
10.77±0.05
|
Cellulose (%)
|
18.40±0.17
|
12.25±0.23
|
10.90±0.07
|
14.45±0.15
|
19.57±0.23
|
13.87±0.53
|
9.59±0.19
|
9.25±0.34
|
|
Hemicellulose(%)
|
12.26±0.05
|
5.70±0.07
|
6.13±0.27
|
6.88±0.16
|
9.79±0.09
|
7.38±0.66
|
5.56±0.19
|
6.23±0.02
|
|
Lignin (%)
|
22.27±0.13
|
15.26±0.16
|
14.73±0.08
|
18.95±0.04
|
21.28±0.14
|
17.21±0.06
|
13.82±0.24
|
14.80±0.47
|
|
Mono-digestion of SMS with 1% and 3% TS content
Eight kinds of SMS were used as a mono-substrate for AD in batch tests with 1% and 3% TS to evaluate the performance of SMS mono-digestion. The pH of all of the reactions was in the range of 6.5–8.0, which is within the suitable range (6.5–8.2) for digestion (Fig. 3a and 3b).
Usually, an increase in the volume of biogas corresponds to an increase in the volume of methane [20]. The highest cumulative methane yield from eight kinds of SMS were all obtained with 3% TS. The methane yield of CK SMS was the highest (687.82 mL/g TS), but not significantly higher than that of T4 SMS (628.65 mL/g TS). The methane yields of T5, T6, and T7 SMSs significantly decreased by 1.5- to 2-fold compared to that of CK and T4 SMSs. These SMSs exhibited the same methane-producing performance at 1% TS (Table 4).
Table 4 Biogas yield and methane yield for the digestate obtained after mono-digestion
SMS
|
TS
(%)
|
Biogas yield
(mL/g TS)
|
Methane yield
(mL/g TS)
|
CK
|
1
|
847.88
|
366.93
|
|
3
|
1511.71
|
687.82
|
T1
|
1
|
666.96
|
212.75
|
|
3
|
825.84
|
381.32
|
T2
|
1
|
696.85
|
230.37
|
|
3
|
1017.92
|
416.01
|
T3
|
1
|
690.40
|
207.81
|
|
3
|
1215.92
|
510.36
|
T4
|
1
|
774.77
|
297.84
|
|
3
|
1304.38
|
628.65
|
T5
|
1
|
672.42
|
215.65
|
|
3
|
834.73
|
353.90
|
T6
|
1
|
585.65
|
187.51
|
|
3
|
668.70
|
291.95
|
T7
|
1
|
624.43
|
186.77
|
|
3
|
758.99
|
371.88
|
The methane yield of CK and T4 SMSs were the highest, likely due to appropriate C/N ratio of SMSs, 22.75 ± 0.82 and 21.34 ± 0.48, respectively. The C/N ratio of feedstock for biogas production should be in the range of 20 to 30 [28], which is not only beneficial for long-lasting and stable production of biogas, but also for the regulation of acidity and alkalinity of reactants, which is conducive to biogas residue.
The methane yields produced by SMSs from P. tuoliensis cultivation in our study were 10-80% higher than that reported by [20], who showed that the methane production of P. tuoliensis SMS via AD was only 159.25 ml/g TS at 3% TS. The difference in the methanogenic performance of P. tuoliensis SMS in different studies is mainly due to differences in substrate ingredients and their proportions. At present, thirteen species of edible fungi SMSs have been used as materials for the generation of biogas and methane, and SMS from Pleurotus spp. have been demonstrated to be excellent lignincellulose biomass for biogas production [19, 29, 30].
Co-digestion of SMS and dairy manure at 3% TS
Co-digestion can increase biogas production, balance the whole system, and diluted inhibitors [31, 32]. Dairy manure (DM) is an excellent co-digestion material, due to its superior buffering capacity and rich microbial and nutrient content [33, 34]. To compare the mono-digestion and co-digestion performance of SMS from P. tuoliensis, further batch tests were performed using co-substrates (SMS and DM with the ratio of 1:1) at 3% TS.
Similar to mono-digestion of SMS, the pH of all co-digestion reactions were in the range of 6.5-8.0, demonstrating the suitable systemic buffering capacity of these reactions (Fig. 3c). As shown in Fig. 4, the cumulative methane production of mono-digestion using DM only was 70.72 mL/g TS, which was the lowest among these tests. The high nitrogen content of DM likely caused ammonia poisoning during the anaerobic digestion [35]. The methane yields for co-digestions using T1, T5, T6, or T7 SMS and DM were 457.18 mL/g TS, 424.47 mL/g TS, 364.30 mL/g TS, and 379.75 mL/g TS, respectively. These methane yields were 19.89%, 19.94%, 24.78%, and 2.11% higher than digestion using SMS alone. However, the co-digestion methane yields for CK, T2, T3, or T4 SMSs and DM were not higher than the mono-digestion with these SMSs.
There are many confounding factors influencing the co-digestion effects, including the C/N ratio, dilution of toxic compounds, buffering capacity of the anaerobic system, and changes in the structure of the microbial community [36]. The positive synergistic effects of SMS and DM were probably due to the substrates providing adequate nutrition for the microorganism present in each component[37]. The addition of DM probably improved the buffering capacity of the reaction and reduced the influence of VFA accumulation[38]. While the appearance of negative synergistic effects was likely due to an improper C/N ratio in the reaction system after the addition of DM, which is an important parameter determining the synergistic effect in AD [39].
Based on the gas-productivity results of mono- and co-digestion of SMS, we found that the methane yield of SMS was gradually decreased with the increase of Korpb content in substrate, showing that the presence of Korpb in SMS is not conducive to the production of biogas. Although the methane yield of SMS containing Korpb were lower than the control SMS with 65% cotton seed hull in our study, but higher than that in other reported research, in which only 286 mg/mL methane produced from P. tuoliensis SMS [20].
Benefit analysis
Our study demonstrated that Korpb can be efficiently converted to high-valued edible products. Meanwhile, anaerobic digestion experiments indicate that SMS, containing Korpb, can produce higher yield of methane than that reported in the previous study. Therefore, the utilization of Korpb for mushroom and biogas production via an integrated P. tuoliensis cultivation and AD process would have remarkable economic, ecological, and social benefits.
Economic benefit analysis
The cultivation formula with the top yield (T2) demonstrates that the addition of 1 kg Korpb in the substrate can produce 1.12 kg P. tuoliensis, which brings a direct economic benefit of RMB 33.6 Yuan and generates 5.6 kg SMS. If these SMS is anaerobic digested, 8578 L methane will be generated for a direct economic benefit of RMB 22.25 Yuan. In general, removing the raw material and labor cost, each kg of Korpb consumed can generate economic benefit of RMB 27.5 Yuan.
The yearly quantity of Korpb in China is estimated to be around 550,000 t, and the utilization rate is just 10%-20%. If 400 large-scale mushroom farms were to adopt the optimal formula for the production of P. tuoliensis, 5% of annual Korpb will be utilized, 48125 t edible mushroom products will be produced, resulting in an enormous economic benefit of RMB 1.44×109 Yuan. Meanwhile, 240625 t SMS will be generated that can be converted into 3.4 × 108 L methane with an economic benefit of RMB 8.8×105 Yuan. The total economic value after removing production costs is estimated as 9.5×108 (Table 5).
Table 5 The economic value of Korshinsk peashrub branches for P. tuoliensis cultivation and anaerobic digestion of SMS
P. tuoliensis cultivation and anaerobic digestion
|
Small-scale cultivation
|
Korpb (kg)
|
1.0
|
Fruiting body (kg)
|
1.12
|
Direct economic value (yuan)
|
33.6
|
Pt SMS (kg)
|
5.6
|
Methane (L)
|
8578
|
Direct economic value (yuan)
|
22.25
|
Actual economic value (yuan)
|
27.5
|
Large-scale cultivation
|
Korshinsk peashrub branches (t)
|
27500
|
Fruiting body (t)
|
48125
|
Direct economic value (yuan)
|
1.44×109
|
Pt SMS (t)
|
240625
|
Methane (L)
|
3.4×108
|
Direct economic value (yuan)
|
8.8×105
|
Actual economic value (yuan)
|
9.5×108
|
Korpb: Korshinsk peashrub branches
Ecological benefit analysis
At present, the utilization rate of chopped Korpb is extremely low. A great number of Korpb are burned or incorporating into the soil and sand. The conversion of Korpb into high quality edible health food can not only avoid the waste of Korshinsk peashrub branche resources, but can also solve the environmental pollution problem. In addition, spent mushroom substrate, the remaining lignocellulose biomass, can be used as a biological energy source for biogas production, to meet the energy needs of people in their daily lives. Therefore, the utilization of Korpb for mushroom and biogas production, via an integrated P. tuoliensis cultivation and AD process, results in minimum energy waste and substance discharge. This integrated process meets the objectives of agriculture in a sustainable economic society by saving energy and reducing emissions.
Social benefit analysis
Korpb are cheap and easy to obtain, and the high efficient conversion capability of Korpb into the P. tuoliensis product can greatly arouse the enthusiasm of the edible fungi-producing enterprises and peasants. Meanwhile, the resulting edible fungi residue can be used as a biological energy source for biogas production, which can solve the environmental pollution problem caused by discarding the SMS. An integrated P. tuoliensis cultivation and biogas production process, with the utilization of Korpb, can form a firm circular chain by promoting mutual development. This new avenue can efficiently solve employment problems for surplus rural labors and significantly increase peasants' incomes to build a new socialist countryside and realize agricultural modernization.