Bioresource Technology https://doi.org/10.1016/j.biortech.2019.121633 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com Short Communication Evaluation of a wet processing strategy for mixed phumdi biomass conversion to bioethanol Anoop Puthiyamadam ⁠a⁠, ⁠2, Velayudhanpillai Prasannakumari Adarsh ⁠a⁠, ⁠2, Kiran Kumar Mallapureddy ⁠a, Anil Mathew ⁠a⁠, ⁠1, Jitendra Kumar ⁠b, Sudhakara Reddy Yenumala ⁠b, Thallada Bhaskar ⁠b, Ummalyama Sabeela Beevi ⁠c, Dinabandhu Sahoo ⁠c, Rajeev K Sukumaran ⁠a⁠, ⁎⁠ a Biofuels and Biorefineries Section, Microbial Processes and Technology Division (MPTD), CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram 695019,India b Biomass Conversion Area (BCA), Materials Resource Efficiency Division (MRED), CSIR-Indian Institute of Petroleum (IIP), of Scientific and Innovative Research (AcSIR), Dehradun 248005, India c Institute of Bioresources and Sustainable Development, Takyelpat, Imphal 795001, India ARTICLE INFO ABSTRACT Keywords: Biorefineries typically use dry feedstock due to technical and logistic issues, but in unique cases where climatic Wet processing conditions are unfavorable and where the biomass has to be processed without a holding time, wet processing Biomass might be advantageous. The present study evaluated the possibility of using the fresh (non-dried) mixed biomass Phumdi harvested from Phumdis; which are floating vegetation unique to Loktak lake in Manipur, India, for bioethanol Loktak production. Pretreatment with dilute alkali (1.5% at 120 °C for 60 min) resulted in 36% lignin removal and an Bioethanol enhancement of cellulose content to 48% from 37%, and enzymatic hydrolysis released 25 g/L glucose. Fermen- Lignocellulose tation of the hydrolysates was highly efficient at 95% attained in 36 h and 80% in just 12 h. The newwetpro- cessing strategy could help in value addition of mixed phumdi biomass. 1. Introduction again has led to a disposal problem and a solution to the perpetual issue would be value addition the biomass so that it is converted quickly to a Loktak Lake in Manipur, India is home to a unique ecosystem called product in demand, so that there is local income generation along with the Phumdis which are floating mats of vegetation, soil and organic disposal of the otherwise waste biomass (Singh, 2015). matter under various stages of decomposition (Singh and Khundrakpam, We had previously described the potential of using biomass from 2011). Anthropogenic activities have led to increased organic content selected invasive species like Paragrass (Brachairia mutica) and wild and nutrient levels- especially phosphorous and nitrogen in the lake, –rice-grass (Zizania latifolia) from the Loktak lake for production of resulting in rapid proliferation of invasive species like paragrass bioethanol (Sahoo et al., 2018, 2017). These studies involved use of a (Brachairia mutica) in the phumdis (Sahoo et al., 2017). Also there is single variety of plant in dry form as the feedstock for bioethanol pro- an increased proliferation of aquatic weeds like Water hyacinth and duction. However, the harvesting of phumdis performed mechanically Salvinia. The changes in vegetation has significantly affected the health yields a mixed vegetation with high water content which is practically of phumdis, leading to rapid disintegration and sinking, further adding difficult remove. A strategy that uses wet (fresh/non-dried) biomass as to the nutrient levels in the lake and increased proliferation of the in- feedstock for conversion to bioethanol or any other value added product vasive plant varieties. The annual biomass production of Loktak lake would be more beneficial in this scenario. Also, the use of an unsegre- is estimated to be ∼2 million tons (Singh, 2015). The lake contributes gated (mixed) biomass as obtained from the harvesting would be highly to the livelihood of a significant population and the local govern- desirable as it is practically difficult to segregate large quantities of wet ment is spending significant amount of money and effort to period- biomass. ically remove the overgrown floating vegetation from the lake. This While is generally accepted that dry biomass possesses several ad- vantages with respect to preventing damage while storing, economics ⁎ Corresponding author at: Microbial Processes and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Thiruvananthapuram 695 019, India. Email address: [email protected] (R.K. Sukumaran) 1 Present Address: Department of Chemical Engineering, Hanyang University, Seoul, Republic of Korea. 2 Both contributed equally to the work. https://doi.org/10.1016/j.biortech.2019.121633 Received 14 April 2019; Received in revised form 7 June 2019; Accepted 9 June 2019 Available online 12 June 2019 0960-8524/ © 2019. A. Puthiyamadam et al. Bioresource Technology xxx (xxxx) xxx-xxx Table 1 Changes in mixed phumdi biomass composition on pretreatment. Temp (°C) Acid/Alkali Conc. (%w/w) Acid pretreated Biomass (%) Presoaked and Alkali Pretreated Biomass (%) Cellulose Hemi-cellulose Lignin Cellulose Hemi-cellulose Lignin 80 0.5 38.7 ± 0.9 19.6 ± 1.1 22.3 ± 1.5 39.4 ± 0.0 22.9 24.0 ± 0.4 1.0 37.9 ± 1.5 18.4 ± 3.3 24.1 ± 0.7 41.0 ± 0.5 29.3 21.4 ± 0.4 1.5 38.5 ± 0.5 18.1 ± 4.0 25.1 ± 0.5 45.5 ± 10.1 25.5 18.6 ± 0.5 100 0.5 37.9 12.6 ± 0.2 24.9 ± 0.1 41.7 ± 0.2 24.9 ± 3.6 21.7 ± 0.9 1.0 40.8 13.8 26.3 ± 0.3 44.0 ± 1.7 26.8 ± 7.0 19.4 ± 1.6 1.5 44.0 12.1 ± 0.3 25.1 ± 0.7 46.2 ± 0.6 26.2 ± 1.4 17.6 ± 0.2 120 0.5 41.1 ± 4.5 20.5 ± 1.4 25.8 ± 0.2 41.5 ± 2.1 26.0 ± 0.3 19.5 ± 4.4 1.0 45.3 ± 0.7 17.5 ± 0.2 25.3 ± 1.9 46.1 ± 0.8 23.2 ± 0.9 17.7 ± 0.2 1.5 48.9 ± 2.0 13.6 ± 0.9 26.4 ± 0.3 48.1 ± 0.5 22.3 ± 0.3 16.0 ± 0.1 Untreated biomass composition (%): Cellulose −36.7 ± 0.9, Hemicellulose – 22.6 ± 1.7, Lignin – 25 ± 0.8. Fig. 1. Comparison of the composition of alkali pretreated samples processed with or without pre-soaking. Fig. 2. Sugar yields from enzymatic hydrolysis of differently pretreated MPB. Table 2 Sugar yields on hydrolysis of acid or alkali pretreated MPB. of moisture. Weight, moisture content and bulk densities were measured as per standard protocols. Before pretreatment, the MPD was chopped Glucose Released for Alkali and wet ground in a food blender. The slurry obtained was analyzed for Temp Glucose Released for Acid presoaked and Pretreated Sample dry solids content and used directly as feedstock for pretreatment. (°C) Pretreated Sample (mg/ml) (mg/ml) 24 h 48 h 24 h 48 h 2.2. Pretreatment 0.5 80 3.2 ± 0.60 4.3 ± 0.01 8.6 ± 0.60 12.0 ± 0.50 Dilute acid (H SO and alkaline (NaOH) pretreatments were car- 100 3.6 ± 0.30 5.3 ± 0.30 13.4 ± 1.20 13.3 ± 1.00 ⁠2 ⁠4) 120 5.7 ± 1.00 7.8 ± 0.90 14.3 ± 1.49 17.7 ± 2.16 ried out using modified methods previously optimized at CSIR-NIIST 1.0 80 4.3 ± 0.10 5.7 ± 0.40 11.0 ± 0.10 13.4 ± 3.00 for other feedstock (Kuttiraja et al, 2013; Christopher et al, 2017). Op- 100 5.81 ± 1.00 5.9 ± 0.30 16.5 ± 1.80 17.6 ± 0.80 timization of pretreatment was carried out by varying the acid/alkali 120 11.5 ± 1.00 15.4 ± 1.00 13.1 ± 1.40 19.8 ± 2.44 loading from 0.5 to 1.5% (w/w) at three different temperatures – 80 °C, 1.5 80 4.9 ± 1.00 7.2 ± 0.20 11.0 ± 2.00 12.9 ± 0.70 100 8.4 ± 1.00 11.7 ± 0.80 19.9 ± 4.20 15.2 ± 1.10 100 °C and 120 °C. Biomass loading was constant at 15% w/w. After pre- 120 14.6 19.3 20.1 ± 2.14 25.1 ± 2.00 treatment, the slurry was adjusted to a pH of 6–6.5 and filtered using a nylon sieve. The pretreated biomass was either used directly for enzy- matic hydrolysis or air dried and used later. For evaluation of presoak- of transportation/logistics and handling (Ewanick and Bura, 2011), the ing as a pre-pretreatment strategy, fresh MPD was soaked in 0.25% w/ context of harvested phumdi biomass presents a different challenge and v NaOH solution overnight followed by pretreatment at different alkali it seems that wet processing may be of advantage in this unique con- loadings and temperature as above. text. As could be expected, there is very less information on wet/fresh processing of lignocellulosic biomass for bioethanol production due to 2.3. Hydrolysis obvious reasons. In this study, a wet processing strategy was evaluated for processing of the mixed wet phumdi biomass for bioethanol produc- Hydrolysis was carried out in 100 ml screw-capped conical flasks at tion. 50 °C and at 200 rpm agitation in a water bath shaker. The total reac- tion volume was kept constant at 20 ml, and the biomass loading was 2. Materials and methods 10% (w/w). Enzymatic hydrolysis was carried out using a commercial acid cellulase (Zytex India Pvt. Ltd, Mumbai, India), used at a loading 2.1. Biomass collection and properties of 15 FPU/g. The commercial acid cellulase had a total cellulase ac- tivity of 75.7 FPUs/ml, endoglucanase (carboxy methyl cellulase/CM- Mixed phumdi biomass (MPD) was collected fresh from Loktak lake Case) activity of 2003 IU/ml, xylanase activity of 889 IU/ml, beta –glu in Manipur, India and transported to laboratory ensuring minima loss 2 A.