Accepted Manuscript

An integrated bioprocess for bioethanol and biomanure production from pineapple

leaf waste

Anjani Devi Chintagunta, Subhabrata Ray, Rintu Banerjee

PII: S0959-6526(17)31625-6
DOI: 10.1016/j.jclepro.2017.07.179
Reference: JCLP 10182

To appear in: Journal of Cleaner Production

Received Date: 18 February 2017

Revised Date: 19 July 2017
Accepted Date: 22 July 2017

Please cite this article as: Chintagunta AD, Ray S, Banerjee R, An integrated bioprocess for bioethanol
and biomanure production from pineapple leaf waste, Journal of Cleaner Production (2017), doi:
10.1016/j.jclepro.2017.07.179.

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1 Word count : 7,171

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3 An integrated bioprocess for bioethanol and biomanure production from pineapple
4 leaf waste
5 Anjani Devi Chintagunta , Subhabrata Rayb and Rintu Banerjeec*
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6 Advanced Technology Development Centre, Indian Institute of Technology,

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7 Kharagpur- 721302, West Bengal, India
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8 Chemical Engineering Department, Indian Institute of Technology,

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9 Kharagpur- 721302, West Bengal, India
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10 Agricultural & Food Engineering Department, Indian Institute of Technology,
11 Kharagpur- 721302, West Bengal, India

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25 Corresponding Author

26 Rintu Banerjee

27 Tel: +91-3222-283104(O)/+91-3222-283105(R).

28 Fax: +91-3222-282244

29 Email: rb@iitkgp.ac.in

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30 Abstract

31 Indiscriminate disposal of the solid waste generated from various agricultural practices and

32 agro-based industries causes detrimental effects on the environment. Utilisation of the waste

33 biomass for production of value added products through biotechnological intervention not

34 only helps to combat environmental pollution but also contributes significantly to the

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35 economy. The present work focuses on integrated production of bioethanol and biomanure

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36 from pineapple leaf waste for its complete utilisation leading to zero waste generation.

37 Bioethanol production from pineapple leaf waste was carried out through simultaneous

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38 saccharification and fermentation (SSF) using cellulolytic enzyme (cellulase-xylanase

39 concoction) and yeast. The SSF of pineapple leaf waste resulted in bioethanol production of

40
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7.12 %(v/v). The residue obtained after bioethanol production was inoculated with five
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41 different strains of blue-green algae and their concoction for nitrogen (N), phosphorus (P) and
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42 potassium (K) enrichment. Among them, Fischerella muscicola was found to enrich N, P and

43 K content of the residue by nearly 6.84, 8.78 and 14.17 folds than that of the initial content,
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44 leading to an improved NPK ratio of approximately 3.5:1:2. Use of biomanure not only
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45 enriches naturally the soil but also reduces the application of chemical fertilizer, generally

46 causing secondary pollution. Thus, this process is more suited to eco-friendly way of waste
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47 disposal.
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48 Keywords: Bioethanol, biomanure, blue-green alga, pineapple leaf waste

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49 Abbreviations
50 m3 - cubic meter, (v/v) - volume by volume, rpm - revolutions per minute, IU/mL -
51 International Units per millilitre, w/v - weight by volume, g/(L·h) - gram per litre per hour,
52 mL/kg - millilitre per kilogram, MJ/kg - megajoules per kilogram, TS - total solid, ATP
53 Adenosine triphosphate, nif - nitrogen fixation, N - nitrogen, P - phosphorus, K - potassium,
54 µg - microgram, kg/ha - kilogram per hectare, LCA - Life Cycle Assessment, GHG -green
55 house gas, Mt - million ton, t - metric ton, Mha - million hectares, mm - millimetre, Pa -
56 pascal.

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57 1. Introduction

58 Energy crisis due to declining fossil fuels, escalating population, rising pollution and global

59 warming triggered the society to comprehend the need for renewable sources. Biomass is one

60 of the promising renewable energy resources for the production of biofuels such as

61 bioethanol. In the global scenario, the production of bioethanol has increased from 29.2

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62 million m3 in 2000 to 115.1 million m3 in 2015 of which 85 % is contributed by the United

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63 States and Brazil from corn and sugarcane (Ethanol industry outlook, 2016). In developing

64 countries, production of bioethanol from lignocellulosic biomass, especially from wastes, is

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65 preferred to resolve the food vs. fuel issues.

66 During the year 2013-14 (FAOSTAT, 2015), pineapple production for the whole world was

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estimated to be 24.80 Mt from 1.02 Mha of which India's contribution was of the order of
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68 1.74 Mt from 0.11 Mha (Saxena and Gandhi, 2014). The leaf portion that remains in the field
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69 after harvesting the fruit accounts for around 2,708.22 Mt, a part of which is generally used

70 for making ropes, home textiles, apparels and in paper industries. Utilization of pineapple
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71 leaves, rich in holocellulose content (60-85 %, w/w), for bioethanol production is an attempt
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72 towards valorisation of the waste (Daud et al., 2014).

73 Bioethanol is in great demand as an engine fuel, octane enhancer in unleaded gasoline and
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74 oxygenated fuel mix for cleaner combustion of gasoline thereby reducing CO2 emission
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75 (Elfasakhany, 2016). Utilization of energy crops, agro-industrial and forest waste, and
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76 municipal solid waste for ethanol production has greatly reduced the green house gas (GHG)

77 emissions (Singh et al., 2010). The impact of biofuel production process on GHG emission

78 and energy balance is being evaluated through life cycle assessment (LCA) which is an

79 internationally recognized methodology (Rathore et al., 2016). The sustainability of any

80 liquid biofuel needs to be assessed with respect to economic, environmental and social

81 sustainability (Nizami et al., 2016).

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82 One of the concerns in ethanol production from lignocellulosic feedstock is the presence of

83 lignin which prevents the accessibility of hydrolysing enzymes to the holocellulosic fractions

84 of the biomass for the production of reducing sugar. Lignin degradation is accomplished by

85 various pretreatment methods such as physical, chemical, physico-chemical and biological

86 including both whole cells and enzymes. Enzymatic pretreatment is a viable and sustainable

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87 method for lignin removal which improves the conversion of holocelluloses to reducing sugar

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88 production during the saccharification process (Banerjee et al., 2017).

89 Production of bioethanol in short incubation time is another challenge and a key factor for

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90 commercialisation of lignocellulosic bioethanol. Several techniques such as simultaneous

91 saccharification and fermentation (SSF), prehydrolysis simultaneous saccharification and

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fermentation (PSSF), simultaneous saccharification and co-fermentation (SSCF) and
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93 consolidated bioprocessing (CBP) have been developed by the researchers to meet this
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94 challenge (Tomas-Pejo et al., 2008). An ideal fermentation technique should reduce the cost

95 and fermentation time, increase the amount of substrate processed per given volume of
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96 hydrolyzing enzymes and fermenting cells, and improve the ethanol yield (Xiros and Olsson,
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97 2014). The cost associated with the bioethanol refinery can be reduced by improving ethanol

98 yield, utilising the by-products as animal feed, substrate for yeast production and, for
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99 generation of electricity and steam (Kawa-Rygielska and Pietrzak, 2014). Apart from this,
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100 some value added products such as biomethane, organic acids, alcohols and aromatic
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101 chemical intermediates are also produced (Ouda et al., 2016). Utilizing the left over residue

102 as soil fertilizer can replenish humus and micronutrients of the soil, substituting the need for

103 synthetic fertilizers (Farrell et al., 2006; Sadef et al., 2016).

104 The bioethanol refinery in the present study includes bioethanol production from pineapple

105 leaf waste followed by enrichment of fermented biomass by cyanobacteria for its application

106 as biomanure to accomplish soil to soil technology. Utilization of dried cyanobacteria for soil

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107 enrichment in rice field has already been reported (Kaushik 2014; Mishra and Pabbi, 2004).

108 Cyanobacteria are gram negative autotrophic bacteria which contain chlorophyll and other

109 accessory pigments such as phycobilins, carotenoids and xanthophylls. These secrete amino

110 acids, vitamins and hormones such as auxins and gibberilins involved in enhancement of

111 plant growth (Ananya et al., 2014). Singh et al. (2016) reported that cyanobacteria are

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112 involved in fixing atmospheric nitrogen, producing growth regulators, reducing soil erosion

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113 and enhancing soil organic matter. Owing to its CO2 assimilation potential, cyanobacteria can

114 fix 10-15 times more carbon than that of the terrestrial plants (Kumar et al., 2011). In

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115 association with methanotrophs, cyanobacteria play an important role in removing significant

116 amount of methane (Tiwari et al., 2015). Thus, cyanobacteria are excellent bioagents for CO2

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sequestration, mitigation of GHG emissions and global warming (Kumar et al., 2017).
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118 Hence, the present paper deals with the optimization of ethanol concentration from pineapple
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119 leaf waste along with the reduction in reaction time. Moreover, the emphasis has been given

120 on utilisation of residual solid waste after ethanol recovery to biomanure production for
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121 organic farming.

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122 2. Materials and Methods

123 2.1. Substrate and its chemical characterisation

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124 Pineapple (Ananas comosus, Giant Kew variety) leaf waste was collected (during September,
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125 2016) from the agriculture farm of Indian Institute of Technology, Kharagpur, India. It was
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126 chopped, sun-dried, pulverised (Hammer mill pulverizer, SWLY300 Murhopye Scientific

127 Company, Mysore, India) to 0.2 mm particle size, delignified with laccase and used for the

128 present study. Cellulose (Viles and Silverman, 1949), hemicellulose (Marlett and Lee, 2006),

129 ash (TAPPI Standard and Suggested Methods, 1991), pectin (Sadasivam and Manickam,

130 1992) and lignin (Hussain et al., 2002) contents of the delignified substrate were measured.

131 The ethanol and reducing sugar contents were measured by potassium dichromate (Seo et al.,

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132 2009) and dinitrosalicylic acid (DNS) (Miller, 1959) methods. Nitrogen, phosphorus and

133 potassium contents of lignocellulosic biomass were measured by Kjeldahl (Kjeldahl, 1883),

134 spectrophotometric (Chapmann and Pratt, 1961) and flame photometric (Hegedus and

135 Pungor, 1955) methods. Organic carbon (C) content of the biomass was measured by

136 Walkley and Black (1934) method.

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137 2.2. Enzyme and microorganism

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138 Delignification of biomass was carried out by laccase obtained from Pleurotus djamor. In a

139 separate optimum study, maximum delignification of 78.57 % was obtained under optimum

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140 conditions of solid loading 25 %(w/v), incubation time 5.30 h, 45 oC, pH 6.16 and enzyme

141 concentration 582.82 IU/mL. Simultaneous saccharification and fermentation of delignified

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biomass was performed by using cellulase-xylanase concoction produced from Trichoderma
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143 reesei Rut-C30 along with Saccharomyces cerevisiae. Saccharifying enzyme was produced

by inoculating the spores (~3.6 × 106 spores per 1 mL, spores counted under
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145 haemocytometer) of T. reesei to wheat bran mixed with Czapek-Doc medium (pH 5) in 1:1
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146 ratio (w/v) and incubated at 30 oC for 5 days. The enzyme was extracted in water and
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147 centrifuged at approximately 18,000 g at 4 °C for 10 min in cold centrifuge to remove the

148 spores and other insolubles for activity measurement. The activities of cellulase and xylanase
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149 were measured following the standard assay protocol (Mandels and Andreotti, 1976; Bailey,
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150 1988). One unit (IU/mL) of enzyme activity was defined as the amount of enzyme required to
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151 produce 1 µmol of reducing sugar per minute under the assay conditions. S. cerevisiae was

152 maintained in the medium containing glucose (2 %, w/v) and yeast extract (0.5 %, w/v) at

153 pH 6.5 and 37 ºC.

154 The cyanobacteria viz., Nostoc muscorum, Anabaena variabilis, Fischerella muscicola,

155 Aulosira fertilissima and Cylindrospermum muscicola, selected for N, P and K enrichment

156 studies were cultured in BG 11 (Blue Green) medium according to Rippka et al. (1979) at 25-

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157 28 ºC and pH 7.5. The medium was prepared by excluding nitrate or ammonium source to

158 induce heterocyst formation in cyanobacteria (Pankaj, 2008).

159 2.3. Methodology and optimization of SSF

160 SSF of the delignified pineapple leaf waste was carried out in an Erlenmeyer flask with

161 required amount of biomass (5-40 %(w/v)), cellulolytic enzyme and yeast (5-30 %(v/v))

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162 under defined reaction conditions. The flasks were sealed with parafilm to provide semi-

anaerobic conditions for yeast and incubated at 37 oC for specific incubation time (12-120 h).

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164 Then the broth containing bioethanol was separated from the biomass, centrifuged at 2,000

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165 rpm for 5 min and measured for ethanol and reducing sugar concentration. The ethanol

166 productivity rate and conversion efficiency were calculated as given in equation 1 and 2:

Ethanol productivity rate (g/(L·h)) =

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final ethanol concentration ( g / L)
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167 (1)
fermentation time (h)

ethanol produced (mL)

Ethanol conversion efficiency (%) = × 100
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168 (2)
theoretical ethanol (mL)

Selection of influential parameters on ethanol production was done by one variable at a time
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170 approach. The effect of various concentrations (levels) of first variable on ethanol production
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171 was studied by fixing the other variables. Then effect of second variable was studied by
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172 taking into account the previously optimized factor and by fixing the rest of the variables. In

173 this way, effect of five parameters such as solid loading, incubation time, temperature,
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174 inoculum age and inoculum volume on ethanol production were studied. Thereafter,
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175 optimization and evaluation of simultaneous saccharification and fermentation of delignified

176 pineapple leaf waste was carried out using three-level (-1, 0, +1), 25 full factorial CCD using

177 MINITAB 16 software. An experimental design was prepared by considering an upper and

178 lower limit of the selected value of each parameter i.e., substrate concentration (25-35

179 %,w/v), incubation time (12-36 h), 35-40 °C, inoculum age (12-36 h), and inoculum volume

180 (10-20 %,v/v). The experimental run conditions [Table S1, SI (Supplementary Information)]
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181 were designed to study the effect of interactions among various parameters on ethanol

182 concentration. The ethanol concentrations obtained (experimental) against each combination

183 of experimental parameters were used by the software to get the predicted ethanol

184 concentration.

185 2.4. Experimental procedure for biomanure production

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186 After bioethanol production, the biomass and the fermentation broth containing bioethanol

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187 were separated by squeezing through a cheese cloth. The separated biomass was collected,

188 measured for nutrient content and then treated with cyanobacteria for nutrient enrichment.

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189 The process of enrichment of fermented biomass with cyanobacteria was as follows:

190 A set of six treatments, each containing 100 g of residual biomass was prepared. To five

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treatments, 10 %(v/w) A. variabilis, A. fertilissima, C. muscicola, N. muscorum and F.
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192 muscicola were inoculated. The sixth treatment was inoculated with concoction of all the five
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193 cyanobacteria in equal proportions by volume and a control was maintained without addition

194 of inoculum. The treatments were incubated at 25-27 oC and analysed for NPK enrichment at
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195 regular intervals of time. The integrated process for bioethanol and biomanure production
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196 from pineapple leaf waste was schematically represented in Fig. 1.

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206
Laccase pretreated pineapple
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leaf waste
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cellulolytic enzyme and
yeast
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Simultaneous saccharification and

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210
fermentation
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incubation followed by solid-

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liquid separation
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213 Bioethanol Residual biomass
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nutrient enrichment through
215 cyanobacteria

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Biomanure
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218 Fig. 1. Schematic representation of integrated bioethanol and biomanure production from
pineapple leaf waste.
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2.5. Energy density measurement
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222 Energy density of the delignified and residual pineapple leaf waste after SSF was measured
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223 using Bomb calorimeter (Oxygen Bomb Calorimeter, Eastern Instruments, Kolkata, India).
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224 The samples were dried overnight in an oven at 60 oC to remove the moisture. The dried

225 samples were cast into pellets. In case of liquid sample, a known mass of the sample was
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226 filled in the crucible of the calorimeter and then ignited. The heat content of the sample
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227 (solid/liquid) was determined by considering the temperature difference in the presence of

228 high pressure (2.75 × 106 Pa) and excess oxygen. The energy density of the samples was

229 determined using the following equation (3):

W (kJ / oC ) × (T2 − T1 ) (oC )

230 Energy density (kJ / g ) = (3)
M (g)

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231 Where, W= water equivalent of calorimeter (9.748 kJ/oC), M= mass of the sample (g) and

232 (T2-T1) = rise in the temperature (oC)

233 2.6. Sample preparation for scanning electron microscopy (SEM)

234 The change in structural morphology of pineapple leaf waste after simultaneous

235 saccharification and fermentation was studied with respect to the structure of pretreated

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236 biomass. The lignocellulosic biomass sample was mounting on a metallic stub and coated

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237 with gold to view it under JEOL JSM 5800 SEM (Jeol Ltd., Tokyo, Japan).

238 3. Results and Discussion

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239 3.1. Chemical characterisation of delignified pineapple leaf waste

240 The chemical composition of delignified pineapple leaf waste was studied to determine its

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suitability as substrate for bioethanol production. The study revealed that pineapple leaf
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242 waste contains high holocellulose content (cellulose 46.17 % ± 0.76 (w/w), hemicellulose
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243 19.28 % ± 0.54 (w/w)), meagre lignin (2.8 % ± 0.70, w/w), ash (18.82 % ± 1.46, w/w) and

244 pectin (2.92 % ± 0.50, w/w). As the substrate is rich in holocelluloses, it was utilized for
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245 ethanol production.

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246 3.2. Parameters affecting SSF of delignified biomass and its optimization

247 A range of various parameters were considered for studying their effect on SSF of
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248 delignified pineapple leaf waste. The effect of these parameters on ethanol production was

shown in Fig. 2. Response surface methodology is a statistical technique used for

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249
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250 optimization studies (Kumar and Banerjee, 2013). Optimisation study based on one

251 variable at a time, substrate concentration 30 %(w/v), incubation time 24 h, 37.5 oC,

252 inoculum age 24 h and yeast volume 15 %(v/v) showed maximum ethanol production.

253 Using the experimental data of ethanol production, a second order polynomial equation

254 was fitted for the SSF of delignified pineapple leaf waste and is shown as:

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Y1 = − 175.73 + 0.22 X 1 + 0.61X 2 + 9.05 X 3 + 0.02 X 4 + 0.18 X 5 − 0.01X 2 X 2 − 0.12 X 3 X 3 + 0.002 X 4 X 4
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+ 0.003 X 1 X 3 + 0.001 X 1 X 4 + 0.002 X 1 X 5 + 0.002 X 4 X 5
256 (4)

257 Where Y1 is ethanol concentration %(v/v) and X1, X2, X3, X4, X5 represent substrate

258 concentration, incubation time, temperature, inoculum age and inoculum volume

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259 respectively.

260 In the above equation (4), substrate concentration, incubation time, temperature, inoculum

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261 age and inoculum volume were represented in %(w/v), h, oC, h and %(v/v) respectively. In

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262 the experimental response, the minimum and maximum ethanol production was observed in

263 run 18 and 12 respectively (Table S1, SI).

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264 The analysis of variance (ANOVA) of regression equation for SSF of pineapple leaf waste
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265 was presented in Table S2, SI. At 20 degrees of freedom, the F and P values were found to be

266 48.08 and <0.001 respectively, where the low P value determines the significance of the
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267 quadratic regression model. The F and P values (1.68 and 0.29) of lack of fit indicated that

the experimental data were in good agreement with the model. There being no significant
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269 difference between R2 and adjusted R2 value, it was suggested that the regression model was
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270 reliable and competent for SSF of pineapple leaf waste.

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271 The 3D response surface plots for the model were shown in Fig. S1, SI. Increase in inoculum

272 volume and inoculum age does not show any significant effect on ethanol production,
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273 whereas an increase in temperature and incubation time have an adverse effect on the yeast
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274 involved in the fermentation process. Raines-Casselman (2005) reported that the temperature

275 above 37 °C inhibits the metabolic growth of yeast and has negative effect on bioethanol

276 production.

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278

279 Fig. 2. Effect of a) substrate concentration b) incubation time c) temperature d) inoculum age
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280 and e) inoculum (containing yeast) volume on ethanol production from pineapple leaf waste
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281 through simultaneous saccharification and fermentation process.

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283 Upon validation, the optimum conditions for SSF of delignified pineapple leaf waste were

284 observed to be substrate concentration 25 %(w/v), incubation time 24 h, 37 oC, inoculum age
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285 28.73 h and inoculum volume 15 %(v/v). Under these optimum conditions, the predicted
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286 ethanol production was 6.87 %(v/v) which was in close proximity with that of the

287 experimental ethanol production i.e., 7.12 %(v/v). Through optimization, maximum ethanol

288 production was obtained from the delignified biomass at high solid loading (25 %,w/v) in a

289 short incubation time (24 h). The final ethanol concentration and productivity rate were

290 calculated to be 56.02 g/L and 2.33 g/(L·h) respectively. The ethanol conversion efficiency

291 was found to be 75.53 % and total ethanol obtained was 355 mL/kg of delignified biomass.
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292 Paschos et al. (2015) reported the ethanol concentration of 58 g/L from SSF of

293 hydrothermally pretreated wheat straw using co-culture of Fusarium oxysporum and S.

294 cerevisiae after 120 h of incubation time. The final ethanol concentration from the present

295 work was in close proximity with the reported ethanol concentration, but the entire ethanol

296 production process in the present study was completed in shorter incubation time i.e., 30 h

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297 (5.30 h for pretreatment and 24 h SSF) than the reported work (120 h). Stream pretreated

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298 wheat straw and corn stover through SSF produced ethanol concentration of 38.1 g/L and

299 36.8 g/L respectively which was lesser than the ethanol concentration (56.02 g/L) obtained in

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300 the present study (Olofsson et al., 2008). Wi et al. (2015) reported ethanol of 351 mL/kg and

301 224 mL/kg from hydrogen peroxide-acetic acid (HPAC) pretreated oak wood and rice straw

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respectively in 24 h. The amount of ethanol produced in the present study (355 mL/kg
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303 delignified biomass) was higher than the reported work. Moreover, temperature required for

HPAC pretreatment was 80 oC which was higher than the temperature required for laccase
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305 pretreatment (45 oC).

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306 3.3. Production of reducing sugar and ethanol during SSF

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307 During SSF, the delignified biomass was hydrolyzed enzymatically for the production of

308 reducing sugar that was simultaneously utilized by the S.cerevisiae for the generation of
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309 ethanol. The reducing sugar and ethanol production profile was shown in the Fig. 3. A sharp
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310 increase in reducing sugar production was observed since the initiation of SSF due to
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311 effective action of cellulase upto 6 h, beyond which a prominent decline in reducing sugar

312 production was observed which might be due to its conversion into ethanol. The cellulose and

313 hemicellulose content of pineapple leaf waste before SSF were 45.20 %(w/w) and 19.80

314 %(w/w) respectively and after SSF were 5.57 %(w/w) and 4.86 %(w/w) respectively. The

315 decrease in the holocellulose content signifies its conversion into simple sugars. The reducing

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316 sugar (502 mg/g) obtained during hydrolysis of pineapple leaf waste contains 21.59 % xylose

317 (including arabinose), 39.29 % glucose and 39.11 % cellobiose.

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318

319

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Fig. 3. Ethanol and residual reducing sugar profile of pineapple leaf waste during
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320 simultaneous saccharification and fermentation.
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322 Production of ethanol started slowly and reached the maximum at 24 h but thereafter slight

323 decrease in ethanol production was observed. Sharma et al. (2007) also reported a slight
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324 decrease in ethanol production after 48 h of incubation time from kinnow waste and banana
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325 peels through SSF. Tang et al. (2011) have worked on ethanol production from

lignocellulosic residues (corn kernel) through SSF and SSCF. They have observed maximum
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327 ethanol yield of 83.7 % with the maximum depletion of reducing sugar in 48 h. Generally, the
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328 acid pretreatment methods are associated with furfural formation (due to decomposition of
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329 sugars) which inhibits the yeast growth and ethanol fermentation process resulting in a

330 prolonged lag phase during SSF. In the present study, laccase pretreated biomass which does

331 not contain any harmful inhibitors was used for ethanol production by SSF process. Ethanol

332 production process was completed within 30 h of incubation. The plausible reason for ethanol

333 production in lesser time than the reported work might be due to the lack of lag phase.

334 3.4. Energy density measurement

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335 The energy density of the biomass and broth at various steps of ethanol production were

336 reported in Table 1. The energy density of the delignified biomass was high due to the

337 presence of the cellulose and hemicellulose molecules. Cellulose has a uniform structure with

338 the higher heating value around 18.6 MJ/kg. Despite having similar chemical structure as that

339 of the cellulose, hemicellulose differs in the composition according to the biomass species

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340 and estimation protocol (Graboski and Bain, 1981). As the process (SSF) proceeds, there was

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341 a gradual decrease in the energy density of the fermented biomass due to the conversion of

342 carbohydrate polymers to monomers by the action of carbohydratases. The monomers were

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343 gradually released into the liquid stream and utilized by the yeast for the production of

344 ethanol thereby increasing the energy density of the broth. The energy density of fermented

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broth containing ethanol obtained from the present study (6.53 MJ/kg) is in proximity with
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346 the energy density of the ethanol (7.46 MJ/kg), produced from corn stover (Luo et al., 2009).
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347 Table 1

348 Energy density of solid and liquid samples.

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Substrate Energy density

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MJ/kg

Delignified biomass 15.96

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Fermented biomass 4.13

Fermented broth with ethanol 6.53
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349
350 3.5. Structural characterisation of pineapple leaf waste
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351 The scanning electron microscopy was conducted to observe the changes in structural

352 morphology of pineapple leaf waste due to simultaneous saccharification and fermentation.

353 Fig. 4 illustrates the surface structure of delignified and fermented biomass where the

354 delignified biomass exhibited distorted surface morphology and the intensity of distortion

355 increased in fermented biomass. The plausible reason for the distortion might be the

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356 degradation of lignin, cellulose and hemicellulose moieties of the lignocellulosic biomass due

357 to effective action of laccase (during delignification) and carbohydratases during SSF.

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358
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359 Fig. 4. Scanning electron microscope images of (a) delignified and (b) fermented pineapple
360 leaf waste.
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361
362 3.6. Enrichment of pineapple leaf waste residue after bioethanol production
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363 Prior to the cyanobacterial enrichment, it is highly essential to determine the nutrient content
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364 of residual pineapple leaf waste. The nutrient constitution and results related to the proximate

365 analysis were incorporated in the Table 2. The results illustrate the presence of meagre
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366 nutrient content in the substrate before the enrichment process.

367 Table 2
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368 Proximate and nutrient composition of pineapple leaf waste residue after ethanol production.
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Parameters Content
Volatile solid (%) 47.58 ± 1.54
Ash (%) 52.42 ±1.47
Nitrogen (% TS) 0.36 ± 0.05
Potassium (% TS) 0.08 ± 0.01
Phosphorus (% TS) 0.10 ± 0.04
Organic carbon (% TS) 18.35 ± 0.91

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369 Instead of dumping the fermented biomass with meager nutrient content on the soil, it was

370 subjected to nutrient enrichment so that it can serve the purpose of biomanure. Various

371 cyanobacteria involved and the methodology implemented for enrichment process have been

372 described in the Sections 2.2 and 2.4 respectively.

373 In the present study, NPK enrichment in the residual biomass due to the action of various

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374 blue green algae was studied at a regular interval of 7 days and represented in Table S3, SI.

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375 The NPK enrichment varied according to the inoculating microorganism. Among the six

376 treatments, maximum enrichment was observed in 5th and 6th weeks of incubation time. A.

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377 fertilissima, C. muscicola, N. muscorum, F. muscicola and concoction showed maximum

378 enrichment in the 5th week whereas A. variabilis showed maximum enrichment in 6th week.

379
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The maximum NPK was observed in the biomass enriched with F. muscicola which is around
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380 2.49 %(w/w), 0.72 %(w/w) and 1.45 %(w/w) respectively approximating to a ratio of 3.5:1:2
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381 (Fig. 5).

382 The algae used in the present study are heterocyst forming cyanobacteria whose growth
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383 requires the interdependent action of vegetative cell and heterocyst. The heterocyst synthesis
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384 in cyanobacteria depends on various factors such as low light intensity, high phosphate

385 content in the medium, availability of nitrate, carbon and ATP. The vegetative cells are
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386 involved in the oxygenic photosynthesis process while the heterocysts are engaged in
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387 nitrogen fixation. The vegetative cells transfer the fixed CO2 as reduced carbon to heterocyst
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388 where N2 is fixed by nitrogenase for ammonia production which is incorporated into amino

389 acids through glutamine synthetase-glutamate synthase pathway. Heterocyst establishes the

390 pumps between adjacent cells for the transport of carbon that acts as a reductant (source of

391 ATP) for nitrogenase (Haselkorn, 1978; Brouwer et al., 2017).

392 Nitrogen fixation is an anaerobic process and presence of oxygen inactivates nitrogenase and

393 the remaining nif gene products. Heterocysts restrict the entry of gases, shut down the oxygen

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394 evolving activity of photosystem II (PS II), retain the ATP synthesising efficiency of

395 photosystem I (PS I) and involve in N2 fixation thereby enriching the substrate with N2

396 (Burnat et al., 2014). Enhancement of phosphorus and potassium contents also occurs in the

397 substrate which might be due to the conversion of inorganic and immobilised P and K into

398 soluble forms in the presence of organic acids such as oxalic, succinic, oxaloacetic, tartaric,

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399 lactic and fatty acids released by the blue green algae inoculated in the substrate (Hariprasad

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400 and Niranjana, 2009; Bhatnagar and Roychoudhury, 1992). Another reason for nutrient

401 enrichment in the substrate was the enhanced decomposition of the organic matter which led

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402 to the decrease in organic carbon content (Pramanik et al., 2007).

403 The NPK ratio of A. variabilis, A. fertilissima, C. muscicola, N. muscorum and the

404
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concoction inoculated pineapple leaf waste residue were 3.4:1:2.2, 2.7:1:1.9, 2.7:1:2, 3.1:1:2
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405 and 2.4:1:1.9 respectively which were in near proximity to 3:1:2. There was no significant
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406 difference in NPK ratio of F. muscicola and A. variabilis enriched biomass but showed

407 variation in the time taken for enrichment. The biomass inoculated with concoction of all the
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408 five blue green algae was least enriched with NPK. The probable reason for poor enrichment
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409 is the negative allelopathic interaction of one organism with the other in the concoction. The

410 allelopathic substances released from one organism in the concoction may inhibit either the
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411 growth, cell motility or PSII of the other organism (Kearns and Hunter, 2001). Production of
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412 allelophatic substance that hampers the colonization of other organisms was reported in
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413 Fischerella sp. (Gantar et al., 2008). Various factors that influence the inhibition are the

414 concentration of the substance and the time of exposure. Gantar et al. (2008) reported the

415 accumulation of 4 µg/mL of Fischerellin A toxin by one month old culture of Fischerella that

416 would inhibit the PSII of other cyanobacteria in the concoction. Fischerellin produced from

417 Fischerella sp. strain 52-1, is lipophillic in nature and gets transferred easily from one cell to

418 another causing ultrastructural damage in green algae such as Chlamydomonas sp. (Zanchett

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419 and Oliveira-Filho, 2013). Singh et al. (2001) also reported the inhibition of growth and

420 photosynthesis besides extensive lysis of Nostoc muscarum and Anabaena sp. upon exposure

421 to microcyatin-LR for six days.

422 The quantity of nutrients required and ratio of NPK varies from crop to crop. The organic

423 fertilizer with 3.5:1:2 ratio was recommended for sugarcane crop whereas fertilizer with 3:1:2

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424 ratio was recommended for sequoia, sunflower, snapdragon and sedum (NAAS, 2009). NPK

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425 content of F. muscicola inoculated pineapple leaf waste residue was increased by 6.84, 8.78

426 and 14.17 folds upon comparison with the NPK content of the biomass before enrichment.

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427 Chintagunta et al. (2016) reported the biomanure production from potato waste using blue-

428 green algae where the NPK ratio of A variabilis enriched biomanure was 2:1:1 with an

429
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increase in NPK by 7.66, 21.66 and 15 fold respectively. Thus, the cyanobacteria containing
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430 heterocyst are very important for the production of biofertilizer/biomanure which could
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431 improve the plant productivity by substituting the chemical fertilizers (Shukia et al., 2008).
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432
433 Fig. 5. Increase in nutrient concentration of fermented pineapple leaf waste after enrichment

434 with F. muscicola

435

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436 In a nutshell, the present study puts forth an integrated process for bioethanol and biomanure

437 production, where the initial moisture content of the biomass was found to be 77.94 %(w/w).

438 During delignification, the percentage lignin removal was recorded as 78.57 %. Upon

439 subjecting the delignified biomass to simultaneous saccharification and fermentation process,

440 7.12 %(v/v) of ethanol was obtained. The nutrient enriched biomass with NPK of 3.5:1:2

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441 ratio was obtained after treating with F. musicola.

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442 3.7. Comparison of vermicompost produced from municipal solid waste with biomanure

443 under study

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444 Vermicomposting is an established technique of converting the solid organic waste into

445 compost, which upon application enriches the soil with the nutrients and enhances plant

446
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growth. Vermicompost with NPK ratio of 9.9:1:9.5 and production rate is 5,933 kg/ha per 8
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447 months was reported by Ghosh (2004). Pathma and Sakthivel (2012) have reported the
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448 transformation of garbage into compost by earthworms in 60-90 days. From the present

449 study, F. muscicola enriched lignocellulosic biomass with NPK ratio of 3.5:1:2 and
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450 productivity of 1,770 kg/ha was obtained. The entire process of biomanure production takes
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451 place within 5-6 weeks of incubation time and specifically 35 days for pineapple leaf waste

452 enrichment with F. muscicola. The enrichment process discussed in the present study is less
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453 labour intensive and takes less time when compared with vermicomposting.
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454 3.8. Industrial perspective of integrated bioethanol and biomanure production from
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455 pineapple leaf waste

456 Integrated production of value added products along with the main product is a promising

457 option for utilization of waste biomass in a biorefinery. Simultaneous saccharification and

458 fermentation of ethanol production occurs in a single reactor at low temperature (37 oC) and

459 short incubation time (24 h). In-house production of high titre saccharifying enzyme

460 (cellulase-xylanase concoction) from T. reesei Rut-C30 and its utilisation at high solid

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461 loading (25 %,w/v) reduces the cost of ethanol production. Reduction in greenhouse gas

462 emission take place during the process as it involves ethanol production from lignocellulosic

463 biomass using low dosage of enzyme (Karlsson et al., 2014). Bhutto et al. (2015) reported

464 that promotion of lignocellulosic bioethanol will achieve national target of at least 5 % of

465 total commercial energy supply by 2030. Several lignocellulosic ethanol production plants

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466 have been installed throughout the world and many biorefineries are emerging for

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467 commercial scale lignocellulosic ethanol production (UNCTAD, 2016; European Biofuels

468 Technology Platform, 2016). Some of them have been tabulated in Table 3. These plants also

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469 provide high employment opportunities (Delivand et al., 2012).

470 Table 3

471
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Commercial scale bioethanol plants in the world.
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Bioethanol plant facility and Substrate Production capacity
location (annual)
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Beta Renewables, Crescentino, rice straw, wheat straw and from 40,000 t
Italy Arundo donax, the common
giant reed
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Beta Renewables / Biochemtex, non-food biomass 55,000 t

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Strazke, Slovak Republic, China

Cometha Project, Porto Marghera, Arundo donax and -
Italy agricultural residues
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Clariant - sunliquid cellulosic agricultural residues such as 1,000 t

ethanol demo, Straubing wheat straw
94,640 m3
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Abengoa, Hugoton, Kansas mixture of agricultural waste,

non-feed energy crops and wood
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waste
POET-DSM Project Liberty, corn stover and cob 75,710 m3
Nevada, Lowa, US
DuPont cellulosic ethanol facility, corn stover 113,562 m3
Lowa, US

LS9 , Okeechobee Florida sweet sorghum syrup, Biomass 37,850 m3

hydrolysate
INEOS Bio , Vero Beach Florida municipal solid waste, 3,028 m3
commercial and industrial waste,
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agricultural waste

472

473 The carbon dioxide released during industrial activities can be used for culturing

474 cyanobacteria which are potential organisms for both GHG reduction and fermented biomass

475 enrichment. Application of enriched biomass to the soil is the cleaner method of disposing

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476 the waste generated during the lignocelulosic ethanol production process. Implementation of

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477 this integrated process is profitable to the industries as lignocellulosic biomass can be utilised

478 completely for the production of value added products.

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479 4. Conclusion

An integrated process for bioethanol and biomanure production has been developed through

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480 AN
481 the present study to valorize the pineapple leaf waste obtained from agriculture and industrial

482 sectors. Simultaneous saccharification and fermentation of the biomass was carried out to
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483 achieve significant ethanol production of 7.12 %(v/v) in 24 h with conversion efficiency of

484 75.53 % F. muscicola enriched residue with NPK ratio of 3.5:1:2 can be recommended as
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485 biomanure for sugarcane crop system. The results establish pineapple leaf waste as a potential
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486 substrate for bioethanol and biomanure production. The process undertaken for valorization

487 of the pineapple leaf waste is a viable process which not only yields valuable products in less
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488 time but also addresses the problems of energy crisis and environmental pollution.
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489 Conflict of interest

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490 The authors declare that they have no conflict of interest.

491 Acknowledgment

492 The authors gratefully acknowledge the financial assistance and help provided by the

493 Department of Biotechnology (DBT), New Delhi, to carry out this work under the grant

494 number is BT/PR15280/AGR/26/255/2011.

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664 Figure captions

665

666 Fig. 1. Schematic representation of integrated bioethanol and biomanure production from

667 pineapple leaf waste.

668 Fig. 2. Effect of a) substrate concentration b) incubation time c) temperature d) inoculum age

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669 and e) inoculum (containing yeast) volume on ethanol production from pineapple leaf waste

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670 through simultaneous saccharification and fermentation process.

671 Fig. 3. Ethanol and residual reducing sugar profile of pineapple leaf waste during

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672 simultaneous saccharification and fermentation.

673 Fig. 4. Scanning electron microscope images of (a) delignified and (b) fermented pineapple

674 leaf waste.

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675 Fig. 5. Increase in nutrient concentration of fermented pineapple leaf waste after enrichment
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676 with F. muscicola

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Highlights

• Pineapple leaf waste was utilized for bioethanol and biomanure production
• Ethanol concentration of 7.12 %(v/v) was obtained in 24 h from delignified
biomass
• NPK enrichment of residual biomass was carried out using cyanobacteria in 4-5
weeks

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• Soil application of NPK enriched lignocellulosics accomplish soil to soil approach
• The process implemented in the study and its outcome have industrial significance

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