Accepted Manuscript

Effect of fertilizer prepared from human feces and straw on germination, growth and
development of wheat

Dianlei Liu, Beizhen Xie, Chen Dong, Guanghui Liu, Dawei Hu, Youcai Qin, Hongyan
Li, Hong Liu

PII: S0094-5765(17)31100-1
DOI: 10.1016/j.actaastro.2018.01.014
Reference: AA 6643

To appear in: Acta Astronautica

Received Date: 4 August 2017

Revised Date: 9 November 2017
Accepted Date: 8 January 2018

Please cite this article as: D. Liu, B. Xie, C. Dong, G. Liu, D. Hu, Y. Qin, H. Li, H. Liu, Effect of fertilizer
prepared from human feces and straw on germination, growth and development of wheat, Acta
Astronautica (2018), doi: 10.1016/j.actaastro.2018.01.014.

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 ACCEPTED MANUSCRIPT

1 Effect of fertilizer prepared from human feces and straw on

2 germination, growth and development of wheat

4 Dianlei Liu a,b,c,1, Beizhen Xie a,b,c,1, Chen Dong a,b,c,1, Guanghui Liu a,b,c, Dawei Hu a,b,c,

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5 Youcai Qin a,b,c , Hongyan Li a,b,c, Hong Liu a,b,c,∗
6

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a
7 School of Biological Science and Medical Engineering, Beihang University, Beijing
8 100191, China

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b
9 Institute of Environmental Biology and Life Support Technology, Beihang
10 University, Beijing 100191, China

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c
11 International Joint Research Center of Aerospace Biotechnology&Medical
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12 Engineering, Beihang University, Beijing 100191, China

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1
14 These authors contributed equally to this work.
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15
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16 E-mail address: liudianlei010187@126.com, xiebeizhen@buaa.edu.cn,

17 wenjian_dongchen@163.com, liugh1991@126.com, stuarthu@163.com,
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18 qinyoucai07@163.com, sy1410111@buaa.edu.cn

19
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20 * Corresponding Author.
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21 Tel: 86-10-82339837 Fax: 86-10-82339837

22 E-mail: Hong Liu: LH64@buaa.edu.cn

23

24

25

26

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27 Abstract: Solid waste treatment is one of the most important rate-limiting steps in the

28 material circulation and energy flow of Bioregenerative Life Support System (BLSS).

29 In our previous work, an efficient and controllable solid waste bio-convertor has been

30 built and a solid waste degradation efficiency of 41.0% has been reached during a

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31 105-d BLSS experiment. However, the fermented residues should be further utilized

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32 to fulfill the closure of the system. One solution might be to use the residues as the

33 fertilizer for plant cultivation. Thus in this study, substrates were prepared using

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34 different ratios of the fermented residues to the vermiculite. And the influences of

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35 different ratios of the fermented residues on the seed germination, growth,
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36 photosynthetic characteristics and antioxidant capacity of wheat were studied. The

37 results showed that the optimal rate of the fermented residue was 5%. With this ratio,
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38 the seed germination reached 97.3% with the root length, shoot length and biomass
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39 production as 59 mm, 52 mm and 150 mg, respectively, at the 4th day. Besides, the
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40 highest straw height of 25.1 cm was obtained at the 21st day. The salinity adversely

41 affected the growth and some relevant metabolic processes of wheat. The Group-40%
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42 led to the lowest seed germination of 34.7% and the minimum straw height of 15 cm.
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43 This inhibition might be caused by the high Na content of 2118 mg/kg in the
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44 fermented residues. Chlorophyll b was more sensitive to the mineral nutrition stress

45 and affects the wheat photosynthetic characteristics. Higher reactive oxygen species

46 levels and reduced antioxidant enzymes may contribute, directly and/or indirectly, to

47 the decline in the observed pigment contents in wheat.

48 Keywords: fermented residues, different ratios, wheat growth, salinity

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49 1. Introduction

50 Current strategies to further explore the space, such as NASA’s Mars Program [1]

51 or China's lunar exploration program [2], strongly suggested that the development of

52 bioregenerative life support systems (BLSS) can be fully incorporated into space

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53 stations, transit vehicles, and eventually habitats on the Moon and Mars [3,4]. The

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54 ultimate goal of this technology is to create a sustainable life support Earth-like

55 ecological environment that is open with respect to energy but closed with respect to

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56 mass. Technological innovation of BLSS unit components for biomass production and

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57 waste recycling is of particular interest for a number of researchers from various
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58 countries [5-7]. Within a BLSS, the cultivation of higher plants takes a crucial role, as

59 they can contribute to most of the major functional aspects such as the food
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60 production, carbon dioxide reduction, oxygen production, water recycling, and waste
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61 management, etc [8]. However, plant cultivation in closed environments is

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62 challenging and several key technologies necessary for space-based plant production

63 are not yet space-qualified or remain in early stages of development.

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64 To improve the closure coefficient of BLSS for reducing the stowage and the
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65 resupply of life support materials, the solid wastes including inedible crop biomass
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66 and human feces should be treated and reused as a soil-like substrate (SLS) for higher

67 plant cultivation in space [9,10]. However, the chemical-physical properties including

68 water content, organic matter content, iron oxides, calcium carbonates, mineralogy,

69 and surface roughness, and particle-size distribution of SLS might affect the plants.

70 Especially, the salinity, which is one of the major abiotic stresses, causes alterations in

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71 a wide range of physiological, biochemical and molecular processes in plants. It may

72 cause significant reductions in the rate and percentage of germination, which in turn

73 may lead to uneven stand establishment and reduce crop yields [11]. It may also affect

74 plant growth and development at different levels of plant organization, e.g., the

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75 photosynthetic carbon gain and leaf growth rate may be reduced [12]. Moreover,

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76 under salt stress, plants have to cope with stress imposed by the low external water

77 potential and with ion toxicity due to accumulation of ions inside the plants [13]. Plant

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78 growth is ultimately reduced by salinity stress but plant species differ in their

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79 sensitivity or tolerance to salts [14, 15]. In particular, wheat (Triticum aestivum L.),
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80 which is a core crop in BLSS [16], is often restricted in growth by inappropriate

81 application of nutrient fertilizer.

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82 Thus, due to the complex growth problems associated with the salt stress of the
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83 SLS, there is a tendency toward to use the residues from solid fermentation as
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84 bio-fertilizer for wheat cultivation in BLSS. In this study, mixed fermentation

85 substrates were prepared using different ratios of the fermented residues and the
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86 vermiculite, and the influences of different concentrations of the fermented residues

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87 on the seed germination, the growth, photosynthetic characteristics and antioxidant

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88 capacity of wheat plants at vegetable stages were studied. The morphology, physical

89 characteristics and composition of the fermented residue were also analyzed. The

90 specific objectives of this study were to (1) investigate the feasibility of utilizing the

91 fermented residue as substrate for wheat plant and (2) determine its optimal ratio for

92 the growth, photosynthetic characteristics and antioxidant capacity of wheat plants at

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93 the seed germination and vegetable stages in controlled conditions. The outcomes of

94 this study would be important for waste recycle and higher plant cultivation in the

95 future BLSS experiments.

96 2. Materials and methods

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97 2.1 The preparation and characteristics analyses of the fermented residues

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98 The fermented residues were obtained from a 50-day aerobic fermentation of

99 wheat straw and human feces using a solid waste bio-convertor as described in our

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100 previous work [17]. At the beginning of the experiment, 2850 g wheat straw mixed

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101 with 150 g dry microbial inoculants were fermented for 10 days in the bio-convertor
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102 to rejuvenate the microbial inoculants which were able to degrade plant waste. Then,

103 400 g dry wheat straw and the daily excretion (only feces) from 4 people were added
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104 to the bio-convertor every day. The experiment was conducted continuously for 50
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105 days.
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106 The fermentation was conducted under the temperature of 45 ºC with moisture

107 content of 60%, and carbon to nitrogen ratio (C/N) of 30:1. The bio-convertor was
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108 performed at rotate frequency of 20 Hz with aeration rate of 20 L·min-1. After the
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109 50-day fermentation process, the remained substance was collected and its
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110 morphology and physical characteristics including moisture content, pH, conductivity,

111 organic nitrogen and C/N were measured [18]. The elementary composition of the

112 fermented residues was also analyzed (Vario EL, Elementar, Germany).

113 2.2 Substrate preparation and experimental design

114 In this study, fermentation substance and vermiculite were mixed as Table 1

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115 described. The vermiculite was purchased from a commercial manufacturer. The

116 vermiculite did not contain any nutrition and could not supply any energy for wheat.

117 The vermiculite played a role in fixing wheat root. Experiments were divided into 7

118 groups: Group-1%, Group-2.5%, Group-5%, Group-10%, Group-20%, Group-40%

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119 and CK based on the proportion of fertilizer (dry weight) in the substrate. To test

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120 effect of the different fertilizer concentrations to the seed germination and wheat

121 growth, we used DI water to cultivate the treatment plants all the time. For the CK

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122 group, the modified Hoagland nutrient solution [16, 19] was the basic culture

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123 medium.
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124 2.3 Germination and seedling planting experiment

125 Each germination test was carried out with at least 50 seeds in a set and each set
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126 is repeated at least three times. The seeds were imbibed for 10 h in distilled water and
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127 placed in 200 g dry mixture substrates (Table 1) and cultured at 23 ºC in the plant
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128 growth chamber. After germination, LED lamps were turned on under a 16/8

129 h light/dark cycle for all the treatments. Photosynthetic photon flux density (PPFD)
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130 levels were measured daily at the top of plant canopy with a quantum sensor (Li-250A,
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131 Li-Cor, USA). PPFD was about 500 µmol·m–2·s–1 for all the treatments. Air
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132 temperature and relative humidity were maintained at 21±2.3 °C and 65±5.5%,

133 respectively. There was ventilation in the growth chamber, so the CO2 level was the

134 same as that in the atmosphere outside which was about 400 ppm. The culture vessel

135 was a plant pot with a size of 25 cm×15 cm ×10 cm (Length × Width × Height). Each

136 culture vessel contained 200 g (dry weight) substrate and 50 seedlings.

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137 2.4 Wheat morphological analyses

138 For germination process, the shoot and root length of wheat samples were

139 measured every day by straight scale and vernier caliper. The samples were selected

140 on random within measurement process. Germination percentages as well as root and

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141 shoot growth in terms of lengths were measured. Protuberance of radicals was taken

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142 as an initiation of germination. Shooting and rooting were defined as protrusion of the

143 shoot and root to the extent of at least 5 mm. Root/shoot length data are the mean and

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144 average of pooled 50 seeds for each set. Seed germination was checked daily and

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145 seeds were considered germinated when the radicle was 5 mm long. Controls were set
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146 up by moistening the filter paper with 3.0 mL deionized water. Seed germination and

147 the length of the longest root and shoot produced by the seeds were measured after 72
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148 hours in all the extracts and they were compared with those of the control. The
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149 germination index (GI) was obtained by multiplying germination percentage (G %)

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150 and relative root growth (RRG), both expressed as percentage (%) of control values

151 [20]. The vigor index (VI) was also obtained.

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152 GI = %
× 100, (1)
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153 Where,
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154 G% = × 100,
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155 RRG% = × 100,

156 VI = G% ×

157 After seed germination, ten samples of those wheat plants were selected

158 randomly when the measurement was in process. The height of wheat was measured

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159 every day by straight scale.

160 2.5 Wheat physiological analyses

161 2.5.1 Determination of chlorophyll concentrations of leaves

162 Chlorophyll was extracted from the leaves of 10 plants at a similar position

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163 within each treatment. Leaves were weighed out in 0.1 g (fresh weight), and samples

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164 were ground in a mortar. The optical density was measured with a UV-1200

165 spectrophotometer (SP-75, Shanghai spectrum instruments co., LTD, China) at 663

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166 nm (OD663) for chlorophyll a (Chl. a), and 645 nm (OD645) for chlorophyll b (Chl. b)

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167 [21].
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168 2.5.2 Determination of peroxidase (POD) activity

169 Activities of peroxidase (POD) were measured using the method of Chance and
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170 Maehly [22] with some modification. The POD reaction solution (3 mL) contained 50
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171 mM phosphate buffer (pH 5.0), 20 mM guaiacol, 40 mM H2O2 and 0.1 mL enzyme
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172 extract. Changes in absorbance of the reaction solution at 470 nm were determined

173 every 20 s. One unit POD activity was defined as an absorbance change of 0.01 unit
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174 min-1. The enzyme activity was expressed on protein basis. Protein concentration of
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175 the crude extract was measured by the method of Bradford [23].
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176 2.5.3 Determination of malondialdehyde (MDA) content

177 The determination of malondiadehycde (MDA) depended on the method of

178 Stewart and Bewley [24]. Briefly, 10 mL 0.1% TCA pestled homogenate was used to

179 centrifuge wheat leaves (0.5 g) at 4000 rpm for 10 min. Then 2 mL supernatant was

180 added to 4 mL 5% TBA which was made up by 20% TCA. The mixture was heated at

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181 95 ºC for 30 min and then cooled in ice-bath rapidly. The supernatant was obtained by

182 centrifuging at 3000 rpm for 10 min. When λ=532 nm and λ=600 nm, the specificity

183 optical density was determined. The content of MDA was measured by absorptivity of

184 155 mM-1 cm-1.

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185 2.5.4 Determination of proline content

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186 Three youngest fully expanded leaves from plants of each of the treatments were

187 harvested at the same time. Leaves were immediately frozen in liquid nitrogen,

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188 freeze-dried and stored for the determination of proline content. Proline was extracted

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189 and the content assayed spectrophotometrically according to the method [25].
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190 2.6 Data analyses

191 The results were expressed as the mean and standard deviation of triplicate
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192 studies. Statistical differences were tested with one-way ANOVA with Turkey's
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193 multiple comparison tests. Any p-values less than 0.05 were considered to be
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194 significant.

195 3 Results and discussion

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196 3.1 Characteristics of fermentation substance

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197 The wheat stalks and human feces were co-composted in the digester for 50 days.
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198 After composting, about 69.8% of the substrates were degraded with a pH value of

199 7.96, conductivity of 4.54 mS/cm and carbon to nitrogen ratio (C/N) of 21.3:1. These

200 indexes indicated that the mixed materials of wheat stalks and human feces after

201 composting could basically fulfil the requirement for mature compost and might be

202 supportive for plants growth. In addition, after the composting process, the organic

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203 nitrogen increased from 0.45 g/100 g in wheat straw to 1.88 g/100 g in fertilizer with

204 a moisture content of 53.2%. And the microbe amount reached as high as 1.91×108

205 cfu/g.

206 After long incubation time of fermentation, the microorganisms could fully

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207 degrade the cellulose and other complex organic substances, which would accelerate

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208 the release of nutrients and elements to the fertilizer. From Fig. 1, K content was at

209 the highest level of 39015 mg/kg in the fermentation substance. In particularly, Na

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210 content was 2118 mg/kg and it may be not beneficial to the wheat growth and

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211 development [26].
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212 Organic compounds in soil-like substrate are derived from root exudates, root

213 residues, and microbial metabolism. During germination, activated hydrolytic

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214 enzymes also hydrolyzed large molecular substances, such as starch, non-starch
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215 polysaccharides and proteins, to small molecular compounds [5].

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216 Fermentation is also a very useful process used in waste biomass to increase

217 nutritional qualities and remove undesirable compounds. Fermentation of inedible

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218 biomass involves the action of microorganisms or enzymes that cause desirable
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219 biochemical change and significant modifications in flavour and texture [27]. It serves
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220 as a means of providing a major source of nourishment for higher plants populations

221 and contributes significantly to food security by increasing the range of raw material

222 which can be used in the production of edible products [28]. In plant fermentations,

223 endogenous enzymes, bacteria, yeast and moulds play important roles either one by

224 one or in combination, and contribute to the creation of a great variety of products.

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225 Many biochemical changes occur during fermentation, leading to altered ratio of

226 nutritive components of cereals, which affect product properties such as bioactivity

227 and some chemical compounds [27].

228 3.2 Wheat morphology

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229 Germination has profound effect on nutritional quality of the cereal [29]. In this

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230 study, the responses of all groups to different salt concentrations were found

231 significantly different (Fig. 2). This means that there existed differences among

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232 groups in respect of the tolerance of salt stress. However, increasing salinity decreased

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233 the germination percentage in all groups. After 4 days, the root length of Group-5%
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234 reached 59 mm and the shoot length was 52 mm. On the contrary, Group-40% had

235 minimum value in root and shoot length. During germination, certain changes occur
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236 in terms of quantity and type of nutrients within the seed. These changes can vary
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237 depending on the type of fermentation substance and the condition of germination.
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238 [30-31].

239 Germination is a natural biological process by which the seeds come out of
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240 latency stage [32]. Some of the wheat groups were more tolerant than the others. As
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241 the result of this fact, the germination of Group-40% showed significant differences
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242 with other groups. While there was no significant difference between the other groups

243 (Fig. 3A). For the relative root growth, Group-1%, 2.5%, 5% were the top level and

244 Group-40% was the worst (Fig. 3B). Generally, decreasing salinity levels increased

245 germination index in all of the wheat groups [33]. Biomass of wheat was negatively

246 affected by increasing salt treatments. The average germination indices of Group-10%,

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247 20% and 40% were significantly higher than that of Group-1%, 2.5%, 5% (Fig. 3C).

248 Salt tolerance at germination stage is an important factor, where medium salinity is

249 mostly dominated at surface layer. High concentration of salts have detrimental

250 effects on germination of seeds [34].

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251 Vigor index of Group-40% reached 270.5% and was significantly higher than

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252 others (Fig. 3D). The wheat groups were significantly more tolerant to salt stress at

253 germination than at later stages of growth. High concentration of fermentation

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254 fertilizer in the medium increases its osmotic potential. In addition, high absorption of

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255 Na and Cl ions during seed germination can be due to cell toxicity that finally inhibits
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256 or slows the rate of germination and thus decreases germination percentage [35]. An

257 increase in bioavailability of minerals and weight has been observed during seed
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258 germination. Germinated seeds are good source of ascorbic acid, riboflavin, choline,
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259 thiamine, tocopheroles and pantothenic acid [33].

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260 It was found that the height of the wheat plants was noteworthily influenced

261 under different medium of different growth periods (Fig. 4). Particularly at the early
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262 stage of seedling, straw height of wheat under low concentration was greater than that
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263 under high concentration medium. On the 7th day, the highest group was Group-5%
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264 and it was significantly different with the other groups. As time goes on, the

265 difference of biomass accumulation and biomass allocation in organs of wheat plants

266 under different medium was integrated with performance of efficiency utilization of

267 environmental resources, which reflecting the adaptive capacity and features of wheat

268 plants in different conditions. After 3 weeks, the straw height of Group-5% was also

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269 at the top level and it had more biomass above the ground. A negative relationship

270 was detected between vegetative growth parameters and increasing salinity. Straw

271 height of most wheat plants showed a decline towards increase in salinity level. On

272 the other hand, reduction in plant growth as a result of salt stress has also been

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273 reported in several other plant species [36]. The ability of plants to cope with salinity

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274 stress is an important determinant of crop distribution and productivity in different

275 medium, so it is important to understand the mechanisms that confer tolerance to

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276 saline environment.

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277 3.3 Wheat physiology
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278 Pigments are integrally related to the physiological function of leaves. In

279 seedling stage, sufficient mineral elements were conducive for the accumulation of
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280 chlorophyll (a+b) in leaves (Fig.5A-C); however excessive supply was not beneficial.
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281 In particularly, chlorophyll b was more sensitive to the mineral nutrition stress (Fig.
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282 5B) and affected the chlorophyll a/b ratio significantly (Fig. 5D). Chlorophyll b

283 content of Group-10% reached 0.91 mg/g on the 21st day and Group-20%, 40% were
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284 the lowest samples. Chlorophyll b content of Group-40% was 0.49 mg/g which was
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285 about half of that in Group-10%. Photosynthesis rate and chlorophyll content also
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286 would decrease with the increase of concentration. Role of stomata in exchange of

287 gases between internal leaf surface and atmosphere is well-documented [37].

288 Photosynthesis, the driving force of plant productivity and the ability to maintain the

289 rate of photosynthetic carbon dioxide, is fundamental to the maintenance of plant

290 growth and production under environmental stresses [38]. Reduction in growth and

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291 photosynthesis are among the most conspicuous effects of salinity stress. In addition,

292 stomatal closure, in order to reduce transpiration, appears to be the main cause of the

293 decrease in photosynthetic rate. The effects of the form and concentration of the

294 nutrient nitrogen on photosynthetic carbon dioxide assimilation may arise from the

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295 influence of the nitrogen source on photosynthetic enzymes, photorespiration,

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296 stomatal conductance or other aspects of photosynthetic metabolism.

297 Currently, it is extensively accepted that ROS are responsible for kinds of

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298 damage (stress-induced) to macro-molecules, finally to the cellular structure. On the

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299 7-day, there was a fluctuation for the different medium (Fig. 6A). In the Group-40%,
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300 the POD activity peaked at 12.22±0.21 U/mg prot on the 21-day. It has been generally

301 recorded that salinity adversely affects plant growth and some relevant metabolic
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302 processes of plants [39]. Similarly, MDA contents of wheat plants from 7-day to
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303 21-day was also gradually increased (Fig. 6B). As for the response of antioxidant
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304 enzyme to different N concentrations conditions, the level of POD and MDA in wheat

305 plants was an important index of ageing and death. Thus, higher ROS levels and
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306 reduced antioxidant enzymes may contribute, directly and/or indirectly, to the decline
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307 in the observed pigment contents in wheat. Decrease in photosynthetic pigments

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308 might be due to decrease in total carbohydrates and sucrose content of damaged

309 leaves [40]. From the relationship between proline contents and different times of

310 development in response to different medium, the effects of salinity changes on

311 proline accumulation were clearly revealed (Fig. 6C). The superior capacity for

312 proline accumulation in wheat plants would seem to be an important factor in its

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313 stress tolerance. The proline contents of Group-5%, 10%, 20% and 40% at the 21st

314 day were significantly higher in comparison with the 7th and 14th days and they were

315 no beneficial to the wheat growth. Carbohydrates are accumulated in plant tissues

316 under saline condition and these substances are suspected of contributing to osmotic

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317 adjustment [41]. Restitution of photosynthetic activity after initiation of osmotic

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318 upshock and proline accumulation are connected together [40]. Increasing

319 carbohydrates in the leaves might be a response to excess accumulation of

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320 monovalent ions in the vacuole under saline stresses. Organic solute e.g.

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321 carbohydrates, amino acids and glycinebetaine accumulated in cytoplasm act as an
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322 osmoticum which, is not harmful to the enzyme systems and membranes and thus will

323 balance the lowered osmotic potential of the vacuole [42].

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324 4. Conclusions
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325 Cereals, particularly wheat, are important components for the daily diet in BLSS,
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326 and wheat is one of the major sources for dietary energy and protein for the survival

327 of the crews. The wheat grown in the substrate with fermentation residue of 5%
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328 (Group-5%) presented the optimal morphological, physiological and antioxidant

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329 characteristics. Under this ratio, the seed germination was as high as 97.3% with the
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330 root length, shoot length and biomass production as 59 mm, 52 mm and 150 mg,

331 respectively, at the 4th day. In addition, the greatest straw height of 25.1 cm was

332 achieved at the 21st day. However, the salinity could significantly inhibit the wheat

333 growth and some relevant metabolic processes of the wheat. The Group-40% resulted

334 in the lowest seed germination of 34.7% and the minimum straw height of 15 cm. In

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335 seedling stage, enough mineral elements was conducive to increase the chlorophyll in

336 leaves; while an excess supply was not beneficial. Chlorophyll b content of Group-40%

337 was 0.49 mg/g which was about half of that in Group-10%. Our findings could be

338 used to design specifically balanced BLSS by supporting plant growth, especially for

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339 specialized applications (e.g. in space). It would play a critical role in further

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340 development of ground, space plant factory and enclosed experiments.

341 5. Acknowledgement

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342 This work was financially supported by Defense Industrial Technology

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343 Development Program (JCKY2016601C010).
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344 Reference

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445 Stage. Pak. J. Bot. 19(1987) 9-15.

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449 Percentage and Seedling Growth in Sweet Sorghum Cultivars. Journal of Biological

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450 Sciences. 7(8) (2007)1492-1495.

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452 Photosynthetic characteristics, antioxidant capacity and biomass yield of wheat

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453 exposed to intermittent light irradiation with millisecond-scale periods. Journal of
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458 ecosystem. Photosynthesis research. 126 (2-3) (2015)351-362.

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460 Genomic Foundation. Annals of Botany. 107(2011)vii-ix.

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462 photosynthetic characteristics, antioxidant capacity, yield and quality of wheat

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467 Exposed to LED Light Sources with Different Spectra Combinations. Journal of

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471 Physiology. 163(2006)847-855.

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472

473

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474

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475
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476

477
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489 Figure captions：

：
490 Fig. 1 Characteristics and elemental composition of fermentation substance.
491

492 Fig. 2 The root length (A), shoot length (B) and the biomass production (C) of
493 different concentrations (1%, 2.5%, 5%, 10%, 20%, 40% and CK) using fermentation

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494 substance and vermiculite. These data was measured every day after seeds planted.
495

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496 Fig. 3 Seed germination (A), relative root growth (B), germination index (C) and
497 vigor index (D) of different concentrations (1%, 2.5%, 5%, 10%, 20%, 40% and CK)

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498 of fermentation substance and vermiculite. Within each graph, bars labeled with the
499 same letters are not significantly different at p≤ 0.05.
500
U
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501 Fig. 4 Response of straw height of wheat plants to different treatments. These data
502 was measured on the 7th day, 14th day and 21st day after seeds planted. Within each
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503 graph, bars labeled with the same letters are not significantly different at p≤ 0.05.

504
D

505 Fig. 5 Response of chlorophyll a content (A), chlorophyll b content (B),chlorophyll

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506 (a+b) content (C) and chlorophyll ratio (a/b) (D) of wheat plants to different
507 concentrations using these substrates. These data was measured on the 7th day, 14th
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508 day and 21st day after seeds planted. Within each graph, bars labeled with the same
509 letters are not significantly different at p≤ 0.05.
C

510
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511 Fig. 6 Response of POD activity (A), MDA content (B) and proline content (C) of
512 wheat plants to different treatments. These data was measured on the 7th day, 14th day
513 and 21st day after seeds planted. Within each graph, bars labeled with the same letters
514 are not significantly different at p≤ 0.05.

515

516

517
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518 Table 1
519 The parameters of different treatments for fertilizer and vermiculite (Dry weight).

Form Group- Group- Group- Group- Group- Group- CK

1% 2.5% 5% 10% 20% 40%

Fermented residue 1% 2.5% 5% 10% 20% 40% 0

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Vermiculite 99% 97.5% 95% 90% 80% 60% 100%

520

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521

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522

523

524
U
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525
M

526

527
D
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528

529
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530

531
C
AC

532

533

534

535

536

537
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538 Fig. 1

Zn

Mn

Fe

Cu
Element types

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Mg

Ca

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Na

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P

U
0 6000 12000 18000 24000 30000 36000 42000
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539 Content (mg/1000g)

540
M

541

542
D

543
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544
EP

545

546
C
AC

547

548

549

550

551

552
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553 Fig. 2

70
1% 2.5% 5% 10% 20%
40% CK
60
(A)
50
Root length (mm)
40

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30

20

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10

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60
(B)

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50
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Shoot length (mm)

40

30
M

20

10
D

0
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160 (C)
EP

140
120
Biomass (mg, FW)
C

100
AC

80
60
40
20
0
0 1 2 3 4
554 Time (day)

555

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556 Fig. 3

120
(A) a (B)
a a a a a a a a a
100 b
100

Relative root growth (%)

80
Seed germination (%)

80
c
60 60

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40 b 40 d

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20 20

0 0

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300 a
1.4 (C) a (D)
250
1.2
b b
bc
Vigor index (%)

c c

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1.0 c 200 b
Germination index

0.8
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150 c c c c
c
0.6
100
0.4
M

50
0.2

0.0 0
D

1% 2.5% 5% 10% 20% 40% CK 1% 2.5% 5% 10% 20% 40% CK

557 Concentrations Concentrations
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558

559
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560
C

561
AC

562

563

564

565

566

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567 Fig. 4

1% a a
25 2.5% ab
5% b b
10% a
20% a c
20 40% b b b
CK
Straw height (cm)

a d

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c c
15 b b
c d

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10 d

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5

U
7th day 14th day 21st day
Time (day)
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568

569
M

570

571
D

572
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573
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574

575
C
AC

576

577

578

579

580

581
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582 Fig. 5
3.0 1.0 a
1% 2.5% 5% (B) b
10% 20% 40%
a b

Chlorophyll b content (mg/g)

Chlorophyll a content (mg/g)

ab b a a bc
2.5 CK bc 0.8
a
(A) b
b c bb c
2.0 b bc a bc
c 0.6 a ab c dd
1.5 a a a a aa d d c bb b b
a c
0.4

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1.0

0.5 0.2

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0.0 0.0
a
3.5 (C) aa 3.5 (D)
Chlorophyll (a+b) content (mg/g)

ab b a c
a a a ba d bc ab b

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a a

Chlorophyll ratio (a/b)

3.0 a 3.0 c
c c bd bc c
a
2.5 bb b 2.5
d a
2.0 a a a a a a c c 2.0
a d

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1.5 1.5
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1.0 1.0
0.5 0.5
0.0 0.0
7th day 14th day 21st day 7th day 14th day 21st day
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583 Time (day) Time (day)

584
D

585
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586
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587

588
C
AC

589

590

591

592

593

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595 Fig. 6
14
1% 2.5% 5% 10% a
20% 40% CK ab
12

POD activity (U/mg prot)

(A) b
bc
10 a c
d
8 ab d
b
a a b

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6 a ab c c bc
b b
4

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2

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35
(B) a
30
MDA content (nmol/g FW)

U
25 c
a c cd
a d
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20 d
b
a a bc c c
15 c
bb b
c
M

10 bc

5
D

0
180
TE

(C) a
160 a
b
Proline content (µg/g FW)

140 a c
b bc cd
d d
EP

120 c
dd d
100
80 a a
b
C

60 c
c c c
AC

40
20
0
7th day 14th day 21st day

596 Time (day)

597

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Highlights
1. Human feces and wheat straw were fermented for plants cultivation in BLSS.
2. The fermented residue was beneficial to wheat germination, growth and
development.
3. Group with fertilizer rate 5% got germination rate of 97.3% and biomass of 150

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mg.
4. Excessive salinity adversely affected wheat growth and some metabolic process.

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