Platform Study on the Development of a Nondetoxified Rice Straw

Subscriber access provided by READING UNIV

Article

A platform study on development of a non-detoxified rice straw hydrolysate to its application in lipid production from Mortierella alpina Batul Diwan, Piyush Parkhey, and Pratima Gupta ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03530 • Publication Date (Web): 13 Nov 2017 Downloaded from http://pubs.acs.org on November 14, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Sustainable Chemistry & Engineering is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Sustainable Chemistry & Engineering

A platform study on development of a non-detoxified rice straw hydrolysate to its application in lipid production from Mortierella alpina Batul Diwana, Piyush Parkheyb,c, Pratima Guptaa,* (*)-Corresponding author Author’s affiliation: a. Department of Biotechnology, National Institute of Technology, Raipur, India b. BEES lab, EEFF Division, CSIR IICT, Hyderabad, India c. Present address: Institute of Biotechnology, Amity University Raipur, India

Alternate short title: Fermentable non-detoxified rice straw hydrolysate for single cell oil (SCO) production

Corresponding author: Dr. Pratima Gupta Assistant Professor Department of Biotechnology, National Institute of Technology, GE Road, Raipur, C.G. 492010 India Email: [email protected], [email protected] Ph No.:7223885552 First author: Batul Diwan Research Scholar Department of Biotechnology, National Institute of Technology, GE Road, Raipur, C.G. 492010, India Email: [email protected] Ph No.: 9893146638 Second author Piyush Parkhey Assistant Professor Institute of Biotechnology, Amity University Raipur, C.G. India Email: [email protected] Ph No.: 9685852321

1 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 30

ABSTRACT In the present work, a mild acid saccharification approach for rice (Oryza Sativa) straw was developed by Response Surface Optimization. A 23 central composite experimental design with variable factors as duration (minutes), mild acid concentration % (v/v) and minimum solid loading % (w/v) was chosen to optimize the hydrolysis process, fixing the operating temperature to a moderate minimum of 121°C. The study determined 5% solid loading, 0.75% acid concentration and 150 (min)-residence duration as optimum, for enhanced reducing sugar release. To compensate the reduced saccharification efficiency under mild operating conditions, a novel approach of multi-tier saccharification was implemented at optimal conditions of saccharification. HPLC analysis revealed the final reducing sugar yield of 0.76 g reducing sugar/gram straw (47.9g/l and 35g/l concentration in two consecutive saccharification cycles) and glucose, galactose, and xylose as major reducing sugars in hydrolysate. On assessing the hydrolysate as a fermentable substrate with and without detoxification for oleaginous strain Mortierella alpina, nondetoxified hydrolysate surpassed detoxified hydrolysate for growth support, sugar consumption, and lipid accumulation. HPLC analysis indicated the complete absence of furfurals and hydroxymethylfurfurals in the non-detoxified hydrolysate. The rice straw hydrolysate produced by multi-tier mild acid saccharification approach was thus suitable for microbial propagation without requiring detoxification suggesting it to be a potential fermentable substrate for microbial lipid production as well as for other prospective bioprocesses. Key words: Mild acid hydrolysis; Multi-tier Saccharification; Non-detoxified hydrolysate; Oleaginous Microorganism; Rice straw; Response surface methodology

INTRODUCTION Lignocelluloses with a total annual production of 150-170 x 109 tons1 are one of the most abundantly occurring plant biomass and promising source of renewable energy. In the past few decades, wide range 2 ACS Paragon Plus Environment

Page 3 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


of lignocellulosic agricultural byproducts have paved the way for cost efficient production of bio-based high value products. As an elemental constituent of lignocelluloses, cellulose has emerged as richest and most attractive renewable carbon reservoir. Almost one third to half of lignocellulosic biomass is constituted of cellulose, trapped under a complex network of hemicelluloses and lignin. While cellulose is a linear homopolymer of β-D glucosic monomers2 hemicelluloses, potentially the second largest biological carbon reservoir after celluloses, are branched heteropolymer of pentoses (xylose and arabinose) and some hexoses (mannose, glucose, galactose). Cellulose and hemicelluloses upon saccharification, can release abundant fermentable sugars which can be processed and converted into diverse high value microbial products like hydrogen, ethanol, propanol, citric acid, phytate, surfactants etc3-5. However, cellulose accessibility for digestion (enzymatic or chemical) is confronted by two major challenges. Firstly, the sugar monomers are entrapped in highly crystalline cellulosic crosslinked chains. Secondly, the celluloses are embedded in complex network of hemicelluloses and lignin. To overcome the structural limitations, a range of physico-chemical pretreatment strategies have been investigated6. In order to optimally utilize heterogeneous feedstocks to derive maximum sugars, appropriate combination of pretreatment and saccharification strategy becomes necessary. However, the pretreatment and enzymatic hydrolysis cumulatively raises the overall production costs. Moreover, the slow reaction rate, sensitivity and stability required during reaction, high cost and extended duration required for enzymatic hydrolysis further encourages the exploration of rapid, economic and efficient alternatives. Acid mediated hydrolysis emerges as a viable substitute for quicker and effective biomass saccharification. Usually sulphuric acid, hydrochloric acid, phosphoric acid, nitric acid, oxalic acid etc are used for hydrolysis, but sulphuric acid is reportedly more efficient7. Strong acids like sulphuric acid, hydrochloric acid etc. solely suffice the need of decrystallization and sugar hydrolysis. They break down the complex mesh of intra and interchain hydrogen bonds (reducing crystallinity) and hydrolyze the glycosidic backbone of cellulosic and hemicellulosic molecules (hydrolysis), releasing sugar moieties8. Although, 3 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 30

saccharification efficiency of acid hydrolysis has achieved remarkable figures9 higher acidic concentration raises numerous concerns like handling hazard, equipment corrosion, maintenance cost elevation, neutralization of generated hydrolysate and complexity of recycling. Moreover, higher the concentration of acid higher will be generation of toxic degradation products like furfurals, hydroxymethylfurfurals (HMF), formic acid, vanillin, syringaldehyde, acetic acid, acid soluble lignin etc10. Most of these compounds are microbial metabolic inhibitors, toxic to successive fermentation processes. The kinetics of acid hydrolysis is complex and higher operating temperature (around 200°C and above) has been quoted to be highly suitable for enhanced sugar release. Moreover hydrolysis requires high activation temperature, which is even higher than activation temperature required for sugar degradation11. Equation-1 clearly shows the reaction dynamics of acid hydrolysis at higher temperature.

-Equation 1

Therefore, with the increasing temperature, although the sugar yield increases but generation of degradation products rises even faster, as sugar decomposition reaction occurs at lower activation temperature compared to depolymerisation (equation-1)11. Several detoxification strategies have been employed lately10 to neutralize and remove the toxicants, but rising stack of treatment methods potentially increase the overall production economy. Also, application of higher temperature for prolonged duration makes the process rather more energy intensive. Hence, development of an acid hydrolysis approach operating at overall mild physico-chemical conditions becomes imperative. In light of the problem statement, the present work aims the development of an acid hydrolysis approach with appreciably mild operating conditions (low temperature, extremely low acid concentration) and 4 ACS Paragon Plus Environment

Page 5 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


reduced solid loading by optimizing the system through statistical approach of Response Surface Methodology (RSM). To compensate the reduced saccharification efficiency under mild operating conditions, a novel approach of multi-tier saccharification has been tested. The application of this hydrolysate was further studied with and without detoxification, as potential substrate for oleaginous strain Mortierella alpina.

EXPERIMENTAL METHODS Procurement, Processing and Pretreatment of Rice Straw Rice straw was collected from local fields of Raipur (Chhattisgarh), India (21.2324° N, 81.6158° E). The biomass was air dried, milled and ground to a size between 200 and 400 sieve mesh before subjecting to microwave assisted alkali pretreatment12. Hydrolysable cellulosic content available in raw and pretreated biomass was determined afterwards as per Updegraff method13 and

increase in availability of

hydrolysable cellulose was calculated in terms of percentage of available cellulose before and after pretreatment. Removal of lignin content from rice straw was also estimated14 so as to confirm that only cellulose and hemicelluloses remain in the biomass for further saccharification.

Structural analysis through XRD Alterations in crystallinity occurred at structural level in native rice straw by pretreatment and saccharification were examined through X-ray diffraction analysis. X-ray diffractometer (X’pert PRO, PANalytical) was set at 45KV, 30 mA; grade range of 10-30°(2θ) and a step size of 2° with Cu Kα radiation source ( λ=0.154 nm) was employed. The crystallinity index of each sample was calculated as per peak intensity method 15,using the formula in equation-2:

CrI = [(Icry- Iam)/ Icry] x 100

- Equation 2

Where: 5 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 30

CrI= Crystallinity index Icry = maximum peak intensity at 2θ = 22.2° Iam= minimum peak intensity corresponding to 2θ = 18.0°

Morphological analysis through SEM imaging The morphological modifications in native rice straw owing to pretreatment and subsequent rounds of hydrolysis were observed through Scanning Electron Microscope (SEM). Microscopic images of gold sputter coated- untreated, pretreated and subsequently saccharified rice straw fibrils were captured by SEM (ZEISS EVO 18) at 1000-1500 X magnification with 10 kV acceleration voltage.

Mild Acid Saccharification of Rice Straw Acid Mediated Hydrolysis Un-optimized System 10 % (w/v) pretreated rice straw was mixed with 1.5% (v/v) sulphuric acid, and the slurry thus obtained was autoclaved for 90 minutes at 121°C16.

Optimized System-Statistical experimental design and analysis Optimization of pretreated rice straw-acid hydrolysis was carried out by RSM. The defined preferred response was reducing sugar yield in mg/g rice straw as a measure of lignocellulosic depolymerisation efficiency of acid hydrolysis. A three factored central composite design (CCD) well suited for process optimization was adapted with fixed temperature 121°C. Residence duration (A), Solid Loading (B) and Acid concentration (C) were chosen as independent variables, at five levels (−2, −1, 0, 1 and 2). The objective of CCD was to study mutual interactions and develop a mathematical correlation amongst chosen independent 6 ACS Paragon Plus Environment

Page 7 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


parameters and sugar release. Data Analysis, experimental design and quadratic model building were performed via software Design Expert (Version 10, State-EaseInc., Minneapolis, MN, USA). All the experiments were conducted in triplicates and the mean were taken as response. For validating the observed response against the predicted one numerical optimization was implemented by setting the response to “maximum” when all parametric values were set “in range”. Table 1 and 2 explains the detailed statistical parameters and design matrix generated for experimental optimization. Multi-Tier Saccharification The experimental conditions which resulted in maximum reducing sugar recovery upon validation by numerical optimization were chosen for further rounds of saccharification. Residual biomass left after primary saccharification was subjected to secondary and tertiary saccharification under same set of experimental parameters.

Detoxification by overliming Rice straw hydrolysates obtained at optimal saccharification condition was stepwise detoxified through overliming10, 17 to obtain Detoxified Liquid hydrolysate (DLH) .

COD determination To quantitatively determine the oxidizable organic content in DLH and NDLH for estimating the reduction in organic matter (preferably toxic degradation products), chemical oxygen demand (COD) of hydrolysates were estimated before and after overliming by closed reflux titrimetric method18.

Assessment of hydrolysate as potential fermentation substrate



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 30

Hydrolysate obtained from optimized system was used as a substrate for Motierella alpina MTCC-6344 maintained in YPD (Yeast peptone dextrose) media. The fungal strain was pre-cultured in media composed of (g/l), Potassium-dihydrogen phosphate-2, Magnesium sulphate-0.5, Ammonium sulphate0.5, Yeast extract-0.5, dextrose- 8 at 25°C, 150 rpm for 48 hours. 10% (v/v) pre-inoculum was transferred to three production media each, formulated by replacing sugar sources in pre-culture media with DLH (M1) and NDLH (M2) and commercial glucose from Himedia (Positive Control- PC) as carbon sources respectively such that, to have equivalent initial carbon to nitrogen ratio of 50:1 by weight and cultivated at 25°C, 160 rpm for 9 days. Growth, biomass multiplication and sugar utilization in different media were examined by withdrawing a culture aliquot periodically (24 hours) for measuring OD550 and residual reducing sugar concentration by Dinitrosalicyclic acid (DNS) method16. To eliminate the possibility of false results on account of hydrolysate’s characteristic color, uninoculated M1, M2 and PC media were taken for blank measurements. Lipid extraction and estimation The production media M1, M2 and PC were centrifuged at 10,000 RPM for 10 minutes to harvest the cells. This was followed by drying at 50°C for 24-48 hrs, weighing and subsequent lipid extraction by Bligh and dyer protocol19. Lipid content was estimated gravimetrically, by repeated drying and weighing the extracted lipids until constant weight was achieved and stored in glass stoppered tubes.

Analysis of hydrolysate The HPLC analysis of rice straw hydrolysates, for quantifying various reducing sugars and estimating toxic furfurals and HMF, was conducted at BEES lab, EEFF division, CSIR-IICT Hyderabad. Reducing sugar analysis and furfural detection were performed using HPLC (Shimadzu LC20A) employing RI detector (RID20A; Shimadzu) and Rezex Monosaccharide (Phenomenex) column by injecting filtered sample of 20 µl with 0.22 µm porosity syringe filter. For elution, a flow rate of 0.3 mL/min was used in 8 ACS Paragon Plus Environment

Page 9 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


an isocratic method with water as the mobile phase and the column temperature was maintained constant at 80º C. Filtered sample (filtered with 0.22 µm porosity syringe filter) was injected. Standards for acids (SUPELCO) and alcohols (SIGMA) were used. Monosaccharide kit (SUPELCO, USA) was used as standards for quantitative estimation of reducing sugars.

RESULTS AND DISCUSSION Pretreatment of Rice Straw Removal of lignin from biomass prior to mild acid hydrolysis was essential in order to increase saccharification efficiency by acid hydrolysis. Hence, rice straw was subjected to microwave assisted alkali pretreatment. Hydrolysable cellulosic content 25.325 % (before pretreatment) in biomass was increased to 83.25% (after pretreatment). In our previous work same approach resulted in an increase in hydrolysable cellulosic content

from 35.8% to 82.08% which is quite comparable to obtained

increment in the present work12. Generally 25% (w/w) of the rice straw biomass is composed of lignin20, while in present case lignin removal was noted to be 25.3% (w/w) of the total biomass, suggesting that almost all the lignin matrix has been separated and removed from rice straw. It clearly indicates that pretreatment was efficient in terms of disrupting lignin and hemicellulosic casing, successfully making cellulosic fibrils accessible for saccharification.

Structural & morphological characterization Structural analysis by XRD Crystalline cellulosic fibers are surrounded by amorphous lignin and hemicellulosic mesh, which restricts the cellulose availability for further saccharification. Changes in crystallinity of rice straw fibrils brought about by pretreatment and saccharification were also examined by X-ray diffraction analysis. The X-ray diffractogram reported an increased crystallinity index from 51.6% before pretreatment to 56.1% after 9 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 30

pretreatment, implying significant removal of amorphous lignin and exposure of crystalline cellulose. Further analyzing the structural transformations in course of saccharification illustrated a peculiar pattern. The crystallinity index of pretreated straw first increased from 56.1 % to 64.33% after first round of saccharification. This increase probably indicates the presence of residual lignin amorphous traces, removal of which might have increased the crystallinity index further. However, crystallinity decreased from this point, and remained more or less same 60.75% and 59.80 % after 2nd and 3rd saccharification respectively. The decrease in crystallinity signifies depolymerization of exposed core cellulosic skeleton because of subsequent rounds of acid hydrolysis which led to gradual reduction in crystallinity index of rice straw. Surface analysis by SEM imaging Microscopic images of untreated, pretreated and subsequently saccharified rice straw fibrils (Figure 1(ae)) show that native rice straw is coarse and crumpled at peripheral level due to casing of amorphous lignin and hemicelluloses. Pretreated straw is smooth indicating the significant removal of amorphous layers, exposing the inner intact crystalline cellulosic fibrils. Figure 1c. shows a further improved exposure of core fibrils as compared to pretreated straw, which eventually appears more permeable and accessible. This could be presumably due to removal of leftover amorphous traces in straw, fitting to XRD results. With subsequent digestion the straw structure visibly seemed disrupted at structural level (figure 1d and e) suggesting the gradual breakdown of core crystalline cellulosic fibrils leading to structural disintegration. The SEM images are in sufficient alignment with XRD data confirming the gradual cellulosic depolymerization on subsequent hydrolysis, resulting in release of reducing sugar monomers.

Mild acid saccharification


Page 11 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In un-optimized acid hydrolysis the reducing sugar recovered was revealed to be 192 mg/g rice straw after 90 minutes of saccharification at 121°C with 10 % biomass load in 1.5% (v/v) sulphuric acid. To further improve the reducing sugar yield per gram straw, the process was optimized by RSM using CCD. Observed responses from experimental runs were analyzed in terms of obtained analysis of variance (ANOVA) and regression coefficients. Consequently a quadratic polynomial regression equation (Equation 3) was established, representing variable interaction profile and their effect on preferred response. Y [Reducing Sugar Yield (mg/g)] = +132.20 + 27.08*A - 22.95*B - 19.83*C - 18.79*AB – 35.15*C + 19.25*BC – 13.65*A2 -10.92*B2 – 53.49*C2

-Equation 3

ANOVA details in Table 3 validate the model equation and show that the CCD model is well fitted. F value of 7.18 implies that the model is significant. A “Prob>F” value less than 0.0500 indicates that model terms are significant and we obtained 0.0025 which is well in alignment suggesting the model to be significant. Further the “Adeq Precision” ratio greater than 4 is said to be desirable and the study deduced value of 9.977 an adequate signal, suggesting that model can be used to navigate design space. After a total of 20 experimental runs at different set of parametric combination the mutual interactions between variables were analyzed by 3D surface and 2D contour plots. All the three factors displayed enough interactions mutually but solid loading and acid percentage were comparatively more significant than residence duration suggested by their relatively high p value of 0.0375 and 0.0651 respectively Lower solid loading and higher hydrolysis duration (figure 2a) appeared suitable for higher reducing sugar release. Higher hydrolysis duration was found to be favorable at lower acid concentration as can be seen from figure 2b. Peculiarly at higher acid concentration, the variations in solid loading ratio did not increase reducing sugar release (figure 2c). With decreasing acid concentration- at maximum solid load, 11 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 30

sugar release increased up to a point and then decreased sharply, while at minimum solid load reducing sugar release constantly increased. Apparently, acid concentration displayed strong co-relation with solid loading and residence time individually. All the interactive plots collectively signified that at lower acid concentration and minimum solid loading, higher residence duration (as per the selected parametric range) was most favorable for enhanced reducing sugar release. The conditions determined by RSM study, adequate for optimal sugar liberation were 5% solid loading, 0.75% acid concentration and 150 minutes residence time yielding 478 mg reducing sugar/g straw with substantial 50% (v/v) hydrolysate recovery. Optimal conditions predicted by model were quite comparable - 5% solid loading, 0.9414% acid concentration and 150 minutes of hydrolysis corresponding to a predicted sugar yield of 502.2 mg reducing sugar/g straw. For numerical optimization, the predicted optimal values were experimentally validated resulting in 499.2 mg reducing sugar / g straw. The obtained reducing sugar yield was sufficiently close to predicted value indicating the desirability of the model. Hike of around 2.5 fold was obtained in optimized system over un-optimized sugar yield displaying remarkable improvement in hydrolysis efficiency.

Multi-tier saccharification Kinetics of acid hydrolysis is very sensitive and no well defined parametric relationship exists for adequate sugar yield. Studies on acid hydrolysis with all the three important parameters (acid concentration, temperature and solid loading) in minimal range are very limited as working simultaneously on lower range of all the parameters happens to be unfavorable for sugar recovery. Higher temperature (> 160° C) preferable around 200°C are reportedly most suitable for mild acid hydrolysis as moderate temperature often results in reduced saccharification yield because of sugar degradation21. Since the focus was to device a safe and energy efficient process, moderate working temperature was 12 ACS Paragon Plus Environment

Page 13 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


chosen. Alternatively, if working at temperature lower than 160° C in batch mode, a higher solid loading between 10-40% has been recommended for suitable sugar yield22. However, with high solid loading, the successive generation of acidic residual biomasses will also multiply, disposal of which raises numerous issues, primarily water expenditure for neutralization. Therefore, for maximizing biomass utilization and minimizing residual waste generation, minimum solid loading was chosen, and conditions for optimum de-polymerization and sugar release was explored. Dilute acid was preferred choice for saccharification, not only to reduce handling hazards but primarily to minimize the generation of degradation products at lower operating temperature. In our previous work we found considerable fraction of cellulose remained unhydrolysed after saccharification12. Therefore, to counterbalance the reduced digestion efficiency at selected parameters, multi-tier saccharification was adapted to exploit unhydrolysed cellulosic fraction. The residual straw biomass from optimal experimental run was subjected to further rounds of saccharification under optimal conditions. While primary saccharification already yielded 0.478 g reducing sugar/ g straw, secondary and tertiary hydrolysis of residual biomass revealed 0.29 g and 0.16 g reducing sugar liberation/gram straw with volumetric recovery of 42% and 40% respectively. Glucose, galactose and xylose were the major reducing sugars detected in hydrolysate (Table-4). Sharp decrease in saccharification yield was observed after 2nd and 3rd hydrolysis certainly because of subsequent decline in cellulosic fraction with consecutive rounds of saccharification. Hence saccharification up to two rounds was visibly efficient yielding a total of 0.76 g reducing sugar/g straw (47.9 g/l and 35g/l reducing sugar concentration in first two saccharification cycle respectively) with collective volumetric recovery of 46% (v/v). Thus the third round can be omitted to retain the process efficiency. This would also be considerable in terms of process economy as up to two saccharification sugar concentrations were significant enough to support any high sugar demanding fermentation process, which would appreciably compensate the energy input and investment of time and acid resource. Apart from parametric conditions, the sugar yield also depends on 13 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 30

source of lignocellulosic materials utilized for saccharification. From sugar cane baggase Dussán et al attained substantial 22.74 g/L sugar recovery at higher parametric range of - solid loading 12.5 % (w/v), acid concentration 2 % (w/v) and temperature 155°C23. While a comparatively low monosaccharide release of 309 mg/g was obtained with a lower solid loading of 6.6% but higher hydrochloric acid concentration of 5%

24

. In case of rice straw as feedstock Huang et al achieved good sugar recovery of

35.2 g/L corresponding to 0.352 g/g yield but the result achieved was again at higher solid loading of 10% (w/v) with 1.5% concentrated acid 25. These reports validate the strong impact of not only substrate employed but also operating conditions (solid loading, acid concentration and temperature) on sugar recovery even from same feedstock. Recently Kshirsagar et al reported sugar recovery of 359 mg sugar/gram rice straw by enzymatic saccharification26. It is interesting to note that enzymatic saccharification which is comparatively better in terms of efficiency to mild acid hydrolysis employed in our case, also reported lower yield compared to the present work.

Organic matter and furfurals removal Overliming is principally a combination of calcium hydroxide treatment of hydrolysate for increasing pH at higher temperature in order to precipitate out dissolved toxicants with calcium hydroxide as gypsum. It has emerged as a promising strategy for detoxification of lignocellulosic hydrolysates. It essentially removes furfurals, phenolics and organic compounds like acetic acid with some loss of sugars from hydrolysate17. In present work optimized rice straw hydrolysates (pool of hydrolysates from first two cycles) were overlimed as per described earlier and COD was estimated to analyze the organic matter reduction. Net 42.30 % COD reduction from an initial value of 2878.72 mg/l in NDLH to 1660.8 mg/l in DLH was observed after overliming. At higher pH (> 7.5) more than 50% furan (Furfurals + HMF) reduction has been reported previously 27 in considerable alignment to reduction percentage obtained in the present study. However, HPLC analysis of detoxified and non-detoxified hydrolysate revealed total absence of furfurals and HMF suggesting that they were not generated during hydrolysis. Moreover some 14 ACS Paragon Plus Environment

Page 15 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


loss of monosaccharide content was also seen (Table-4) in the hydrolysate during overliming. Therefore, the sharp decline in organic matter in DLH was due to reducing sugar loss and reduction in other inhibitory organic compounds generated during saccharification like predominant-phenolics and acetic acid and minor- ferulic acid, syringaldehyde etc28.

Hydrolysate as potential fermentation substrate The OD550 and corresponding cell count in M1, M2 and PC were measured for analyzing variation in growth and cell proliferation trends. Figure 3 and 4 graphically compares the change in growth (with respect to OD550) and reducing sugar utilization of M. alpina periodically cultured in three media. In an un-optimized lipid production setup, synthetic sugar (glucose), DLH and NDLH carbon sources resulted in a total lipid content of 20.5%, 26.6% and 40.23% of M.alpina CDW in PC, M1 and M2 media respectively (Table 5). Growth of M.alpina in three environments was almost similar till 24 hours. Afterwards a sharp increase in biomass multiplication was observed in DLH and NDLH media contrary to PC, which achieved stagnancy after 48 hours. The biomass multiplication in NDLH exceeded DLH marginally during final two days of fermentation (Figure 3). The cells in PC had least sugar utilization in alignment with lower microbial proliferation observed (figure 3). However, significantly higher sugar consumption occurred in DLH and NDLH which further exceeded in NDLH over the course of fermentation (figure 4). It is noteworthy that saccharified rice straw both as DLH and NDLH showed better growth support compared to synthetic glucose, which is most readily utilizable and fermentable source, and surprisingly crude hydrolysate (NDLH) out matched even detoxified in terms of sugar consumption, biomass accumulation and lipid content. The pattern indicates that the carbon source from M2 (NDLH) is probably been more utilized by the fungi for metabolic conversion to lipids rather than just for biomass multiplication, suggestive of the fact that M.alpina is utilizing NDLH as both growth and fermentative substrate. In contemporary study yeast Trichosporon fermentans accumulated 40.1% of its 15 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 30

CDW as lipids on sulphuric acid treated rice straw hydrolysate, similar to present case, but in detoxified hydrolysate, as raw hydrolysate posed inhibitory effects and yeast displayed poor lipid content25. Contrastingly the present work reports almost 2 folds improvement in lipid content in NDLH over DLH in M.alpina in an unoptimized production setup. Despite of range of biomass utilized for generating fermentable substrate, detoxification was found obligatory for supporting microbial growth, sugar utilization and lipid production in majority of mainstream investigations25, 29. But here it was interesting to note that in spite of considerable reduction in organic content in DLH, NDLH supported higher microbial growth, and sugar uptake for lipid accumulation. This might be due to the presence of other reducing sugars in hydrolysate based media unlike PC (comprising of only glucose), which might be readily utilized by the fungi. Furthermore, higher and variant sugar concentration in NDLH compared to DLH (as detoxification lead to marginal reducing sugar loss) could also be accounted for higher proliferation in raw hydrolysate. The dramatic hike in lipid content was also contributed by higher reducing sugar consumption from NDLH during final days of fermentation compared to DLH when the biomass accumulation was nearly same in both. This indicated that the additional sugar consumed was converted into lipids. Higher lipid content in M.alpina cultured in NLDH compared to DLH can be due to various factors contributing synergistically to result in higher accumulation. Mild saccharification conditions could be one of the major reasons as absence of major microbial toxicants-furfural and HMF allowed uninterrupted microbial growth and lipid accumulation. Mild conditions might have also led to reduced generation of other fermentative inhibitors, whose low concentrations might be tolerable to microorganisms via innate defense machinery or adaptation by means of suitable enzymes and coenzymes10. Also the large inoculum size of seed culture might have presumably compensated the inhibitor load in media. The other logical explanation could be the reason that some of the auxiliary metabolites generated during acid hydrolysis might be serving as growth stimulators, or probable substrate itself30 present only in NDLH as detoxification led to their 16 ACS Paragon Plus Environment

Page 17 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


removal from DLH. An investigation recently confirmed this, where acid degradation products like acetic acid, vanillin have been surprisingly reported as probable growth inducers31. Recently a group of investigators also observed this anomalous behavior of higher growth and accumulation in non-detoxified hydrolysate in this particular genus of mold30. The probable reason behind this is consumption of acetic acid (a constituent in non-detoxified hydrolysate) which is said to be totally removed during detoxification28 and hence was only present in NDLH. It reportedly contributes not only in cell growth but also in lipid accumulation32 as acetic acid might be serving as a substrate for intracellular Acetyl CoA formation - a building block for lipid synthesis. To evaluate whether the observed tendency was because of this particular species and restricted to it only, widely known oleaginous yeast Lipomyces starkeyi was also tested under similar conditions. It was found that it also exhibited similar behavior and showed higher growth in NDLH (data not shown) compared to DLH and PC. Since such behavior was not restricted to this mold (M.alpina), it confirms that it is the make of NDLH and not the organism, deciding the fermentability of hydrolysate. In a study Cryptococcus curvatus was interestingly found to exhibit higher lipid content 37.9% CDW when acetate was supplemented with sugar in media, in contrast with lipid content of 33.1% CDW with sole glucose, supporting the findings of present study

33

..

Literature also provides supporting evidences of some oleaginous species to be not only tolerant to NDLH, but also exhibiting notable oleagenicity. C.curvatus on non-detoxified wheat straw acid hydrolysate (10% w/v solid loading and 2% v/v acid load) accumulated 33.5% of CDW as lipids, while another species of genus Mortierella- M. isabellina accumulated 34.4% of its CDW as lipids on nondetoxified acid pretreated corn stover hydrolysate30 comparable to our observations. Lipid production on different lignocellulosic hydrolysates has been explored with several other oleaginous species. A survey of lipid production on switch grass hydrolysate- prepared with higher solid loading (20% w/v) and sulphuric acid concentration (0,936% v/v) compared to present study, by different yeasts of oleaginous Yarrowia clade, revealed highest lipid content of 37.9 % in Yarrowia lipolytica34. In food waste acid 17 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

hydrolysate (1.5% v/v sulphuric acid and 1:2 solid to liquid loading ratio) Rhodosporidium toruloides accumulated 22.9% CDW as lipids35. With wheat straw as lignocellulosic feedstock C. curvatus endured both detoxified and non-detoxified hydrolysate and exhibited higher lipid content in NDLH 33.5% against 22.1% in DLH analogous to current observations17. On diluted raw corn cob hydrolysate Cryptococcus sp. SM5S05 accumulated a significant 60.2% of its CDW as lipids36. While for oleaginous species Y. lipolytica Po1g and Trichosporon fermentans detoxification appeared obligatory in case of sugar cane baggase hydrolysate as a fermentation substrate. In un-optimized setup these yeasts accumulated 58.5% and 39.9% of CDW as lipids respectively37-38. Therefore degree and severity of degradation product’s impact on microbial growth and fermentation cannot be generalized as it varies with microorganism in question, lignocellulosic substrate in use, conditions of saccharification (deciding the concentration of degradation products), desired end product and its biosynthesis in microorganism. The present work reports competitive lipid contents compared to the discussed contemporary reports, with a Non-detoxified acid hydrolysate prepared at comparatively mild acid concentration and lower solid loads. Study suggests that such lignocellulosic NDLH can facilitate the economical large scale lipid production for achieving a potential SCO bio-refinery.

CONCLUSION An increase in 2.5 fold sugar recovery was achieved in optimized system over un-optimized hydrolysis. Multi-tier saccharification of the residual biomass enhanced the cumulative sugar recovery from rice straw to 0.76 g/gram with 46% (v/v) total volumetric recovery. Since furfurals and HMFs were absent on account of the mild hydrolysis conditions, NDLH came up as suitable fermentable substrate in terms of growth support, sugar utilization and lipid synthesis for oleaginous species M.alpina The three exceptional outcomes of the study were (1) Development of an effective mild acid hydrolysis approach safe, simply operable and environmentally restorative resulting in a fermentable non-detoxified 18 ACS Paragon Plus Environment

Page 19 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


hydrolysate (2) Sugar recovery was simultaneously compensated with multiple saccharification, ensuring maximum and resourceful utilization of limited biomass before disposal (3) Ease of rice straw saccharification and successful employment of crude (non-detoxified) hydrolysate for growth and lipid fermentation of a potent oleaginous fungi.

ACKNOWLEDGEMENTS The authors heartily acknowledge Dr. Venkata Mohan S and Mr. Om Prakash from BEES lab, EEFF division CSIR IICT Hyderabad and Dr. Manoj Chopkar, Mr. Suresh Dua and Mr. Viond Kumar Ingole Department of Metallurgical Engineering, NIT-Raipur for their generous support offered in conducting HPLC analysis and XRD and SEM analysis respectively. Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors Conflict of Interest: All the authors mutually declare that there is no conflict of interest. This article does not contain any studies with human participants or animals performed by any of the authors. SYNOPSIS: The work develops an energy efficient mild process for maximum utilization and optimum hydrolysis of lignocellulosic rice straw with minimum residue generation. Study is progressive as the process altogether eliminates the need of entire detoxification step for preparation of a fermentable hydrolysate for microbial bioprocess.

REFERENCES 1. Loqué, D.; Scheller, H. V.; Pauly, M., Engineering of plant cell walls for enhanced biofuel production. Current opinion in plant biology 2015, 25, 151-161.DOI: 10.1016/j.pbi.2015.05.018. 2. Da Vinha, F. N. M.; Gravina-Oliveira, M. P.; Franco, M. N.; Macrae, A.; da Silva Bon, E. P.; Nascimento, R. P.; Coelho, R. R. R., Cellulase production by Streptomyces viridobrunneus SCPE-09 using lignocellulosic 19 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 30

biomass as inducer substrate. Applied biochemistry and biotechnology 2011, 164 (3), 256-267.DOI: 10.1007/s12010-010-9132-8. 3. Cheng, K.-K.; Cai, B.-Y.; Zhang, J.-A.; Ling, H.-Z.; Zhou, Y.-J.; Ge, J.-P.; Xu, J.-M., Sugarcane bagasse hemicellulose hydrolysate for ethanol production by acid recovery process. Biochem Eng J 2008, 38 (1), 105109.DOI: 10.1016/j.bej.2007.07.012. 4. Papanikolaou, S.; Fakas, S.; Fick, M.; Chevalot, I.; Galiotou-Panayotou, M.; Komaitis, M.; Marc, I.; Aggelis, G., Biotechnological valorisation of raw glycerol discharged after bio-diesel (fatty acid methyl esters) manufacturing process: production of 1, 3-propanediol, citric acid and single cell oil. Biomass Bioenergy 2008, 32 (1), 60-71.DOI: 10.1016/j.biombioe.2007.06.007. 5. Gupta, P.; Parkhey, P., Design of a single chambered microbial electrolytic cell reactor for production of biohydrogen from rice straw hydrolysate. Biotechnol lett 2015, 37 (6), 1213-1219.DOI: 10.1007/s10529-0151780-x. 6. Kumar, P.; Barrett, D. M.; Delwiche, M. J.; Stroeve, P., Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuel production. Ind Eng Chem Res 2009, 48 (8), 37133729.DOI: 10.1021/ie801542g. 7. Canilha, L.; Chandel, A. K.; Suzane dos Santos Milessi, T.; Antunes, F. A. F.; Luiz da Costa Freitas, W.; das Graças Almeida Felipe, M.; da Silva, S. S., Bioconversion of sugarcane biomass into ethanol: an overview about composition, pretreatment methods, detoxification of hydrolysates, enzymatic saccharification, and ethanol fermentation. Journal of Biomedicine and Biotechnology 2012, 2012, 1-15.DOI:10.1155/2012/989572. 8. Xiang, Q.; Lee, Y.; Pettersson, P. O.; Torget, R. W., Heterogeneous aspects of acid hydrolysis of αcellulose. In Biotechnology for Fuels and Chemicals, Walt, D. R., Ed. Springer: 2003; Vol. 107, pp 505-514. DOI: 10.1007/978-1-4612-0057-4_42. 9. Farina, G.; Barrier, J.; Forsythe, M., Fuel alcohol production from agricultural lignocellulosic feedstocks. Energy sources 1988, 10 (4), 231-237.DOI: 10.1080/00908318808908931. 10. Chandel, A. K.; Singh, O. V.; da Silva, S. S., Detoxification of lignocellulosic hydrolysates for improved bioethanol production. In Biofuel Production-Recent Developments and Prospects, Bernardes, M. A. d. S., Ed. INTECH Open Access Publisher: 2011; pp 225-246. 11. Lee, Y.; Iyer, P.; Torget, R. W., Dilute-acid hydrolysis of lignocellulosic biomass. In Recent progress in bioconversion of lignocellulosics, Th., S., Ed. Springer: 1999; Vol. 65, pp 93-115.DOI: 10.1007/3-540-49194-5_5. 12. Gupta, P.; Parkhey, P., A two-step process for efficient enzymatic saccharification of rice straw. Bioresour technol 2014, 173, 207-215.DOI: 10.1016/j.biortech.2014.09.101. 13. Updegraff, D. M., Semimicro determination of cellulose inbiological materials. Analytical biochemistry 1969, 32 (3), 420-424.DOI: 10.1016/S0003-2697(69)80009-6.14.Mishra, M.; Thakur, I. S., Isolation and characterization of alkalotolerant bacteria and optimization of process parameters for decolorization and detoxification of pulp and paper mill effluent by Taguchi approach. Biodegradation 2010, 21 (6), 967-978.DOI: 10.1007/s10532-010-9356-x. 15. Segal, L.; Creely, J.; Martin, A.; Conrad, C., An empirical method for estimating the degree of crystallinity of native cellulose using the X-ray diffractometer. Textile Research Journal 1959, 29 (10), 786794.DOI: 10.1177/004051755902901003. 16. Gao, M.; Xu, F.; Li, S.; Ji, X.; Chen, S.; Zhang, D., Effect of SC-CO 2 pretreatment in increasing rice straw biomass conversion. Biosystems engineering 2010, 106 (4), 470-475.DOI: 10.1016/j.biosystemseng.2010.05.011. 17. Yu, X.; Zheng, Y.; Dorgan, K. M.; Chen, S., Oil production by oleaginous yeasts using the hydrolysate from pretreatment of wheat straw with dilute sulfuric acid. Bioresour technol 2011, 102 (10), 6134-6140.DOI: 10.1016/j.biortech.2011.02.081. 18. Yadu, A.; Sahariah, B. P.; Anandkumar, J., Studies on Biological Degradation of 4-Bromophenol Using Anaerobic, Anoxic and Aerobic Bioreactors. Journal of Modern Chemistry & Chemical Technology 2016, 7 (1), 37-41. 19. Bligh, E. G.; Dyer, W. J., A rapid method of total lipid extraction and purification. Canadian journal of biochemistry and physiology 1959, 37 (8), 911-917.DOI: 10.1139/y59-099. 20 ACS Paragon Plus Environment

Page 21 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


20. Binod, P.; Sindhu, R.; Singhania, R. R.; Vikram, S.; Devi, L.; Nagalakshmi, S.; Kurien, N.; Sukumaran, R. K.; Pandey, A., Bioethanol production from rice straw: an overview. Bioresource technology 2010, 101 (13), 4767-4774.DOI: 10.1016/j.biortech.2009.10.079.21. Sun, Y.; Cheng, J., Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource technology 2002, 83 (1), 1-11.DOI: 10.1016/S09608524(01)00212-7. 22. Esteghlalian, A.; Hashimoto, A. G.; Fenske, J. J.; Penner, M. H., Modeling and optimization of the dilutesulfuric-acid pretreatment of corn stover, poplar and switchgrass. Bioresource technology 1997, 59 (2), 129136.DOI: 10.1016/S0960-8524(97)81606-9. 23. Dussan, K. J.; Silva, D.; Moraes, E.; Arruda, P. V.; Felipe, M., Dilute-acid hydrolysis of cellulose to glucose from sugarcane bagasse. Chemical Engineering Transaction 2014, 38, 433-438. DOI: 10.3303/CET1438073. 24. Du Toit, P.; Olivier, S.; Van Biljon, P., Sugar cane bagasse as a possible source of fermentable carbohydrates. I. Characterization of bagasse with regard to monosaccharide, hemicellulose, and amino acid composition. Biotechnology and bioengineering 1984, 26 (9), 1071-1078.DOI: 10.1002/bit.260260909. 25. Huang, C.; Zong, M.-h.; Wu, H.; Liu, Q.-p., Microbial oil production from rice straw hydrolysate by Trichosporon fermentans. Bioresour technol 2009, 100 (19), 4535-4538.DOI: 10.1016/j.biortech.2009.04.022. 26. Kshirsagar, S. D.; Waghmare, P. R.; Loni, P. C.; Patil, S. A.; Govindwar, S. P., Dilute acid pretreatment of rice straw, structural characterization and optimization of enzymatic hydrolysis conditions by response surface methodology. RSC Advances 2015, 5 (58), 46525-46533.DOI: 10.1039/C5RA04430H. 27. Martinez, A.; Rodriguez, M. E.; York, S. W.; Preston, J. F.; Ingram, L. O., Effects of Ca (OH) 2 treatments (overliming) on the composition and toxicity of bagasse hemicellulose hydrolysates. Biotechnology and bioengineering 2000, 69 (5), 526-536. DOI: 10.1016/j.biortech.2006.07.047. 28. Deng, L.-H.; Tang, Y.; Liu, Y., Detoxification of corncob acid hydrolysate with SAA pretreatment and xylitol production by immobilized Candida tropicalis. The Scientific World Journal 2014, 2014, 1-11.DOI: 10.1155/2014/214632. 29. Wei, Z.; Zeng, G.; Huang, F.; Kosa, M.; Sun, Q.; Meng, X.; Huang, D.; Ragauskas, A. J., Microbial lipid production by oleaginous Rhodococci cultured in lignocellulosic autohydrolysates. Appl Microbiol Biotechnol 2015, 99 (17), 7369-7377.DOI: 10.1007/s00253-015-6752-5. 30. Ruan, Z.; Zanotti, M.; Wang, X.; Ducey, C.; Liu, Y., Evaluation of lipid accumulation from lignocellulosic sugars by Mortierella isabellina for biodiesel production. Bioresour technol 2012, 110, 198-205.DOI: 10.1016/j.biortech.2012.01.053. 31. Huang, C.; Wu, H.; Liu, Q.-p.; Li, Y.-y.; Zong, M.-h., Effects of aldehydes on the growth and lipid accumulation of oleaginous yeast Trichosporon fermentans. J Agric Food Chem 2011, 59 (9), 4606-4613.DOI: 10.1021/jf104320b. 32. Chen, X.; Li, Z.; Zhang, X.; Hu, F.; Ryu, D. D.; Bao, J., Screening of oleaginous yeast strains tolerant to lignocellulose degradation compounds. Applied biochemistry and biotechnology 2009, 159 (3), 591-604.DOI: 10.1007/s12010-008-8491-x. 33. Gong, Z.; Zhou, W.; Shen, H.; Yang, Z.; Wang, G.; Zuo, Z.; Hou, Y.; Zhao, Z. K., Co-fermentation of acetate and sugars facilitating microbial lipid production on acetate-rich biomass hydrolysates. Bioresource technology 2016, 207, 102-108.DOI: 10.1016/j.biortech.2016.01.122. 34. Quarterman, J.; Slininger, P. J.; Kurtzman, C. P.; Thompson, S. R.; Dien, B. S., A survey of yeast from the Yarrowia clade for lipid production in dilute acid pretreated lignocellulosic biomass hydrolysate. Applied Microbiology and Biotechnology 2017, 101 (8), 3319-3334.DOI: 10.1007/s00253-016-8062-y. 35. Zeng, Y.; Bian, D.; Xie, Y.; Jiang, X.; Li, X.; Li, P.; Zhang, Y.; Xie, T., Utilization of food waste hydrolysate for microbial lipid and protein production by Rhodosporidium toruloides Y2. Journal of Chemical Technology and Biotechnology 2017, 92 (3), 666-673.DOI: 10.1002/jctb.5049. 36. Chang, Y.-H.; Chang, K.-S.; Lee, C.-F.; Hsu, C.-L.; Huang, C.-W.; Jang, H.-D., Microbial lipid production by oleaginous yeast Cryptococcus sp. in the batch cultures using corncob hydrolysate as carbon source. Biomass and Bioenergy 2015, 72, 95-103.DOI: 10.1016/j.biombioe.2014.11.012. 21 ACS Paragon Plus Environment


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 30

37. Tsigie, Y. A.; Wang, C.-Y.; Truong, C.-T.; Ju, Y.-H., Lipid production from Yarrowia lipolytica Po1g grown in sugarcane bagasse hydrolysate. Bioresour technol 2011, 102 (19), 9216-9222.DOI: 10.1016/j.biortech.2011.06.047. 38. Huang, C.; Wu, H.; Li, R.-f.; Zong, M.-h., Improving lipid production from bagasse hydrolysate with Trichosporon fermentans by response surface methodology. New Biotechnology 2012, 29 (3), 372-378.DOI: 10.1016/j.nbt.2011.03.008.

Table 1: Parametric range and levels defined in 23 central composite experimental design for optimizing Reducing sugar release from rice straw Independent parameters Residence duration (min) Solid Loading %(w/v) Acid concentration%(v/v)

Code

Parametric range and levels -1 0 +1

-α/-2 (Axial min.) 29.3193 3.29552 0.325879

A B C

(Factorial min.) 60 5 0.75

(Central) 105 7.5 1.375

(Factorial max.) 150 10 2

+α/+2 (Axial max.) 180.681 11.7045 2.42612

Table 2: A central composite experimental design matrix developed by RSM for optimizing reducing sugar yield S.N

Run

Space type

A:Residence time (min)

B:Solid loading %(w/v)

C:Acid concentration %(v/v)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

2 4 6 7 17 1 9 10 12 3 8 15 20 11 16 5 13 14 18 19

Centre Axial Axial Axial Axial Factorial Factorial Factorial Factorial Factorial Factorial Factorial Factorial Axial Axial Centre Centre Centre Centre Centre

105 105 105 105 105 60 60 60 60 150 150 150 150 29.3193 180.681 105 105 105 105 105

7.5 7.5 11.7045 3.29552 7.5 10 5 5 10 5 10 10 5 7.5 7.5 7.5 7.5 7.5 7.5 7.5

1.375 2.42612 1.375 1.375 0.325879 0.75 0.75 2 2 2 2 0.75 0.75 1.375 1.375 1.375 1.375 1.375 1.375 1.375

Hydrolysate volume recovered from an initial 50 ml experimental mixture (ml) 32 31.5 22 40 27.5 22 34 33 23 38 30 24.5 25 29.5 35 31 32 31 31 32

Observed reducing sugar yield (mg/g biomass) 145.588 2.7216 74.661 172.765 3.901 3.226 5.954 5.856 9.499 2.128 1.2306 64.944 478 82.521 149.47 139.301 125 121.93 130.48 123.25

Table 3: ANOVA for regression based quadratic RSM model for mild acid hydrolysis of pretreated rice straw 22 ACS Paragon Plus Environment

Page 23 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Sum of

Mean

F

p-value

Source

Squares

df

Square

Value

Prob> F

Model

80851.66

9

8983.52

7.18

0.0025

A-Time

10014.08

1

10014.08

8.00

0.0179

B-Solid Loading

7195.59

1

7195.59

5.75

0.0375

C-Acid Percentage

5371.40

1

5371.40

4.29

0.0651

AB

2824.45

1

2824.45

2.26

0.1640

AC

9884.91

1

9884.91

7.90

0.0185

BC

2963.71

1

2963.71

2.37

0.1549

A

2

2686.44

1

2686.44

2.15

0.1737

B

2

1719.98

1

1719.98

1.37

0.2683

2

C

41238.16

1

41238.16

32.94

0.0002

Cor Total

93372.10

19

significant

Table 4: Reducing sugar composition of rice straw hydrolysates

Components

Reducing Sugars

Glucose Galactose Xylose

Unoptimized Hydrolysate (mg/ml) 12 24 2.4

Optimized condition (mg/ml) Detoxified nd rd Hydrolysate 3 1 2 (mg/ml) saccharification saccharification saccharification 26 18 14 18 20 15 4.6 6.5 1.9 2 1.7 1.9 st

TABLE 5: Biomass concentration, sugar consumption and lipid content profile of M.alpina on PC, M1 and M2 media Total Biomass (cells/ml) PC(Glucose) M1(DLH) M2 (NDLH)

1.41x106 5.217x106 5.427x106

Total Reducing sugar consumed (g/l) 12.24 18.8 32.59


Lipid produced (g/g CDW) 0.21 0.26 0.4


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 30

FIGURE CAPTIONS

Figure 1 (a-e): SEM micrographs of rice straw under 1000X magnification (a) Untreated straw (control), (b) Microwave assisted alkaline pretreated straw, (c) Straw after primary saccharification, (d) Straw after secondary saccharification, (e) Straw after tertiary saccharification

Figure 2(a-c): Response 3D surface plots for optimization of Rice straw mild acid hydrolysis (a) Interaction effects of hydrolysis duration and solid loading at fixed acid concentration (b) Interaction effects of hydrolysis duration and acid percentage at fixed solid loading (c) Interaction effects of solid loading and acid percentage at fixed hydrolysis duration

Figure 3: OD550 of M.alpina cultured in diverse media at different time points. Positive control: Media containing synthetic sugar substrate, DLH: sugar substrate replaced by Detoxified liquid hydrolysate, NDLH: sugar substrate replaced by Non-detoxified liquid hydrolysate

Figure 4: Reducing sugar consumption by M.alpina cultured in diverse media at different time points. Positive control: Media containing synthetic sugar substrate, DLH: sugar substrate replaced by Detoxified liquid hydrolysate, NDLH: sugar substrate replaced by Non-detoxified liquid hydrolysate

Synopsis for Table of Contents (TOC) graphic Safe, efficient, sustainable and environmentally considerate mild acid saccharification process of lignocellulosic rice straw completely bypassed detoxification to become a carbon rich fermentable substrate for microbial SCO production


ABSTRACT Page 25 of 30 GRAPHIC

Synopsis : Safe, efficient, sustainable environmentally considerate mild acid saccharification process of lignocellulosic ACSand Sustainable Chemistry & Engineering rice straw completely bypassed detoxification to become a carbon rich fermentable substrate for microbial SCO production

1 2 3 4 5 6 7 8 9 10 11 12 13 Cellulose 14 15 16 17 18 19 20 Pentoses Hexoses 21 22 23 Fermentation 24 Inhibitors 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

Microwave assisted alkali pretreatment Optimization of mild acid hydrolysis by RSM

Lignin Hemicellulose

Hydrolysate

Detoxification DLH (detoxified)

Multi-tier saccharification at optimized parameters

NDLH (Non-detoxified)

Oleaginous species

Gradual disruption of cellulosic fibrils with multiple hydrolysis

Growth & lipogenic fermentation in DLH & NDLH

ACS Paragon Plus Environment

Recovered lipid

NDLH surpassed DLH in terms of growth support, sugar utilization and lipid accumulation in oleaginous species


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

(a).

Page 26 of 30

(b).

(c).

(d).


Page 27 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(e).

Figure 1 (a-e)


1.2306

250

X1 = A: Time X2 = C: Acid Percentage

200 Actual Factor B: Solid Loading = 7.5

150 100 50 0

150 140 130 120 110 100

5 6 7 8

90

A: Time (Minutes)

80

9

70 60

250 200 150 100 50 0

0.75

150 140 130 120 110 100

1 1.25 1.5

90

B: Solid Loading (% (w/v))

A: Time (Minutes)

10

1.75 C: Acid

80 70 60

(a)

2

(b)

Design-Expert® Software Factor Coding: Actual Reducing sugar concentration (mg/g straw) Design points above predicted value Design points below predicted value 213.45 1.2306 X1 = B: Solid Loading X2 = C: Acid Percentage Actual Factor A: Time = 105

Reducing sugar concentration (mg/g straw)


Page 28 of 30

Design-Expert® Software Factor Coding: Actual Reducing sugar concentration (mg/g straw) Design points above predicted value Design points below predicted value 213.45

ert® Software ng: Actual ugar concentration (mg/g straw) oints above predicted value oints below predicted value


1 2 3 4 5 6 e 7 id Loading or 8 centage = 1.375 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


250 200 150 100 50 0 0.75 10

1 9

1.25 8 1.5

7

B: Solid Loading (% (w/v))

C: Acid Percentage (% (v/v)) 1.75

6 5

2

(c) Figure 2(a-c)


Percentage (% (v/v))

Page 29 of 30

2 1.8

1.6 1.4

OD550

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1.2 Positive control

1

Non detoxified

0.8

Detoxified

0.6 0.4 0.2 0 0

50

100

Hours

150

Figure 3


200

250


40

% Reducing sugar consumption

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 30

35 30 25 20

Positive Control

15

Non detoxified

10

Detoxified

5 0 0

50

100

Hours

150

Figure 4


200

250

Platform Study on the Development of a Nondetoxified Rice Straw

Recommend Documents