Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Sustaining ethanol production from lime pretreated water hyacinth biomass using mono and co-cultures of isolated fungal strains with Pichia stipitis

Sustaining ethanol production from lime pretreated water hyacinth biomass using mono and... Background: The high rate of propagation and easy availability of water hyacinth has made it a renewable carbon source for biofuel production. The present study was undertaken to screen the feasibility of using water hyacinth's hemicelluloses as a substrate for alcohol production by microbial fermentation using mono and co-cultures of Trichoderma reesei and Fusarium oxysporum with Pichia stipitis. Results: In separate hydrolysis and fermentation (SHF), the alkali pretreated water hyacinth biomass was saccharified by crude fungal enzymes of T. reesei, F. oxysporum and then fermented by P. stipitis. In simultaneous saccharification and fermentation (SSF), the saccharification and fermentation was carried out simultaneously at optimized conditions using mono and co-cultures of selected fungal strains. Finally, the ethanol production kinetics were analyzed by appropriate methods. The higher crystalline index (66.7%) and the Fourier transform infrared (FTIR) spectra showed that the lime pretreatment possibly increased the availability of cellulose and hemicelluloses for enzymatic conversion. In SSF, the co-culture fermentation using T. reesei and P. stipitis was found to be promising −1 with a higher yield of ethanol (0.411 g g ) at 60 h. The additional yield comparable with the monocultures was due to the xylanolytic activity of P. stipitis which ferments pentose sugars into ethanol. In SHF, the pretreatment followed by crude enzymatic hydrolysis and fermentation resulted in a significantly lesser yield of ethanol −1 (0.344 g g )at96h. Conclusions: It is evident from the study that the higher ethanol production was attained in a shorter period in the co-culture system containing T. reesei and the xylose fermenting yeast P. stipitis. SSF of pretreated water hyacinth biomass (WHB) with P. stipitis instead of traditional yeast is found to be an effective biofuel production process. Keywords: Water hyacinth; Hemicelluloses; Xylose; T. reesei; F. oxysporum; P. stipitis;SSF Background has posed a threat to the food supply [2], and the cost of The global depletion of fossil fuels that are the dominant these raw materials accounts for up to 40% to 70% of the sources for supplying cheap energy for the world's econ- production cost [3]. Lignocellulosic biomass serves as a omy has prompted recent significant research efforts in cheap and abundant feedstock [4], which has the potential finding viable and sustainable alternatives [1]. Among to produce low-cost bioethanol at a large scale. In recent various options, conversion of abundant lignocellulosic days, screening of such substrates for biofuel has gained biomass to biofuels has received significant attention. Cur- new speed and still there are many factors to be taken into rently, bioethanol production from corn and sugarcane consideration for the large scale production. The performance of enzymatic saccharification is one of the foremost limiting factors which may strongly be * Correspondence: pothi2005@yahoo.com dependent on the diverse species, complex chemical com- PG and Research Department of Botany, Alagappa Government Arts College positions, and structural characteristics of the feedstock (Alagappa University), Karaikudi, Tamilnadu 630 003, India Full list of author information is available at the end of the article © 2014 Pothiraj et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 2 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 materials. The sugar yields from enzymatic hydrolysis vary Methods from plant to plant as a result of the differences mainly in Biomass and culture organisms cellulose content [5]. Like cellulose, hemicellulose is also a Fresh water hyacinth biomass (WHB) was collected from viable source of fermentable sugars such as xylose for a local pond at Karaikudi, Tamilnadu, India (10.07°N, biorefining applications. It was suggested that the produc- 78.78°E). The collected samples were washed to remove tion of fuel-grade ethanol from xylose requires a micro- adhering dirt, cut into small piece (2 or 3 mm) thick- organism capable of producing 50 to 60 g/L ethanol nesses, and dried in sunlight. The proximate analysis for within 36 h with a yield of at least 0.4 g ethanol per gram biomass was done using standard methods for moisture of sugar [6]. But only few xylose-fermenting microorgan- content, ash, crude protein, crude fibre, cellulose, hemi- isms have been reported earlier [7], and it is generally celluloses, and lignin [16,17]. The fungal strains of T. known that Pichia stipitis is superior to all other yeast spe- reesei and F. oxysporum were isolated by primary selec- cies for ethanol production from xylose. tion from a naturally contaminated water hyacinth, and Water hyacinth (Eichhornia crassipes) is a fast growing the isolates were confirmed by their morphology and perennial aquatic weed invasively distributed throughout colony characteristics [18]. The isolated organisms were the world. This tropical plant can cause infestations maintained on modified potato dextrose agar (PDA) over large areas of water resources and consequently slants at 4°C. Fresh colonies were used for saccharification leads to series of problems like reduction of biodiver- and fermentation studies. The pure culture of P. stipitis sity, blockage of rivers and drainage system, depletion (NCIM 3497) was procured from the National Collection of dissolved oxygen, and alteration on water chemistry of Industrial Microorganisms, Pune, India. that leads to severe environmental pollution. In the past, attempts have been geared towards the use of Alkaline pretreatment biological, chemical, and mechanical approaches for The dried WHB (10% w/v) was pretreated with calcium preventing the spread of, or eradication of, water hya- hydroxide solution (0.5% w/v) with a soaking time of 3 h cinth. On the other hand, much attention has been at 100°C. The pretreated WHB washed to neutrality with focused on the potentials and constrains of using distilled water, oven dried to a constant weight, and then water hyacinth for a variety of applications since it has milled to powder was used for enzymatic hydrolysis and a lignocellulosic composition of 48% hemicelluloses, fermentation [19]. 18% cellulose, and 3.5% lignin [8,9]. Since the biomass productivity of this plant is very high, it can be a suit- Experimental design able feedstock for ethanol production. Two modes of bioconversion methodologies for ethanol The technologies for the possible conversion of production were trialed in the present study. Mode I water hyacinth to biogas or fuel ethanol using fungal comprised of a separate hydrolysis and fermentation extracellular enzymes are well documented in a num- (SHF) process using crude fungal enzymes with yeast. ber of developing countries [10-13]. Saccharomyces Mode II was designed to conduct a simultaneous sac- cerevisiae and Zymomonas mobilis are being used as charification and fermentation (SSF) process using mono candidate organisms in the large-scale production of and co-cultures of selected fungal strains. ethanol from cellulosic biomass. These organisms are capable of utilizing hexose sugars efficiently but not Separate hydrolysis and fermentation (SHF) the pentoses, which are the second dominant sugar The cellulolytic enzymes (cellulases and xylanases) were source in lignocellulosic biomass [14]. From earlier produced by growing the isolated fungal strains of T. research, P. stipitis hasbeenidentifiedasanefficient reesei and F. oxysporum separately at 35°C in a simple −1 −1 strain for the conversion of pentose sugars into alco- liquid medium (4.2 g L (NH ) SO ,2gL KH PO , 4 2 4 2 4 −1 −1 hol [15]. Fermentation technologies utilizing strains 0.05 g L yeast extract, 2 mL L Tween-80, 2% (w/v) of P. stipitis instead of traditional yeast have been pro- poultry manure with 1.6% total N, pH 4.8) containing −1 posed by a number of authors [14,15], as they have 100 g L water hyacinth biomass as the chief C source been shown to ferment under fully anaerobic condi- for 5 days as optimized earlier [20]. The culture superna- tions with faster specific rates of pentose sugar uptake tants were separated at the end of the incubation period and ethanol production as well as an ethanol yield from each organism and used as crude enzymes source for close to theoretical yield. The present study, therefore, hydrolysis. Cellulase and xylanase activities were measured was carried out to screen the feasibility of using in the culture supernatant as per standard methods. hexose- and pentose-utilizing fungal strains (Tricho- Cellulase was measured according to the IUPAC derma reesei, Fusarium oxysporum,and Pichia stipitis) methods [21] using Whatman filter paper no. 1 as the for the effective conversion of water hyacinth biomass substrate and glucose as the standard. Xylanase was into ethanol. assayed by the optimized method described by Bailey Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 3 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 et al. [22], using 1% birchwood xylan as the substrate dichromate method [26]. The microbial biomass was de- and xylose as the standard. One unit (IU) of enzyme termined by harvesting cells by centrifugation, drying activity is defined as the amount of enzyme releasing them at 70°C under vacuum to a constant weight, and 1 μmol glucose or xylose/mL per minute expressed as gram dry cell weight (DCW) per liter [27]. Enzymatic hydrolysis was carried out by incubating The kinetic parameters of ethanol fermentation were de- the pretreated WHB (10% w/v) with the crude fungal termined followed by Abate et al. [28] as follows: enzymes (10% (v/v)) of T. reesei and F. oxysporum separ- Ethanol concentrationðÞ Ec ¼ ½ ethanol produced ðÞ g = ately at 35°C for 48 h with agitation at 200 rpm [23]. volume of reaction mixtureðÞ L The pH of the reaction mixture (6.0) was maintained at Ethanol productivity E ¼½ethanol produced ðÞ g = constant. Samples were aliquoted from hydrolysates at a volume of reaction mixtureðÞ L = regular interval (24 and 48 h) to estimate the released time ðÞ h sugar content using standard methods [24,25]. The hydro- Ethanol Yield E ¼½ethanol produced ðÞ g = lysates obtained after 48 h from both the fungal cultures y weight of substrate ðÞ g were centrifuged at 10,000 rpm for 10 min. The superna- tants were collected separately and supplemented with Specific ethanol yield E ¼½ ethanol produced ðÞ g = sy −1 −1 basal medium (1 g L yeast extract; 2 g L (NH )SO ; 4 4 sugar consumed ðÞ g −1 1gL MgSO � 7H O) (pH 6.0) [23]. The culture suspen- 4 2 The results obtained were analyzed by using analysis sion of P. stipitis (10% v/v) was added to initiate the fer- of variance (ANOVA), and the group means were com- mentation by incubating the mixture at 35°C for 48 h with pared with Duncan's Multiple Range Test (DMRT) [29]. agitation at 200 rpm. Fourier transform infrared (FTIR) analysis Simultaneous saccharification and fermentation (SSF) Fourier transform infrared spectra were studied on SSF represents a single step process in which ferment- treated and untreated WHB using a Shimadzu spec- able sugars get released by enzymatic hydrolysis and are trometer (Shimadzu, Kyoto, Japan). For this, 3.0 mg of simultaneously exploited by yeasts for fermentation in the sample was dispersed in 300 mg of spectroscopic the same medium. The microbial fermentation was carried grade KBr and subsequently pressed into disks at out using mono and co-cultures as previously described 10 MPa for 3 min. The spectra were obtained with an [9]. The influences of various parameters such as micro- −1 average of 25 scans and a resolution of 4 cm in the bial biomass (5% to 25%), temperature (25°C to 45°C), and −1 range of 4,000 to 400 cm . incubation time (24, 36, 48, 60, 72 h) on SSF were also optimized by step-wise experiments where the specified X-ray diffraction (XRD) analysis parameters were changed by keeping all other parameters The crystallinity of cellulose in the pretreated and constant. The pH of the reaction mixture in all the treated water hyacinth was analyzed by X-ray diffraction optimization experiments was kept constant at 6.0 method in a PANalytical X'pert PRO Diffractometer (PANalytical B.V., Almelo, Netherlands) set at 40 KV, Mono and co-culture fermentations 30 mA; radiation was Cu Kα(λ = 1.54Ǻ) and the grade For monoculture experiments (F1 and F2), previously 0 0 range between 10 to 30 with a step size of 0.03 . The sterilized (121°C for 60 min) pretreated WHB supple- crystallinity index (CrI) was determined based on the mented with a basal medium (without C source) was equation shown below [30]: inoculated with late log-phase cultures of T. reesei (F1) and F. oxysporum (F2), separately. For co-culture fer- I −I 002 am CrI ¼  100 mentation (F3 and F4), separate sets of reaction mixtures am consisting of pretreated WHB supplemented with basal medium were treated with P. stipitis simultaneously with where I is the intensity of the diffraction from the 002 T. reesei (F3) and F. oxysporum (F4). The fermentation plane at 2θ = 22.6° and I is the intensity of the back- am process was carried out at optimized conditions. ground scatter measured at 2θ = 18.7°. It is known that the I peak corresponds to the crystalline fraction and the Estimations I peak corresponds to the amorphous fraction [31]. am Samples were withdrawn from the fermenting media at regular intervals of time for the determination of etha- Results and discussion nol, residual sugar concentration, and microbial biomass. The lignocellulosic biomass composition of WH includes −1 Estimation of xylose was done by the Trinder method cellulose (20.2 g 100 g dry matter (DM)), hemicellulose −1 −1 [24] and glucose by the DNS method [25]. Ethanol esti- (34.3 g 100 g DM), lignin (4.4 g 100 g DM ), crude −1 −1 mation was done spectrophotometrically by potassium protein (13.3 g 100 g DM), crude fibre (18.2 g 100 g Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 4 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 Table 1 Proximate composition of water hyacinth biomass (WHB) comparable with earlier literatures Content Present study Previous literature −1 [g 100 g DM] −1 −1 −1 [g 100 g DM] [8] [g 100 g DM] [12] [g 100 g DM] [32] Moisture (%) 92.8 - - - Ash 15.4 20.0 - 20.0 Crude protein 13.3 18.0 10.2 18.0 Crude fibre 18.2 - - - Cellulose 20.2 25.0 19.02 25.0 Hemicellulose 34.3 35.0 32.69 35.0 Lignin 4.4 10.0 4.37 10.0 DM, dry matter. −1 DM), and ash (15.4 g 100 g DM) (Table 1). The results increased the crystallinity of cellulose in water hyacinth. obtained in the present study on the proximate compos- Similar results were reported earlier by Kim and Holtzapple ition of WHB are basically consistent with previous [39] whofound that thedegreeof crystallinityofcorn literatures [8,12,32]. The digestibility of lignocelluloses is stover slightly increased from 43% to 60% through delignifi- hindered by many physicochemical, structural, and com- cation with calcium hydroxide and by Li et al. [40] who positional factors which required a suitable pretreatment have reported high cellulose CrI of 70.6% in Metasequoia in order to enhance the susceptibility of biomass for chipsbynitricacid-ethanol method. TheincreaseinCrI of hydrolysis. It is highly essential for the economical produc- alkali-treated WHB might be due to the removal of tion of ethanol that both the cellulose and hemicellulosic amorphous components including lignin during the pre- sugars present in the biomass should be utilized efficiently. treatment process [41,42]. According to Satyanagalakshmi The FTIR and XRD data in the present study clearly sug- et al. [33], the amorphous cellulose portions in aquatic gested that the pretreatment with lime could increase the plants are more prone to recrystallization to form crystal- availability of polysaccharide for enzymatic hydrolysis. line cellulose, resulting in greater increases in CrI. Among different pretreatment methods used in earlier re- searches for water hyacinth, maximum reducing sugar was −1 observed in diluted H SO (0.342 g g biomass) [33], HCl 2 4 −1 −1 140 Untreated WHB (0.277 g g biomass), acetic acid (0.097 g g biomass), −1 and formic acid (0.088 g g biomass) [31,34]. In compari- son with the above reports, it is evident that the lime pretreatment used in the present study is a promising method for higher sugar yield. The pretreatment with Ca (OH) is preferable because it is less expensive, more safe as compared to NaOH, and it can be easily recovered from the hydrolysate by reaction with CO . Lime has been used 20 to pretreat many lignocellulosic materials such as wheat straw [35], poplar wood [36], and corn stover [37]. XRD - cellulose crystallinity Alkali pretreated WHB Cellulose crystallinity, usually measured as CrI, is con- sidered an important parameter determining the enzym- atic hydrolysis susceptibility of cellulose. The CrI of a cellulose sample is an indication of the degree of formed crystallinity in the sample when the cellulose aggregates. The crystallinity has been found to have a greater impact on enzymatic hydrolysis than other structural characteris- tics such as the degree of polymerization (DP) of cellulose or the specific surface area (SSA) [38]. The XRD profile of 5 101520 2530354045 WHB indicated that the CrI of untreated WHB is 28.6% 2 Theta and alkali-treated WHB is 66.7% (Figure 1). The X-ray dif- Figure 1 XRD analysis of untreated and lime pretreated WHB. fractogram clearly revealed that the lime pretreatment Intensity Intensity Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 5 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 FTIR analysis p-coumeric acids of lignin and/or hemicelluloses [44]. A −1 FTIR spectra of the untreated and treated samples indi- sharp band at 896 cm , corresponding to the C1 group cated structural changes in the biomass upon pretreat- frequency or ring frequency, was attributed to the glyco- ment (Figure 2). The increased absorption bands at sidic linkages between xylose units in hemicelluloses −1 1,000 to 1,200 cm were related to structural features of [45]. The peaks in the pretreated sample had the highest cellulose and hemicelluloses [43]. The spectra of alkali- absorbance suggesting increase in cellulose and hemicel- treated WHB sample (Figure 3) showed increase in lulose content. In the FTIR spectrum, the peaks ob- −1 absorbance in the above-mentioned range. The peak at served at 1,092 and 842 cm were attributed to C-O −1 1,635 cm was observed due to either the acetyl and ur- stretching and C-H rocking vibration of the cellulose onic ester linkage of carboxylic group of the ferulic and structure. Untreated WHB Alkali treated WHB Figure 2 FTIR spectra of untreated and alkali (Ca (OH) )-treated WHB. 2 Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 6 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 0.5 0.4 0.3 F1 F2 0.2 F3 0.1 F4 12 24 36 48 60 72 Fermentation period (h) Figure 3 Effect of fermentation period on ethanol yield from pretreated WHB using mono and co-cultures. F1, T. reesei;F2, F. oxysporum; F3, T. reesei + P. stipitis;F4, F. oxysporum + P. stipitis. Enzyme activity Sugar yield In recent decades, the use of fungi in bioprocesses has The yield of sugars from enzymatic hydrolysis of WHB growninimportancebecause of theproductionofnu- using crude enzymes produced by fungal isolates was sum- merous enzymes with different biochemical properties marized in Table 2. The saccharification was significantly and excellent potential for biotechnological applica- higher (40.8%) while using crude enzyme from T. reesei tion. The cellulase and xylanase activities reached when compared to F. oxysporum (38.2%). The release of their maximum values on the 6th day of incubation total sugars by the crude enzymes of both monocultures in- for both the fungal isolates. Cellulase production creased slowly to reach a peak value at 48 h of incubation. −1 on WHB with nutrient supplements indicated higher The maximum yield of total sugar (0.531 g g WHB) in- −1 −1 cellulase production by T. reesei (0.923 IU/mL) com- cluding glucose (0.444 g g WHB) and xylose (0.057 g g pared to F. oxysporum (0.432 IU/mL). However, it WHB) was observed after 48 h of hydrolysis using crude is less than the value of 1.35 IU/mL reported by enzymes of T. reesei. The crude enzymes obtained from F. Deshpande et al. [46] on the substrate water hyacinth oxysporum produced comparably lower reducing sugar −1 −1 with Toyama-Ogawa medium [47]. The xylanase produc- (0.428 g g WHB) and xylose (0.038 g g WHB). Thus, it tion was slight, but significantly higher in F. oxysporum substantiates that the amount of sugar released increases (0.764 IU/mL), compared to T. reesei (0.611 IU/mL). Ac- with time which may be due to the increased action of cording to Kang et al. [48], high xylanase production in cellulolytic and xylanolytic enzymes of T. reesei and F. some fungi has been shown to be linked strictly to the oxysporum [51]. The cellulolytic fungus T. reesei looks ratio of cellulose to xylan of the growth substrate and promising for on-site cellulase production due to its substrate degradation due to time course or incubation superior features, i.e., capability to produce all components period. of cellulase complex, endocellulase, exocellulase, and β- According to Polizeli et al. [49], filamentous fungi are glucosidase in good proportions as well as production of widely utilized as enzyme producers and are generally other enzymes such as xylanases or laccases in comparison considered more potent xylanase producers than bacteria to other enzyme producers [52]. or yeast. Several mesophilic fungal species have been evaluated in relation to xylanase production, including Ethanol members of Aspergillus, Trichoderma,and Penicillium. The optimization studies in SSF showed that the yield of Currently, most commercial xylanolytic preparations ethanol is found to be proportional to fermentation time are produced by genetically modified Trichoderma or where the yield increases with the increase in time up to Aspergillus strains [50]. 60 h and then declines (Figure 3). Maximum yield of −1 Table 2 Sugar composition (g g WHB) of enzymatic hydrolysates of pretreated WHB at 48 h Enzyme source Glucose Xylose Total sugar Saccharification % a a a T. reesei 0.444 ± 0.12 0.057 ± 0.09 0.531 ± 0.12 40.2 b b b F. oxysporum 0.428 ± 0.31 0.038 ± 0.11 0.488 ± 0.17 38.2 Values are the mean of three replicates ± SE. Means followed by the same letter within treatment do not differ significantly (p = 0.05). -1 Ethanol Yield (g g WHB) Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 7 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 0.5 0.4 F1 0.3 F2 0.2 F3 F4 0.1 25 30 35 40 45 Temperature C Figure 4 Effect of temperature on ethanol yield from pretreated WHB using mono and co-cultures. F1, T. reesei;F2 F. oxysporum;F3 T. reesei + P. stipitis;F4, F. oxysporum + P. stipitis. −1 ethanol is 0.413, 0.378, 0.194, and 0.187 g g of WHB fermentation of pretreated water hyacinth biomass at 60 h of fermentation for F3, F4, F1, and F2, respect- using monocultures of T. reesei and F. oxysporum was ively. After 60 h of time, the yield of ethanol decreases due to the inability of these organisms to convert pen- in all treatments, and therefore, fermentation time of tose sugars into ethanol. A similar finding was reported 60 h is taken as the optimum time for ethanol fermenta- earlier where 0.11 g ethanol was obtained from alkali- tion. With the increase in temperature, the yield of pretreated water hyacinth through SHF [23]. According ethanol increased up to 35°C and then it decreased to Preez et al. [53] P. stipitis is known to produce etha- (Figure 4). At high temperature (>35°C), death rate ex- nolupto33to57g/L;however,30g/L is known as a ceeds the growth rate, which causes a net decrease in critical concentration above which cells cannot grow at concentration of viable fungal populations with lower 30°C. generation of ethanol. With the increase in loading of In SSF, monocultures of T. reesei (F1) and F. oxysporum −1 biomass, the yield of ethanol increased up to 10% and (F2) produced 19.3 and 17.8 g L ethanol, respectively, then decreased in all the samples (Figure 5). The de- after 60-h fermentation (Table 4). Simultaneous co- crease in ethanol yield with the increase in biomass culturing of T. reesei (F3) and F. oxysporum (F4) with P. loading can be attributed to the inhibitory effect of ei- stipitis resulted in a higher ethanol production (40.8 and −1 ther the product or the biomass. Inhibitory compounds 36.8 g L , respectively) at the same time. The maximal −1 limit efficient utilization of hydrolysates by the ferment- ethanol yield was 0.411 g g WHB when P. stipitis was ing organism resulting in less ethanol production [33]. used along with T. reesei which is positively correlated to −1 In the SHF process, a maximum of 14.3 g L ethanol the theoretical yield 0.429 g on the basis of biomass. Since was produced at the end of the process (96 h) which is xylose was present as a predominant sugar in the WHB −1 equivalent to 0.143 g g WHB (Table 3). The mini- hydrolysate, P. stipitis was used to make the biomass-to- mum production of ethanol observed in the submerged ethanol process more economical. Mishima et al. [34], on 0.5 0.4 0.3 F1 F2 0.2 F3 F4 0.1 510 15 20 25 Microbial biomass (% v/v) Figure 5 Effect of microbial biomass on ethanol yield from pretreated WHB using mono and co-cultures. F1, T. reesei;F2, F. oxysporum;F3, T. reesei + P. stipitis;F4, F. oxysporum + P. stipitis. 1 1 Ethanol Yield (g g WHB) Ethanol Yield (g g WHB) Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 8 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 Table 3 Ethanol production in SHF process using crude fungal enzymes and P. stipitis with pretreated WHB at 96 h Sample (s)E E E E E c p y sy TE c a c c c S 14.3 ± 0.12 0.24 ± 0.09 0.143 ± 0.11 0.322 ± 0.11 0.261 ± 0.11 d a d d d S 12.8 ± 0.13 0.21 ± 0.17 0.128 ± 0.08 0.299 ± 0.08 0.243 ± 0.17 −1 −1 −1 −1 −1 E , ethanol concentration (g L ); E , ethanol productivity (g L h ); E , ethanol yield (g g WHB); E , specific ethanol yield (g g sugar); E , theoretical yield c p y sy TE −1 (g g WHB); S1, hydrolysate from T. reesei; S2, hydrolysate from F. oxysporum. Values are the mean of three replicates ± SE. Mean followed by the same letter within treatment do not differ significantly (p = 0.05). the other hand, reported a lesser ethanol yield of 0.14 obtained in the co-culture of T. reesei and P. stipitis at −1 gg dry substrate through SSF of pretreated water hya- 60-h fermentation (Table 4). Inoculation of P. stipitis cinth using commercial cellulase and S. cerevisiae.The with F. oxysporum resulted in a biomass content of −1 overall production could be enhanced by co-culture rather 2.64 g DCW L over the monocultures. Statistically, a than monoculture of test organisms. Similarly, direct mi- less significant difference was observed with monocul- crobial conversion of cellulosic or lignocellulosic biomass ture's fermentation when compared with co-culture [58]. into ethanol using co-cultures had been reported by sev- eral authors [32,34,54]. S. cerevisiae or Z. mobilis utilize Conclusions glucose or sucrose efficiently but their inability to utilize The fermentation of bioethanol from pretreated water pentose sugars make them inappropriate candidates for hyacinth biomass with mono and co-cultures of fungal refineries, but the candidate organism P. stipitis used in strains along with P. stipitis is found to be an effective bio- the present study showed efficient conversion of pentose fuel production process. The yield of ethanol recovered sugars into alcohol. Among the pentose-fermenting organ- from WHB through enzymatic hydrolysis and fermentation isms, P. stipitis has been shown to have the most promise from simultaneous inoculation of co-cultures of fungal for industrial applications [55]. Earlier reports showed isolates with P. stipitis was significantly higher than that re- that the hemicellulosic hydrolysates of Prosopis juliflora covered through monocultures. The optimum parameters (18.24 g sugar/L broth) when fermented with P. stipitis for bioethanol fermentation are as follows: time 60 h, −1 produced 7.13 g/L ethanol [56]. Kuhad et al. [57] observed temperature 35°C, and WHB loading 100 g L .The max- −1 0.33 g g ethanol yield from detoxified xylose-rich hydrol- imum yield of ethanol in the fermentation process was −1 ysate of Lantana camara fermented with P. stipitis at found to be 0.411 g g of WHB which is equivalent to −1 pH 5 for 36 h. Similarly, the detoxified water hyacinth a specific yield of 0.456 g g total sugar consumed. hemicellulose acid hydrolysate (rich in pentose sugars) fer- The use of crude fungal enzymes produced on-site mented with P. stipitis NCIM-3497 at pH 6.0 and 30°C re- would be a cost-effective approach towards enzymatic sulted in 0.425 g ethanol/g lignocelluloses [15]. The yield hydrolysis of alkali-pretreated WHB biomass instead of ethanol per unit biomass of water hyacinth obtained of using commercial cellulases. The aquatic menace through the bioprocess in the present study was compar- water hyacinth, which is currently being used in waste able to or even better than those reported earlier. The water treatment for its unique ability to absorb heavy current results clearly demonstrated the saccharification metal pollutants, could also be utilized as abundant potential of T. reesei and F. oxysporum, where the per- cheap feedstock for the production of fuel ethanol. formance of both strains in co-cultures with P. stipitis was This study proved that water hyacinth has a potential significantly higher than their respective single culture. renewable and low-cost biomass for alcohol produc- tion on the commercial scale. Present cost effective- Microbial biomass ness of respective process at a commercial scale All the co-culture processes reached a higher value of needs to be standardized, and the water hyacinth microbial biomass than the single fermentation process. biomass could be a better substrate source for alco- −1 A maximum of 3.12 g DCW L biomass content was hol production. Table 4 Ethanol production in mono and co-culture fermentation process (SSF) using pretreated WHB at 60 h −1 Culture (s)E E E E Microbial biomass (g DCW L ) c p y sy c c c c c T. reesei 19.3 ± 0.12 0.32 ± 0.09 0.196 ± 0.11 0.377 ± 0.11 2.14 ± 0.01 d d d d c F. oxysporum 17.8 ± 0.13 0.29 ± 0.17 0.176 ± 0.08 0.348 ± 0.08 2.06 ± 0.08 a a a a a T. reesei + P. stipitis 40.8 ± 0.09 0.68 ± 0.14 0.411 ± 0.03 0.798 ± 0.11 3.12 ± 0.12 b b b b b F. oxysporum + P. stipitis 36.8 ± 0.06 0.61 ± 0.12 0.371 ± 0.07 0.720 ± 0.08 2.64 ± 0.20 −1 −1 −1 −1 −1 E , ethanol concentration (g L ); E , ethanol productivity (g L h ); E , ethanol yield (g g WHB); E , specific ethanol yield (g g sugar). Values are the mean of c p y sy three replicates ± SE. Mean followed by the same letter within treatment do not differ significantly (p = 0.05). Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 9 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 Competing interests 15. Kumari N, Bhattacharya A, Dey A, Ganguly A, Chatterjee PK (2014) The authors declare that they have no competing interests. Bioethanol production from water hyacinth biomass using isolated fungal strain from local environment. Biolife 2(2):516–522 16. Association of Official Analytical Chemists (AOAC) (1975) Methods of Authors' contributions analysis of the Association of Official Analytical Chemists. Association of CP carried out the submerged fermentation processes and helped design Official Analytical Chemists, Washington DC the whole study. RA carried out proximate analysis of biomass and helped in 17. Robertson JB, van Soest PJ (1981) The Detergent System of Analysis and its manuscript preparation. RMG helped in characterization of biomass using application to human foods. In: James WPT, Thiander O (eds) The analysis of FTIR and XRD. All authors read and approved the final manuscript. dietary fibers in food. Marcel Dekker, New York, pp 123–158 18. Alexopoulos CJ, Beneke ES (1962) Laboratory manual for introductory mycology. 1, 2 Burgess publishing Co, Minneapolis Acknowledgements 19. Chang VS, Burr B, Holtzapple MT (1997) Lime pretreatment of switchgrass. The authors are thankful to all faculty members of the PG and Research Appl Biochem Biotechnol 63(65):3–19 Department of Botany, Alagappa Government Arts College (Alagappa University), Karaikudi. We also acknowledge the Head, Department of 20. Mukhopadhyay S, Nandi B (2001) Cellulase production by Trichoderma reesei physics, Alagappa University, Karaikudi and the Department of Chemistry, on pretreated water hyacinth: effect of nutrients. J Mycopathol Res VHNSN College, Virudhunagar, Tamilnadu for helping us with XRD and FTIR 39(1):57–60 analysis, respectively. 21. Ghose TK (1987) Measurement of cellulose activities. Pure Applied Chem 59(257):268 Author details 22. Bailey M, Buchert J, Viikari L (1993) Effect of pH on production of xylanase PG and Research Department of Botany, Alagappa Government Arts College of Trichoderma reesei on xylan- and cellulose-based media. Appl Microbiol (Alagappa University), Karaikudi, Tamilnadu 630 003, India. Department of Biotechnol 40:224–229 Microbiology, VHNSN College, Virudhunagar, Tamilnadu 626 001, India. 23. Mukhopadhyay S, Mukherjee PS, Chatterjee NC (2008) Optimization of enzymatic hydrolysis of water hyacinth by Trichoderma reesei vis-à-vis Received: 30 July 2014 Accepted: 10 November 2014 production of fermentable sugars. Acta Aliment 37(3):367–377 24. Trinder P (1975) Micro-determination of xylose in plasma. Analyst 100:12–15 25. Miller GL (1959) Use of DNS reagent for the determination of reducing sugars. Anal Chem 31:426–428 References 26. Caputi A, Veda M, Brown T (1968) Spectrophotometric determination of 1. Chang KL, Thitikorn-amorn J, Hsieh JF, Ou BM, Chen SH, Ratanakhanokchai ethanol in wine. American J Enol Viticul 19:160–165 K, Huang PJ, Chen ST (2011) Enhanced enzymatic conversion with freeze 27. Doelle HW, Greenfield PF (1985) The production of ethanol from sucrose pretreatment of rice straw. Biomass Bioenergy 35(1):90–95 using Zymomonas mobilis. Appl Microbial Biotechnol 22:405–441 2. Guragain YN, De Coninck J, Husson F, Durand A, Rakshit SK (2011) 28. Abate C, Callieri D, Rodriguez E, Garro O (1996) Ethanol production by Comparison of some new pretreatment methods for second generation mixed culture of flocculent strains of Zymomonas mobilis and bioethanol production from wheat straw and water hyacinth. Bioresour Saccharomyces sp. Appl Microbial Biotechnol 45:580–583 Technol 102(6):4416–4424 29. Duncan BD (1957) Multiple range tests for correlated and heteroscedastic 3. Quintero JA, Montoya MI, Sánchez OJ, Giraldo OH, Cardona CA (2008) Fuel means. Biometrics 13(2):359–364 ethanol production from sugarcane and corn: comparative analysis for a 30. Segal L, Creely JJ, Martin AE, Conrad CM (1959) An empirical method for Colombian case. Energy 33(3):385–399 estimating the degree of crystallinity of native cellulose using X –Ray 4. Balat M (2011) Production of bioethanol from lignocellulosic materials via diffractometer. Text Res J 29:786–794 the biochemical pathway: a review. Energy Convers Manage 52(2):858–875 31. Wang LS, Zhang YZ, Gao PJ, Shi DX, Liu HW, Gao HJ (2006) Changes in the 5. Sukumaran RK, Singhania RR, Mathew GM, Pandey A (2009) Cellulase structural properties and rate of hydrolysis of cotton fibers during extended production using biomass feed stock and its application in lignocelluloses enzymatic hydrolysis. Biotechnol Bioeng 93(3):443–456 saccharification for bio-ethanol production. Renew Energy 34(2):421–424 32. Aswathy US, Sukumaran RK, Lalitha Devi G, Rajasree KP, Singhania RR, 6. Lee Y, Kim M, Kim K, Park K, Ryu Y, Seo J (2000) A parametric study on Pandey A (2010) Bio-ethanol from water hyacinth biomass: an evaluation of ethanol production from xylose Pichia stipitis. Biotechnol Bioprocess Eng enzymatic saccharification strategy. Biores Technol 101:925–930 5:27–31 33. Satyanagalakshmi K, Sindhu R, Binod P, Janu KU, Sukumaran RK, Pandey A 7. Ganguly A, Das S, Bhattaacharya A, Dey A, Chatterjee PK (2013) Enzymatic (2011) Bioethanol production from acid pretreated water hyacinth by hydrolysis of water hyacinth biomass for the production of ethanol: separate hydrolysis and fermentation. J Sci Ind Res 70:156–161 optimization of driving parameters. Ind J Exp Biol 51:556–566 34. Mishima D, Kuniki M, Sei K, Soda S, Ike M, Fujita M (2008) Ethanol 8. Gunnarsson CC, Petersen CM (2007) Water hyacinth as a resource in production from candidate energy crops: water hyacinth (Eichhornia agriculture and energy production: a literature review. Waste Man crassipes) and water lettuce (Pistia stratiotes L.). Biores Technol 27:117–129 99:2495–2500 9. Nigam JN (2002) Bioconversion of water hyacinth (Eichhornia crassipes) 35. Chang VS, Nagwani M, Holtzapple MT (1998) Lime pretreatment of crop hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting residues bagasse and wheat straw. Appl Biochem Biotechnol 74:135–159 yeast. J Biotech 97:107–116 36. Chang VS, Nagwani M, Kim CH, Holtzapple MT (2001) Oxidative lime 10. Zabala I, Ferrer A, Ledesma A, Aiello C (1994) Microbial protein production pretreatment of high-lignin biomass. Appl Biochem Biotechnol 94:1–28 by submerged fermentation of mixed cellulolytic cultures In: Galindo E, 37. Karr WE, Holtzapple MT (1998) The multiple benefits of adding non-ionic Ramirez OT (eds) Advances in Bioprocess Engineering. Kluwer Academic surfactant during the enzymatic hydrolysis of corn stover. Biotechnol Bioeng Publishers, The Netherlands, pp 455-460 59:419–427 11. Singhal V, Rai JP (2003) Biogas production from water hyacinth and channel 38. Peng HD, Li HQ, Luo H, Xu J (2013) A novel combined pretreatment of ball grass used for phytoremediation of industrial effluents. Bioresour Technol milling and microwave irradiation for enhancing enzymatic hydrolysis of 86:221–225 microcrystalline cellulose. Bioresour Technol 130:81–87 12. Sornvoraweat B, Kongkiattikajorn J (2010) Separated hydrolysis and 39. Kim S, Holtzapple MT (2006) Effect of structural features on enzyme fermentation of water hyacinth leaves for ethanol production. KKU Res J digestibility of corn stover. Bioresource Technol 97:583–591 15(9):794–802 40. Li L, Wenbing Z, Hongwei W, Yun Y, Fen L, Duanwei Z (2014) Relationship 13. Idrees M, Adman A, Sheikh S, Qureshi FA (2013) Optimization of dilute acid between crystallinity index and enzymatic hydrolysis performance of pretreatment of water hyacinth biomass for enzymatic hydrolysis and cellulose separated from aquatic and terrestrial plant materials. Bioresources ethanol production. EXCLI J 12:30–40 9(3):3993–4005 14. Bhattacharya A, Ganguly A, Das S, Chatterjee PK, Dey A (2013) Fungal isolates from local environment: isolation, screening and application for the 41. Converse AO, Kwartneg IK, Grethlein HE, Ooshima H (1989) Kinetics of production of ethanol from water hyacinth. Int J Emerg Tech Adv Engi thermochemical pretreatment of lignocellulosic materials. Appl Biochem 3(3):58–65 Biotechnol 20(21):63–78 Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 10 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 42. Maeda RB, Serpa VI, Rocha RAL, Mesquita RAA, Anna LMMS, De Carlo AM, Driemeier CE, Pereira N, Polikarpov I (2011) Enzymatic hydrolysis of pretreated sugar cane baggase using Penicillium funiculosum and Trichoderma harzianum cellulases. J Process Biochem 30:5–8 43. Hu J, Arantes J, Saddler JN (2011) The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect. Biotechnol Biofuels 4:36 44. Langkilde FW, Svantesson A (1995) Identification of celluloses with Fourier- transform (FT) mid-infrared, FT-Raman and near-infrared spectrometry. J Pharm Biomed Anal 13:409 45. Marimuthu TS, Atmakuru R (2012) Isolation and characterization of cellulose nanofibers from the aquatic weed water hyacinth-Eichhornia crassipes. Carbohydr Polym 87:1701 46. Deshpande SK, Bhotmange MG, Chakrabarti T, Shastri PN (2008) Production of cellulase and xylanase by Trichoderma reesei (QM 9414 mutant), Aspergillus niger and mixed culture by solid state fermentation (SSF) of water hyacinth (Eichhornia crassipes). Ind J Chem Tech 15:449–456 47. Toyama M, Ogawa K (1977) Cellulase production of Trichoderma viride in solid and submerged culture methods. In: Ghosh TK, Ghosh TK (eds) Proc. symp. On bioconversion of cellulosic substrates into energy, chemical and microbial protein. IIT, New Delhi, India, pp 305–312 48. Kang SW, Park YS, Lee JS, Hong SI, Kim SW (2004) Production of cellulose and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresour Technol 91:153–156 49. Polizeli MLT, Rizzati ACS, Monti R, Terenzi HF, Jorge JA, Amorin DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67(5):577–591 50. Mussatto SI, Teixeira JA (2010) Lignocellulose as raw material in fermentation processes. In: Méndez-Vilas A (ed) Current research, technology and education topics in applied microbiology and microbial biotechnology. Formatex Research Center, Badajoz, pp 897–907 51. Sun YC, Weu JL, Xu F, Sun RC (2011) Structure and thermal characterization of hemicelluloses isolated by organic solvents and alkaline solutions from Tamarix austromongolica. Biores Technol 102:5947 52. Arantes V, Saddler JN (2010) Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis. Biotechnol Biofuels 3(4):1–11 53. Preez JC, Bosch M, Prior BA (1987) Temperature profiles of growth and ethanol tolerance of xylose fermenting yeasts Candida shehatae and Pichia stipitis. Appl Microbiol Biotechnol 25:521–525 54. Mukhopadhyay S, Chatterjee NC (2010) Bioconversion of water hyacinth hydrolysate into ethanol. BioResourses 5(2):1301–1310 55. Agbogbo FK, Coward-Kelly G, Torry-Smith M, Wenger KS (2006) Fermentation of glucose/xylose mixtures using Pichia stipitis. Process Biochem 41:2333–2336 56. Gupta R, Sharma KK, Kuhad RC (2009) Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, woody substrate for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498. Bioresour Technol 100:1214–1220 57. Kuhad RC, Gupta R, Khasa YP, Singh A (2010) Bioethanol production from Lantana camara (red sage): pretreatment, saccharification and fermentation. Bioresour Technol 101:8348–8354 58. Manilal VB, Narayanan CS, Balagopalan C (1991) Cassava starch effluent treatment with concomitant SCP production. World J Microbiol Biotechnol 7:185–190 doi:10.1186/s40643-014-0027-3 Cite this article as: Pothiraj et al.: Sustaining ethanol production from lime pretreated water hyacinth biomass using mono and co-cultures of isolated fungal strains with Pichia stipitis. Bioresources and Bioprocessing Submit your manuscript to a 2014 1:27. journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png "Bioresources and Bioprocessing" Springer Journals

Sustaining ethanol production from lime pretreated water hyacinth biomass using mono and co-cultures of isolated fungal strains with Pichia stipitis

Download PDF
10 pages

Loading next page...
 
/lp/springer-journals/sustaining-ethanol-production-from-lime-pretreated-water-hyacinth-MRufvft8g4

References (69)

Publisher
Springer Journals
Copyright
2014 Pothiraj et al.; licensee Springer.
eISSN
2197-4365
DOI
10.1186/s40643-014-0027-3
Publisher site
See Article on Publisher Site

Abstract

Background: The high rate of propagation and easy availability of water hyacinth has made it a renewable carbon source for biofuel production. The present study was undertaken to screen the feasibility of using water hyacinth's hemicelluloses as a substrate for alcohol production by microbial fermentation using mono and co-cultures of Trichoderma reesei and Fusarium oxysporum with Pichia stipitis. Results: In separate hydrolysis and fermentation (SHF), the alkali pretreated water hyacinth biomass was saccharified by crude fungal enzymes of T. reesei, F. oxysporum and then fermented by P. stipitis. In simultaneous saccharification and fermentation (SSF), the saccharification and fermentation was carried out simultaneously at optimized conditions using mono and co-cultures of selected fungal strains. Finally, the ethanol production kinetics were analyzed by appropriate methods. The higher crystalline index (66.7%) and the Fourier transform infrared (FTIR) spectra showed that the lime pretreatment possibly increased the availability of cellulose and hemicelluloses for enzymatic conversion. In SSF, the co-culture fermentation using T. reesei and P. stipitis was found to be promising −1 with a higher yield of ethanol (0.411 g g ) at 60 h. The additional yield comparable with the monocultures was due to the xylanolytic activity of P. stipitis which ferments pentose sugars into ethanol. In SHF, the pretreatment followed by crude enzymatic hydrolysis and fermentation resulted in a significantly lesser yield of ethanol −1 (0.344 g g )at96h. Conclusions: It is evident from the study that the higher ethanol production was attained in a shorter period in the co-culture system containing T. reesei and the xylose fermenting yeast P. stipitis. SSF of pretreated water hyacinth biomass (WHB) with P. stipitis instead of traditional yeast is found to be an effective biofuel production process. Keywords: Water hyacinth; Hemicelluloses; Xylose; T. reesei; F. oxysporum; P. stipitis;SSF Background has posed a threat to the food supply [2], and the cost of The global depletion of fossil fuels that are the dominant these raw materials accounts for up to 40% to 70% of the sources for supplying cheap energy for the world's econ- production cost [3]. Lignocellulosic biomass serves as a omy has prompted recent significant research efforts in cheap and abundant feedstock [4], which has the potential finding viable and sustainable alternatives [1]. Among to produce low-cost bioethanol at a large scale. In recent various options, conversion of abundant lignocellulosic days, screening of such substrates for biofuel has gained biomass to biofuels has received significant attention. Cur- new speed and still there are many factors to be taken into rently, bioethanol production from corn and sugarcane consideration for the large scale production. The performance of enzymatic saccharification is one of the foremost limiting factors which may strongly be * Correspondence: pothi2005@yahoo.com dependent on the diverse species, complex chemical com- PG and Research Department of Botany, Alagappa Government Arts College positions, and structural characteristics of the feedstock (Alagappa University), Karaikudi, Tamilnadu 630 003, India Full list of author information is available at the end of the article © 2014 Pothiraj et al.; licensee Springer. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 2 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 materials. The sugar yields from enzymatic hydrolysis vary Methods from plant to plant as a result of the differences mainly in Biomass and culture organisms cellulose content [5]. Like cellulose, hemicellulose is also a Fresh water hyacinth biomass (WHB) was collected from viable source of fermentable sugars such as xylose for a local pond at Karaikudi, Tamilnadu, India (10.07°N, biorefining applications. It was suggested that the produc- 78.78°E). The collected samples were washed to remove tion of fuel-grade ethanol from xylose requires a micro- adhering dirt, cut into small piece (2 or 3 mm) thick- organism capable of producing 50 to 60 g/L ethanol nesses, and dried in sunlight. The proximate analysis for within 36 h with a yield of at least 0.4 g ethanol per gram biomass was done using standard methods for moisture of sugar [6]. But only few xylose-fermenting microorgan- content, ash, crude protein, crude fibre, cellulose, hemi- isms have been reported earlier [7], and it is generally celluloses, and lignin [16,17]. The fungal strains of T. known that Pichia stipitis is superior to all other yeast spe- reesei and F. oxysporum were isolated by primary selec- cies for ethanol production from xylose. tion from a naturally contaminated water hyacinth, and Water hyacinth (Eichhornia crassipes) is a fast growing the isolates were confirmed by their morphology and perennial aquatic weed invasively distributed throughout colony characteristics [18]. The isolated organisms were the world. This tropical plant can cause infestations maintained on modified potato dextrose agar (PDA) over large areas of water resources and consequently slants at 4°C. Fresh colonies were used for saccharification leads to series of problems like reduction of biodiver- and fermentation studies. The pure culture of P. stipitis sity, blockage of rivers and drainage system, depletion (NCIM 3497) was procured from the National Collection of dissolved oxygen, and alteration on water chemistry of Industrial Microorganisms, Pune, India. that leads to severe environmental pollution. In the past, attempts have been geared towards the use of Alkaline pretreatment biological, chemical, and mechanical approaches for The dried WHB (10% w/v) was pretreated with calcium preventing the spread of, or eradication of, water hya- hydroxide solution (0.5% w/v) with a soaking time of 3 h cinth. On the other hand, much attention has been at 100°C. The pretreated WHB washed to neutrality with focused on the potentials and constrains of using distilled water, oven dried to a constant weight, and then water hyacinth for a variety of applications since it has milled to powder was used for enzymatic hydrolysis and a lignocellulosic composition of 48% hemicelluloses, fermentation [19]. 18% cellulose, and 3.5% lignin [8,9]. Since the biomass productivity of this plant is very high, it can be a suit- Experimental design able feedstock for ethanol production. Two modes of bioconversion methodologies for ethanol The technologies for the possible conversion of production were trialed in the present study. Mode I water hyacinth to biogas or fuel ethanol using fungal comprised of a separate hydrolysis and fermentation extracellular enzymes are well documented in a num- (SHF) process using crude fungal enzymes with yeast. ber of developing countries [10-13]. Saccharomyces Mode II was designed to conduct a simultaneous sac- cerevisiae and Zymomonas mobilis are being used as charification and fermentation (SSF) process using mono candidate organisms in the large-scale production of and co-cultures of selected fungal strains. ethanol from cellulosic biomass. These organisms are capable of utilizing hexose sugars efficiently but not Separate hydrolysis and fermentation (SHF) the pentoses, which are the second dominant sugar The cellulolytic enzymes (cellulases and xylanases) were source in lignocellulosic biomass [14]. From earlier produced by growing the isolated fungal strains of T. research, P. stipitis hasbeenidentifiedasanefficient reesei and F. oxysporum separately at 35°C in a simple −1 −1 strain for the conversion of pentose sugars into alco- liquid medium (4.2 g L (NH ) SO ,2gL KH PO , 4 2 4 2 4 −1 −1 hol [15]. Fermentation technologies utilizing strains 0.05 g L yeast extract, 2 mL L Tween-80, 2% (w/v) of P. stipitis instead of traditional yeast have been pro- poultry manure with 1.6% total N, pH 4.8) containing −1 posed by a number of authors [14,15], as they have 100 g L water hyacinth biomass as the chief C source been shown to ferment under fully anaerobic condi- for 5 days as optimized earlier [20]. The culture superna- tions with faster specific rates of pentose sugar uptake tants were separated at the end of the incubation period and ethanol production as well as an ethanol yield from each organism and used as crude enzymes source for close to theoretical yield. The present study, therefore, hydrolysis. Cellulase and xylanase activities were measured was carried out to screen the feasibility of using in the culture supernatant as per standard methods. hexose- and pentose-utilizing fungal strains (Tricho- Cellulase was measured according to the IUPAC derma reesei, Fusarium oxysporum,and Pichia stipitis) methods [21] using Whatman filter paper no. 1 as the for the effective conversion of water hyacinth biomass substrate and glucose as the standard. Xylanase was into ethanol. assayed by the optimized method described by Bailey Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 3 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 et al. [22], using 1% birchwood xylan as the substrate dichromate method [26]. The microbial biomass was de- and xylose as the standard. One unit (IU) of enzyme termined by harvesting cells by centrifugation, drying activity is defined as the amount of enzyme releasing them at 70°C under vacuum to a constant weight, and 1 μmol glucose or xylose/mL per minute expressed as gram dry cell weight (DCW) per liter [27]. Enzymatic hydrolysis was carried out by incubating The kinetic parameters of ethanol fermentation were de- the pretreated WHB (10% w/v) with the crude fungal termined followed by Abate et al. [28] as follows: enzymes (10% (v/v)) of T. reesei and F. oxysporum separ- Ethanol concentrationðÞ Ec ¼ ½ ethanol produced ðÞ g = ately at 35°C for 48 h with agitation at 200 rpm [23]. volume of reaction mixtureðÞ L The pH of the reaction mixture (6.0) was maintained at Ethanol productivity E ¼½ethanol produced ðÞ g = constant. Samples were aliquoted from hydrolysates at a volume of reaction mixtureðÞ L = regular interval (24 and 48 h) to estimate the released time ðÞ h sugar content using standard methods [24,25]. The hydro- Ethanol Yield E ¼½ethanol produced ðÞ g = lysates obtained after 48 h from both the fungal cultures y weight of substrate ðÞ g were centrifuged at 10,000 rpm for 10 min. The superna- tants were collected separately and supplemented with Specific ethanol yield E ¼½ ethanol produced ðÞ g = sy −1 −1 basal medium (1 g L yeast extract; 2 g L (NH )SO ; 4 4 sugar consumed ðÞ g −1 1gL MgSO � 7H O) (pH 6.0) [23]. The culture suspen- 4 2 The results obtained were analyzed by using analysis sion of P. stipitis (10% v/v) was added to initiate the fer- of variance (ANOVA), and the group means were com- mentation by incubating the mixture at 35°C for 48 h with pared with Duncan's Multiple Range Test (DMRT) [29]. agitation at 200 rpm. Fourier transform infrared (FTIR) analysis Simultaneous saccharification and fermentation (SSF) Fourier transform infrared spectra were studied on SSF represents a single step process in which ferment- treated and untreated WHB using a Shimadzu spec- able sugars get released by enzymatic hydrolysis and are trometer (Shimadzu, Kyoto, Japan). For this, 3.0 mg of simultaneously exploited by yeasts for fermentation in the sample was dispersed in 300 mg of spectroscopic the same medium. The microbial fermentation was carried grade KBr and subsequently pressed into disks at out using mono and co-cultures as previously described 10 MPa for 3 min. The spectra were obtained with an [9]. The influences of various parameters such as micro- −1 average of 25 scans and a resolution of 4 cm in the bial biomass (5% to 25%), temperature (25°C to 45°C), and −1 range of 4,000 to 400 cm . incubation time (24, 36, 48, 60, 72 h) on SSF were also optimized by step-wise experiments where the specified X-ray diffraction (XRD) analysis parameters were changed by keeping all other parameters The crystallinity of cellulose in the pretreated and constant. The pH of the reaction mixture in all the treated water hyacinth was analyzed by X-ray diffraction optimization experiments was kept constant at 6.0 method in a PANalytical X'pert PRO Diffractometer (PANalytical B.V., Almelo, Netherlands) set at 40 KV, Mono and co-culture fermentations 30 mA; radiation was Cu Kα(λ = 1.54Ǻ) and the grade For monoculture experiments (F1 and F2), previously 0 0 range between 10 to 30 with a step size of 0.03 . The sterilized (121°C for 60 min) pretreated WHB supple- crystallinity index (CrI) was determined based on the mented with a basal medium (without C source) was equation shown below [30]: inoculated with late log-phase cultures of T. reesei (F1) and F. oxysporum (F2), separately. For co-culture fer- I −I 002 am CrI ¼  100 mentation (F3 and F4), separate sets of reaction mixtures am consisting of pretreated WHB supplemented with basal medium were treated with P. stipitis simultaneously with where I is the intensity of the diffraction from the 002 T. reesei (F3) and F. oxysporum (F4). The fermentation plane at 2θ = 22.6° and I is the intensity of the back- am process was carried out at optimized conditions. ground scatter measured at 2θ = 18.7°. It is known that the I peak corresponds to the crystalline fraction and the Estimations I peak corresponds to the amorphous fraction [31]. am Samples were withdrawn from the fermenting media at regular intervals of time for the determination of etha- Results and discussion nol, residual sugar concentration, and microbial biomass. The lignocellulosic biomass composition of WH includes −1 Estimation of xylose was done by the Trinder method cellulose (20.2 g 100 g dry matter (DM)), hemicellulose −1 −1 [24] and glucose by the DNS method [25]. Ethanol esti- (34.3 g 100 g DM), lignin (4.4 g 100 g DM ), crude −1 −1 mation was done spectrophotometrically by potassium protein (13.3 g 100 g DM), crude fibre (18.2 g 100 g Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 4 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 Table 1 Proximate composition of water hyacinth biomass (WHB) comparable with earlier literatures Content Present study Previous literature −1 [g 100 g DM] −1 −1 −1 [g 100 g DM] [8] [g 100 g DM] [12] [g 100 g DM] [32] Moisture (%) 92.8 - - - Ash 15.4 20.0 - 20.0 Crude protein 13.3 18.0 10.2 18.0 Crude fibre 18.2 - - - Cellulose 20.2 25.0 19.02 25.0 Hemicellulose 34.3 35.0 32.69 35.0 Lignin 4.4 10.0 4.37 10.0 DM, dry matter. −1 DM), and ash (15.4 g 100 g DM) (Table 1). The results increased the crystallinity of cellulose in water hyacinth. obtained in the present study on the proximate compos- Similar results were reported earlier by Kim and Holtzapple ition of WHB are basically consistent with previous [39] whofound that thedegreeof crystallinityofcorn literatures [8,12,32]. The digestibility of lignocelluloses is stover slightly increased from 43% to 60% through delignifi- hindered by many physicochemical, structural, and com- cation with calcium hydroxide and by Li et al. [40] who positional factors which required a suitable pretreatment have reported high cellulose CrI of 70.6% in Metasequoia in order to enhance the susceptibility of biomass for chipsbynitricacid-ethanol method. TheincreaseinCrI of hydrolysis. It is highly essential for the economical produc- alkali-treated WHB might be due to the removal of tion of ethanol that both the cellulose and hemicellulosic amorphous components including lignin during the pre- sugars present in the biomass should be utilized efficiently. treatment process [41,42]. According to Satyanagalakshmi The FTIR and XRD data in the present study clearly sug- et al. [33], the amorphous cellulose portions in aquatic gested that the pretreatment with lime could increase the plants are more prone to recrystallization to form crystal- availability of polysaccharide for enzymatic hydrolysis. line cellulose, resulting in greater increases in CrI. Among different pretreatment methods used in earlier re- searches for water hyacinth, maximum reducing sugar was −1 observed in diluted H SO (0.342 g g biomass) [33], HCl 2 4 −1 −1 140 Untreated WHB (0.277 g g biomass), acetic acid (0.097 g g biomass), −1 and formic acid (0.088 g g biomass) [31,34]. In compari- son with the above reports, it is evident that the lime pretreatment used in the present study is a promising method for higher sugar yield. The pretreatment with Ca (OH) is preferable because it is less expensive, more safe as compared to NaOH, and it can be easily recovered from the hydrolysate by reaction with CO . Lime has been used 20 to pretreat many lignocellulosic materials such as wheat straw [35], poplar wood [36], and corn stover [37]. XRD - cellulose crystallinity Alkali pretreated WHB Cellulose crystallinity, usually measured as CrI, is con- sidered an important parameter determining the enzym- atic hydrolysis susceptibility of cellulose. The CrI of a cellulose sample is an indication of the degree of formed crystallinity in the sample when the cellulose aggregates. The crystallinity has been found to have a greater impact on enzymatic hydrolysis than other structural characteris- tics such as the degree of polymerization (DP) of cellulose or the specific surface area (SSA) [38]. The XRD profile of 5 101520 2530354045 WHB indicated that the CrI of untreated WHB is 28.6% 2 Theta and alkali-treated WHB is 66.7% (Figure 1). The X-ray dif- Figure 1 XRD analysis of untreated and lime pretreated WHB. fractogram clearly revealed that the lime pretreatment Intensity Intensity Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 5 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 FTIR analysis p-coumeric acids of lignin and/or hemicelluloses [44]. A −1 FTIR spectra of the untreated and treated samples indi- sharp band at 896 cm , corresponding to the C1 group cated structural changes in the biomass upon pretreat- frequency or ring frequency, was attributed to the glyco- ment (Figure 2). The increased absorption bands at sidic linkages between xylose units in hemicelluloses −1 1,000 to 1,200 cm were related to structural features of [45]. The peaks in the pretreated sample had the highest cellulose and hemicelluloses [43]. The spectra of alkali- absorbance suggesting increase in cellulose and hemicel- treated WHB sample (Figure 3) showed increase in lulose content. In the FTIR spectrum, the peaks ob- −1 absorbance in the above-mentioned range. The peak at served at 1,092 and 842 cm were attributed to C-O −1 1,635 cm was observed due to either the acetyl and ur- stretching and C-H rocking vibration of the cellulose onic ester linkage of carboxylic group of the ferulic and structure. Untreated WHB Alkali treated WHB Figure 2 FTIR spectra of untreated and alkali (Ca (OH) )-treated WHB. 2 Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 6 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 0.5 0.4 0.3 F1 F2 0.2 F3 0.1 F4 12 24 36 48 60 72 Fermentation period (h) Figure 3 Effect of fermentation period on ethanol yield from pretreated WHB using mono and co-cultures. F1, T. reesei;F2, F. oxysporum; F3, T. reesei + P. stipitis;F4, F. oxysporum + P. stipitis. Enzyme activity Sugar yield In recent decades, the use of fungi in bioprocesses has The yield of sugars from enzymatic hydrolysis of WHB growninimportancebecause of theproductionofnu- using crude enzymes produced by fungal isolates was sum- merous enzymes with different biochemical properties marized in Table 2. The saccharification was significantly and excellent potential for biotechnological applica- higher (40.8%) while using crude enzyme from T. reesei tion. The cellulase and xylanase activities reached when compared to F. oxysporum (38.2%). The release of their maximum values on the 6th day of incubation total sugars by the crude enzymes of both monocultures in- for both the fungal isolates. Cellulase production creased slowly to reach a peak value at 48 h of incubation. −1 on WHB with nutrient supplements indicated higher The maximum yield of total sugar (0.531 g g WHB) in- −1 −1 cellulase production by T. reesei (0.923 IU/mL) com- cluding glucose (0.444 g g WHB) and xylose (0.057 g g pared to F. oxysporum (0.432 IU/mL). However, it WHB) was observed after 48 h of hydrolysis using crude is less than the value of 1.35 IU/mL reported by enzymes of T. reesei. The crude enzymes obtained from F. Deshpande et al. [46] on the substrate water hyacinth oxysporum produced comparably lower reducing sugar −1 −1 with Toyama-Ogawa medium [47]. The xylanase produc- (0.428 g g WHB) and xylose (0.038 g g WHB). Thus, it tion was slight, but significantly higher in F. oxysporum substantiates that the amount of sugar released increases (0.764 IU/mL), compared to T. reesei (0.611 IU/mL). Ac- with time which may be due to the increased action of cording to Kang et al. [48], high xylanase production in cellulolytic and xylanolytic enzymes of T. reesei and F. some fungi has been shown to be linked strictly to the oxysporum [51]. The cellulolytic fungus T. reesei looks ratio of cellulose to xylan of the growth substrate and promising for on-site cellulase production due to its substrate degradation due to time course or incubation superior features, i.e., capability to produce all components period. of cellulase complex, endocellulase, exocellulase, and β- According to Polizeli et al. [49], filamentous fungi are glucosidase in good proportions as well as production of widely utilized as enzyme producers and are generally other enzymes such as xylanases or laccases in comparison considered more potent xylanase producers than bacteria to other enzyme producers [52]. or yeast. Several mesophilic fungal species have been evaluated in relation to xylanase production, including Ethanol members of Aspergillus, Trichoderma,and Penicillium. The optimization studies in SSF showed that the yield of Currently, most commercial xylanolytic preparations ethanol is found to be proportional to fermentation time are produced by genetically modified Trichoderma or where the yield increases with the increase in time up to Aspergillus strains [50]. 60 h and then declines (Figure 3). Maximum yield of −1 Table 2 Sugar composition (g g WHB) of enzymatic hydrolysates of pretreated WHB at 48 h Enzyme source Glucose Xylose Total sugar Saccharification % a a a T. reesei 0.444 ± 0.12 0.057 ± 0.09 0.531 ± 0.12 40.2 b b b F. oxysporum 0.428 ± 0.31 0.038 ± 0.11 0.488 ± 0.17 38.2 Values are the mean of three replicates ± SE. Means followed by the same letter within treatment do not differ significantly (p = 0.05). -1 Ethanol Yield (g g WHB) Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 7 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 0.5 0.4 F1 0.3 F2 0.2 F3 F4 0.1 25 30 35 40 45 Temperature C Figure 4 Effect of temperature on ethanol yield from pretreated WHB using mono and co-cultures. F1, T. reesei;F2 F. oxysporum;F3 T. reesei + P. stipitis;F4, F. oxysporum + P. stipitis. −1 ethanol is 0.413, 0.378, 0.194, and 0.187 g g of WHB fermentation of pretreated water hyacinth biomass at 60 h of fermentation for F3, F4, F1, and F2, respect- using monocultures of T. reesei and F. oxysporum was ively. After 60 h of time, the yield of ethanol decreases due to the inability of these organisms to convert pen- in all treatments, and therefore, fermentation time of tose sugars into ethanol. A similar finding was reported 60 h is taken as the optimum time for ethanol fermenta- earlier where 0.11 g ethanol was obtained from alkali- tion. With the increase in temperature, the yield of pretreated water hyacinth through SHF [23]. According ethanol increased up to 35°C and then it decreased to Preez et al. [53] P. stipitis is known to produce etha- (Figure 4). At high temperature (>35°C), death rate ex- nolupto33to57g/L;however,30g/L is known as a ceeds the growth rate, which causes a net decrease in critical concentration above which cells cannot grow at concentration of viable fungal populations with lower 30°C. generation of ethanol. With the increase in loading of In SSF, monocultures of T. reesei (F1) and F. oxysporum −1 biomass, the yield of ethanol increased up to 10% and (F2) produced 19.3 and 17.8 g L ethanol, respectively, then decreased in all the samples (Figure 5). The de- after 60-h fermentation (Table 4). Simultaneous co- crease in ethanol yield with the increase in biomass culturing of T. reesei (F3) and F. oxysporum (F4) with P. loading can be attributed to the inhibitory effect of ei- stipitis resulted in a higher ethanol production (40.8 and −1 ther the product or the biomass. Inhibitory compounds 36.8 g L , respectively) at the same time. The maximal −1 limit efficient utilization of hydrolysates by the ferment- ethanol yield was 0.411 g g WHB when P. stipitis was ing organism resulting in less ethanol production [33]. used along with T. reesei which is positively correlated to −1 In the SHF process, a maximum of 14.3 g L ethanol the theoretical yield 0.429 g on the basis of biomass. Since was produced at the end of the process (96 h) which is xylose was present as a predominant sugar in the WHB −1 equivalent to 0.143 g g WHB (Table 3). The mini- hydrolysate, P. stipitis was used to make the biomass-to- mum production of ethanol observed in the submerged ethanol process more economical. Mishima et al. [34], on 0.5 0.4 0.3 F1 F2 0.2 F3 F4 0.1 510 15 20 25 Microbial biomass (% v/v) Figure 5 Effect of microbial biomass on ethanol yield from pretreated WHB using mono and co-cultures. F1, T. reesei;F2, F. oxysporum;F3, T. reesei + P. stipitis;F4, F. oxysporum + P. stipitis. 1 1 Ethanol Yield (g g WHB) Ethanol Yield (g g WHB) Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 8 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 Table 3 Ethanol production in SHF process using crude fungal enzymes and P. stipitis with pretreated WHB at 96 h Sample (s)E E E E E c p y sy TE c a c c c S 14.3 ± 0.12 0.24 ± 0.09 0.143 ± 0.11 0.322 ± 0.11 0.261 ± 0.11 d a d d d S 12.8 ± 0.13 0.21 ± 0.17 0.128 ± 0.08 0.299 ± 0.08 0.243 ± 0.17 −1 −1 −1 −1 −1 E , ethanol concentration (g L ); E , ethanol productivity (g L h ); E , ethanol yield (g g WHB); E , specific ethanol yield (g g sugar); E , theoretical yield c p y sy TE −1 (g g WHB); S1, hydrolysate from T. reesei; S2, hydrolysate from F. oxysporum. Values are the mean of three replicates ± SE. Mean followed by the same letter within treatment do not differ significantly (p = 0.05). the other hand, reported a lesser ethanol yield of 0.14 obtained in the co-culture of T. reesei and P. stipitis at −1 gg dry substrate through SSF of pretreated water hya- 60-h fermentation (Table 4). Inoculation of P. stipitis cinth using commercial cellulase and S. cerevisiae.The with F. oxysporum resulted in a biomass content of −1 overall production could be enhanced by co-culture rather 2.64 g DCW L over the monocultures. Statistically, a than monoculture of test organisms. Similarly, direct mi- less significant difference was observed with monocul- crobial conversion of cellulosic or lignocellulosic biomass ture's fermentation when compared with co-culture [58]. into ethanol using co-cultures had been reported by sev- eral authors [32,34,54]. S. cerevisiae or Z. mobilis utilize Conclusions glucose or sucrose efficiently but their inability to utilize The fermentation of bioethanol from pretreated water pentose sugars make them inappropriate candidates for hyacinth biomass with mono and co-cultures of fungal refineries, but the candidate organism P. stipitis used in strains along with P. stipitis is found to be an effective bio- the present study showed efficient conversion of pentose fuel production process. The yield of ethanol recovered sugars into alcohol. Among the pentose-fermenting organ- from WHB through enzymatic hydrolysis and fermentation isms, P. stipitis has been shown to have the most promise from simultaneous inoculation of co-cultures of fungal for industrial applications [55]. Earlier reports showed isolates with P. stipitis was significantly higher than that re- that the hemicellulosic hydrolysates of Prosopis juliflora covered through monocultures. The optimum parameters (18.24 g sugar/L broth) when fermented with P. stipitis for bioethanol fermentation are as follows: time 60 h, −1 produced 7.13 g/L ethanol [56]. Kuhad et al. [57] observed temperature 35°C, and WHB loading 100 g L .The max- −1 0.33 g g ethanol yield from detoxified xylose-rich hydrol- imum yield of ethanol in the fermentation process was −1 ysate of Lantana camara fermented with P. stipitis at found to be 0.411 g g of WHB which is equivalent to −1 pH 5 for 36 h. Similarly, the detoxified water hyacinth a specific yield of 0.456 g g total sugar consumed. hemicellulose acid hydrolysate (rich in pentose sugars) fer- The use of crude fungal enzymes produced on-site mented with P. stipitis NCIM-3497 at pH 6.0 and 30°C re- would be a cost-effective approach towards enzymatic sulted in 0.425 g ethanol/g lignocelluloses [15]. The yield hydrolysis of alkali-pretreated WHB biomass instead of ethanol per unit biomass of water hyacinth obtained of using commercial cellulases. The aquatic menace through the bioprocess in the present study was compar- water hyacinth, which is currently being used in waste able to or even better than those reported earlier. The water treatment for its unique ability to absorb heavy current results clearly demonstrated the saccharification metal pollutants, could also be utilized as abundant potential of T. reesei and F. oxysporum, where the per- cheap feedstock for the production of fuel ethanol. formance of both strains in co-cultures with P. stipitis was This study proved that water hyacinth has a potential significantly higher than their respective single culture. renewable and low-cost biomass for alcohol produc- tion on the commercial scale. Present cost effective- Microbial biomass ness of respective process at a commercial scale All the co-culture processes reached a higher value of needs to be standardized, and the water hyacinth microbial biomass than the single fermentation process. biomass could be a better substrate source for alco- −1 A maximum of 3.12 g DCW L biomass content was hol production. Table 4 Ethanol production in mono and co-culture fermentation process (SSF) using pretreated WHB at 60 h −1 Culture (s)E E E E Microbial biomass (g DCW L ) c p y sy c c c c c T. reesei 19.3 ± 0.12 0.32 ± 0.09 0.196 ± 0.11 0.377 ± 0.11 2.14 ± 0.01 d d d d c F. oxysporum 17.8 ± 0.13 0.29 ± 0.17 0.176 ± 0.08 0.348 ± 0.08 2.06 ± 0.08 a a a a a T. reesei + P. stipitis 40.8 ± 0.09 0.68 ± 0.14 0.411 ± 0.03 0.798 ± 0.11 3.12 ± 0.12 b b b b b F. oxysporum + P. stipitis 36.8 ± 0.06 0.61 ± 0.12 0.371 ± 0.07 0.720 ± 0.08 2.64 ± 0.20 −1 −1 −1 −1 −1 E , ethanol concentration (g L ); E , ethanol productivity (g L h ); E , ethanol yield (g g WHB); E , specific ethanol yield (g g sugar). Values are the mean of c p y sy three replicates ± SE. Mean followed by the same letter within treatment do not differ significantly (p = 0.05). Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 9 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 Competing interests 15. Kumari N, Bhattacharya A, Dey A, Ganguly A, Chatterjee PK (2014) The authors declare that they have no competing interests. Bioethanol production from water hyacinth biomass using isolated fungal strain from local environment. Biolife 2(2):516–522 16. Association of Official Analytical Chemists (AOAC) (1975) Methods of Authors' contributions analysis of the Association of Official Analytical Chemists. Association of CP carried out the submerged fermentation processes and helped design Official Analytical Chemists, Washington DC the whole study. RA carried out proximate analysis of biomass and helped in 17. Robertson JB, van Soest PJ (1981) The Detergent System of Analysis and its manuscript preparation. RMG helped in characterization of biomass using application to human foods. In: James WPT, Thiander O (eds) The analysis of FTIR and XRD. All authors read and approved the final manuscript. dietary fibers in food. Marcel Dekker, New York, pp 123–158 18. Alexopoulos CJ, Beneke ES (1962) Laboratory manual for introductory mycology. 1, 2 Burgess publishing Co, Minneapolis Acknowledgements 19. Chang VS, Burr B, Holtzapple MT (1997) Lime pretreatment of switchgrass. The authors are thankful to all faculty members of the PG and Research Appl Biochem Biotechnol 63(65):3–19 Department of Botany, Alagappa Government Arts College (Alagappa University), Karaikudi. We also acknowledge the Head, Department of 20. Mukhopadhyay S, Nandi B (2001) Cellulase production by Trichoderma reesei physics, Alagappa University, Karaikudi and the Department of Chemistry, on pretreated water hyacinth: effect of nutrients. J Mycopathol Res VHNSN College, Virudhunagar, Tamilnadu for helping us with XRD and FTIR 39(1):57–60 analysis, respectively. 21. Ghose TK (1987) Measurement of cellulose activities. Pure Applied Chem 59(257):268 Author details 22. Bailey M, Buchert J, Viikari L (1993) Effect of pH on production of xylanase PG and Research Department of Botany, Alagappa Government Arts College of Trichoderma reesei on xylan- and cellulose-based media. Appl Microbiol (Alagappa University), Karaikudi, Tamilnadu 630 003, India. Department of Biotechnol 40:224–229 Microbiology, VHNSN College, Virudhunagar, Tamilnadu 626 001, India. 23. Mukhopadhyay S, Mukherjee PS, Chatterjee NC (2008) Optimization of enzymatic hydrolysis of water hyacinth by Trichoderma reesei vis-à-vis Received: 30 July 2014 Accepted: 10 November 2014 production of fermentable sugars. Acta Aliment 37(3):367–377 24. Trinder P (1975) Micro-determination of xylose in plasma. Analyst 100:12–15 25. Miller GL (1959) Use of DNS reagent for the determination of reducing sugars. Anal Chem 31:426–428 References 26. Caputi A, Veda M, Brown T (1968) Spectrophotometric determination of 1. Chang KL, Thitikorn-amorn J, Hsieh JF, Ou BM, Chen SH, Ratanakhanokchai ethanol in wine. American J Enol Viticul 19:160–165 K, Huang PJ, Chen ST (2011) Enhanced enzymatic conversion with freeze 27. Doelle HW, Greenfield PF (1985) The production of ethanol from sucrose pretreatment of rice straw. Biomass Bioenergy 35(1):90–95 using Zymomonas mobilis. Appl Microbial Biotechnol 22:405–441 2. Guragain YN, De Coninck J, Husson F, Durand A, Rakshit SK (2011) 28. Abate C, Callieri D, Rodriguez E, Garro O (1996) Ethanol production by Comparison of some new pretreatment methods for second generation mixed culture of flocculent strains of Zymomonas mobilis and bioethanol production from wheat straw and water hyacinth. Bioresour Saccharomyces sp. Appl Microbial Biotechnol 45:580–583 Technol 102(6):4416–4424 29. Duncan BD (1957) Multiple range tests for correlated and heteroscedastic 3. Quintero JA, Montoya MI, Sánchez OJ, Giraldo OH, Cardona CA (2008) Fuel means. Biometrics 13(2):359–364 ethanol production from sugarcane and corn: comparative analysis for a 30. Segal L, Creely JJ, Martin AE, Conrad CM (1959) An empirical method for Colombian case. Energy 33(3):385–399 estimating the degree of crystallinity of native cellulose using X –Ray 4. Balat M (2011) Production of bioethanol from lignocellulosic materials via diffractometer. Text Res J 29:786–794 the biochemical pathway: a review. Energy Convers Manage 52(2):858–875 31. Wang LS, Zhang YZ, Gao PJ, Shi DX, Liu HW, Gao HJ (2006) Changes in the 5. Sukumaran RK, Singhania RR, Mathew GM, Pandey A (2009) Cellulase structural properties and rate of hydrolysis of cotton fibers during extended production using biomass feed stock and its application in lignocelluloses enzymatic hydrolysis. Biotechnol Bioeng 93(3):443–456 saccharification for bio-ethanol production. Renew Energy 34(2):421–424 32. Aswathy US, Sukumaran RK, Lalitha Devi G, Rajasree KP, Singhania RR, 6. Lee Y, Kim M, Kim K, Park K, Ryu Y, Seo J (2000) A parametric study on Pandey A (2010) Bio-ethanol from water hyacinth biomass: an evaluation of ethanol production from xylose Pichia stipitis. Biotechnol Bioprocess Eng enzymatic saccharification strategy. Biores Technol 101:925–930 5:27–31 33. Satyanagalakshmi K, Sindhu R, Binod P, Janu KU, Sukumaran RK, Pandey A 7. Ganguly A, Das S, Bhattaacharya A, Dey A, Chatterjee PK (2013) Enzymatic (2011) Bioethanol production from acid pretreated water hyacinth by hydrolysis of water hyacinth biomass for the production of ethanol: separate hydrolysis and fermentation. J Sci Ind Res 70:156–161 optimization of driving parameters. Ind J Exp Biol 51:556–566 34. Mishima D, Kuniki M, Sei K, Soda S, Ike M, Fujita M (2008) Ethanol 8. Gunnarsson CC, Petersen CM (2007) Water hyacinth as a resource in production from candidate energy crops: water hyacinth (Eichhornia agriculture and energy production: a literature review. Waste Man crassipes) and water lettuce (Pistia stratiotes L.). Biores Technol 27:117–129 99:2495–2500 9. Nigam JN (2002) Bioconversion of water hyacinth (Eichhornia crassipes) 35. Chang VS, Nagwani M, Holtzapple MT (1998) Lime pretreatment of crop hemicellulose acid hydrolysate to motor fuel ethanol by xylose-fermenting residues bagasse and wheat straw. Appl Biochem Biotechnol 74:135–159 yeast. J Biotech 97:107–116 36. Chang VS, Nagwani M, Kim CH, Holtzapple MT (2001) Oxidative lime 10. Zabala I, Ferrer A, Ledesma A, Aiello C (1994) Microbial protein production pretreatment of high-lignin biomass. Appl Biochem Biotechnol 94:1–28 by submerged fermentation of mixed cellulolytic cultures In: Galindo E, 37. Karr WE, Holtzapple MT (1998) The multiple benefits of adding non-ionic Ramirez OT (eds) Advances in Bioprocess Engineering. Kluwer Academic surfactant during the enzymatic hydrolysis of corn stover. Biotechnol Bioeng Publishers, The Netherlands, pp 455-460 59:419–427 11. Singhal V, Rai JP (2003) Biogas production from water hyacinth and channel 38. Peng HD, Li HQ, Luo H, Xu J (2013) A novel combined pretreatment of ball grass used for phytoremediation of industrial effluents. Bioresour Technol milling and microwave irradiation for enhancing enzymatic hydrolysis of 86:221–225 microcrystalline cellulose. Bioresour Technol 130:81–87 12. Sornvoraweat B, Kongkiattikajorn J (2010) Separated hydrolysis and 39. Kim S, Holtzapple MT (2006) Effect of structural features on enzyme fermentation of water hyacinth leaves for ethanol production. KKU Res J digestibility of corn stover. Bioresource Technol 97:583–591 15(9):794–802 40. Li L, Wenbing Z, Hongwei W, Yun Y, Fen L, Duanwei Z (2014) Relationship 13. Idrees M, Adman A, Sheikh S, Qureshi FA (2013) Optimization of dilute acid between crystallinity index and enzymatic hydrolysis performance of pretreatment of water hyacinth biomass for enzymatic hydrolysis and cellulose separated from aquatic and terrestrial plant materials. Bioresources ethanol production. EXCLI J 12:30–40 9(3):3993–4005 14. Bhattacharya A, Ganguly A, Das S, Chatterjee PK, Dey A (2013) Fungal isolates from local environment: isolation, screening and application for the 41. Converse AO, Kwartneg IK, Grethlein HE, Ooshima H (1989) Kinetics of production of ethanol from water hyacinth. Int J Emerg Tech Adv Engi thermochemical pretreatment of lignocellulosic materials. Appl Biochem 3(3):58–65 Biotechnol 20(21):63–78 Pothiraj et al. Bioresources and Bioprocessing 2014, 1:27 Page 10 of 10 http://www.bioresourcesbioprocessing.com/content/1/1/27 42. Maeda RB, Serpa VI, Rocha RAL, Mesquita RAA, Anna LMMS, De Carlo AM, Driemeier CE, Pereira N, Polikarpov I (2011) Enzymatic hydrolysis of pretreated sugar cane baggase using Penicillium funiculosum and Trichoderma harzianum cellulases. J Process Biochem 30:5–8 43. Hu J, Arantes J, Saddler JN (2011) The enhancement of enzymatic hydrolysis of lignocellulosic substrates by the addition of accessory enzymes such as xylanase: is it an additive or synergistic effect. Biotechnol Biofuels 4:36 44. Langkilde FW, Svantesson A (1995) Identification of celluloses with Fourier- transform (FT) mid-infrared, FT-Raman and near-infrared spectrometry. J Pharm Biomed Anal 13:409 45. Marimuthu TS, Atmakuru R (2012) Isolation and characterization of cellulose nanofibers from the aquatic weed water hyacinth-Eichhornia crassipes. Carbohydr Polym 87:1701 46. Deshpande SK, Bhotmange MG, Chakrabarti T, Shastri PN (2008) Production of cellulase and xylanase by Trichoderma reesei (QM 9414 mutant), Aspergillus niger and mixed culture by solid state fermentation (SSF) of water hyacinth (Eichhornia crassipes). Ind J Chem Tech 15:449–456 47. Toyama M, Ogawa K (1977) Cellulase production of Trichoderma viride in solid and submerged culture methods. In: Ghosh TK, Ghosh TK (eds) Proc. symp. On bioconversion of cellulosic substrates into energy, chemical and microbial protein. IIT, New Delhi, India, pp 305–312 48. Kang SW, Park YS, Lee JS, Hong SI, Kim SW (2004) Production of cellulose and hemicellulases by Aspergillus niger KK2 from lignocellulosic biomass. Bioresour Technol 91:153–156 49. Polizeli MLT, Rizzati ACS, Monti R, Terenzi HF, Jorge JA, Amorin DS (2005) Xylanases from fungi: properties and industrial applications. Appl Microbiol Biotechnol 67(5):577–591 50. Mussatto SI, Teixeira JA (2010) Lignocellulose as raw material in fermentation processes. In: Méndez-Vilas A (ed) Current research, technology and education topics in applied microbiology and microbial biotechnology. Formatex Research Center, Badajoz, pp 897–907 51. Sun YC, Weu JL, Xu F, Sun RC (2011) Structure and thermal characterization of hemicelluloses isolated by organic solvents and alkaline solutions from Tamarix austromongolica. Biores Technol 102:5947 52. Arantes V, Saddler JN (2010) Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis. Biotechnol Biofuels 3(4):1–11 53. Preez JC, Bosch M, Prior BA (1987) Temperature profiles of growth and ethanol tolerance of xylose fermenting yeasts Candida shehatae and Pichia stipitis. Appl Microbiol Biotechnol 25:521–525 54. Mukhopadhyay S, Chatterjee NC (2010) Bioconversion of water hyacinth hydrolysate into ethanol. BioResourses 5(2):1301–1310 55. Agbogbo FK, Coward-Kelly G, Torry-Smith M, Wenger KS (2006) Fermentation of glucose/xylose mixtures using Pichia stipitis. Process Biochem 41:2333–2336 56. Gupta R, Sharma KK, Kuhad RC (2009) Separate hydrolysis and fermentation (SHF) of Prosopis juliflora, woody substrate for the production of cellulosic ethanol by Saccharomyces cerevisiae and Pichia stipitis-NCIM 3498. Bioresour Technol 100:1214–1220 57. Kuhad RC, Gupta R, Khasa YP, Singh A (2010) Bioethanol production from Lantana camara (red sage): pretreatment, saccharification and fermentation. Bioresour Technol 101:8348–8354 58. Manilal VB, Narayanan CS, Balagopalan C (1991) Cassava starch effluent treatment with concomitant SCP production. World J Microbiol Biotechnol 7:185–190 doi:10.1186/s40643-014-0027-3 Cite this article as: Pothiraj et al.: Sustaining ethanol production from lime pretreated water hyacinth biomass using mono and co-cultures of isolated fungal strains with Pichia stipitis. Bioresources and Bioprocessing Submit your manuscript to a 2014 1:27. journal and benefi t from: 7 Convenient online submission 7 Rigorous peer review 7 Immediate publication on acceptance 7 Open access: articles freely available online 7 High visibility within the fi eld 7 Retaining the copyright to your article Submit your next manuscript at 7 springeropen.com

Journal

"Bioresources and Bioprocessing" – Springer Journals

Published: Dec 1, 2014

Keywords: Biochemical Engineering; Environmental Engineering/Biotechnology; Industrial and Production Engineering

There are no references for this article.