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

Learn More →

Ethanol production from Kinnow mandarin (Citrus reticulata) peels via simultaneous saccharification and fermentation using crude enzyme produced by Aspergillus oryzae and the thermotolerant Pichia kudriavzevii strain

Ethanol production from Kinnow mandarin (Citrus reticulata) peels via simultaneous... Ann Microbiol (2012) 62:655–666 DOI 10.1007/s13213-011-0302-x ORIGINAL ARTICLE Ethanol production from Kinnow mandarin (Citrus reticulata) peels via simultaneous saccharification and fermentation using crude enzyme produced by Aspergillus oryzae and the thermotolerant Pichia kudriavzevii strain Simanjeet Kaur Sandhu & Harinder Singh Oberoi & Sandeep Singh Dhaliwal & Neha Babbar & Ujjal Kaur & Dhiraj Nanda & Dinesh Kumar Received: 12 March 2011 /Accepted: 16 June 2011 /Published online: 8 July 2011 # Springer-Verlag and the University of Milan 2011 . . Abstract The aim of this study was to assess the potential Keywords Aspergillus oryzae Ethanol . . of using the crude filtrate extract (CFE) produced by a Pichia kudriavzevii Kinnow peels newly isolated strain of Aspergillus oryzae and fermentation Simultaneous saccharification and fermentation with a novel thermotolerant strain of Pichia kudriavzevii for the production of ethanol from kinnow peel waste (KP) via Introduction simultaneous saccharification and fermentation (SSF). High-performance liquid chromatography determination Demand for fuels produced from renewable sources has showed that pre-hydrolysis of KP with CFE at 3 cellulase increased in recent years due to increased oil prices, filter paper units/g dry substrate (FPU/g-ds) at 50°C -1 -1 concerns about greenhouse gas production and increasing resulted in 24.87±0.75 g l glucose, 21.98±0.53 g l -1 -1 dependence on foreign sources of energy (Hill et al. 2006). fructose, 10.86±0.34 g l sucrose and 6.56±0.29 g l The development of energy from renewable sources has the galacturonic acid (GA) along with insignificant amounts of potential to provide domestic energy supplies, while arabinose, galactose and xylose. Simultaneous saccharifi- reducing net greenhouse gas emission and developing a cation and fermentation of hydrothermally pretreated KP at -1 more favorable energy balance than traditional petroleum a substrate concentration of 15% (w v ) was conducted in a production (Farrell et al. 2006). Unlike fossil fuels, ethanol 2.5-l laboratory fermentor with P. kudriavzevii at 40°C after is a renewable energy source produced through the a 3-h pre-hydrolysis. Oligosaccharides were not produced fermentation of sugars. It is currently used as a partial during the SSF process. Ethanol production leveled off after gasoline replacement in a number of countries in the world. 12 h, resulting in an ethanol concentration and productivity -1 -1 -1 Kinnow mandarin (Citrus reticulata) belongs to the citrus of 33.87 g l and 2.82 g l h , respectively. These results family of fruits and is an economically important fruit demonstrate the potentiality of SSF using crude enzymes grown in India and Pakistan. According to Food and and P. kudriavzevii for the scale-up production of ethanol Agriculture Organization of the United Nations statistics, from KP. India produces around 7.13 million tonnes of citrus fruits annually, of which kinnow mandarin accounts for about 15%. Because of its high juice content and sweet taste, : : : S. Kaur Sandhu H. Singh Oberoi (*) S. Singh Dhaliwal there has been an upsurge in kinnow production in India N. Babbar U. Kaur during the past few years. Kalra et al. (1989) reported that Central Institute of Post Harvest Engineering and Technology, P.O. PAU, Ludhiana 141 004 Punjab, India kinnow mandarin processing residues, composed of peels, e-mail: hari_manu@yahoo.com pulp and seeds, are rich in carbohydrates and account for about 55–60% of the weight of the raw fruit. Dhillon et al. D. Nanda D. Kumar (2004) proposed that citrus peel waste could be used to National Bureau of Animal Genetic Resources, Karnal, India produce important commercial products, such as enzymes, 656 Ann Microbiol (2012) 62:655–666 ethanol, microbial biomass, volatile flavoring compounds, was to assess the potential of using crude enzymes obtained organic acids and antioxidants. The residues obtained from from a newly isolated A. oryzae strain and a novel citrus fruits are rich in fermentable sugars, such as glucose, thermotolerant P. kudriavzevii isolate in a SSF process for fructose, sucrose and galactose, as well as in insoluble ethanol production using kinnow peel waste (KP) in a 2.5- polysaccharides, such as cellulose, hemicellulose and pectin l laboratory fermentor. (Grohmann et al. 1995; Oberoi et al. 2011). Despite this richness in sugars, proteins and minerals, fruit residues do not have any significant commercial use or value in India Materials and methods and are disposed off in municipal bins, leading to environmental pollution problems. Materials The low lignin content in citrus fruit residues relative to other lignocellulosic substrates renders the former attractive Kinnow mandarin (Citrus reticulata) residues composed of substrates for ethanol production as there is no need for peels, pulp and seeds were procured from a fruit processing expensive and energy-intensive pretreatment. Fruit residues unit located in Ludhiana, Punjab, India. The peels were may also offer substrate flexibility in the biomass-to- manually separated and washed with distilled water to ethanol conversion process for use as biofuel in countries remove any extraneous material, cut into small pieces and producing substantial quantities of fruits. The successful dried at 70°C in a hot-air oven until completely dry. The hydrolysis of citrus peel waste into sugars and their dried peels were ground to a fine powder using an electric subsequent conversion into ethanol have been reported mill. Celluclast (C-2730), Novozyme 188 (C-6105), and (Grohmann et al. 1994; Oberoi et al. 2011; Wilkins et al. pectinase (P-2611) were procured from Sigma-Aldrich (St. 2007a, b). Previous studies conducted on the production of Louis, MO). The fungal isolate used for cellulase produc- ethanol from citrus fruit residues used commercial enzymes tion was isolated from rice straw, while the yeast strain used and wild-type strains of Saccharomyces cerevisiae or for fermentation was isolated from sugarcane juice. Details recombinant Escherichia coli (Grohmann et al. 1994; on the isolation of fungal and yeast strains are presented in Wilkins et al. 2007a). The total cost of cellulosic ethanol the following subsections. The dehydrated media and media production is very high, but the cost can be drastically ingredients and analytical grade chemicals were purchased reduced if in-house produced enzymes are used for from Hi-Media Laboratories Pvt Ltd (Mumbai, India) and saccharification (Kadam 1996). According to Limtong et Fisher Scientific, India, respectively. The standards used for al. (2007), an ideal microorganism used for ethanol high-performance liquid chromatography (HPLC) determi- production must have rapid fermentation potential, appre- nations, such as those for glucose, fructose, sucrose, xylose, ciable thermotolerance, ethanol tolerance and high osmo- arabinose, galactose and galacturonic acid (GA), were tolerance. Simultaneous saccharification and fermentation procured from Sigma-Aldrich, and those for oligosacchar- (SSF) increases the hydrolysis rate by reducing the product ides, such as cellobiose, cellotriose, xylobiose and xylo- inhibition of enzymes, reduces tank usage by combining the triose, were procured from Megazyme International (Bray, saccharification and fermentation tanks into one tank, County Wicklow, Ireland). simplifies the operational process and decreases the processing time, thereby improving process economics Isolation of cellulase-producing fungal strains (Faga et al. 2010; Shen et al. 2008). Different optimum temperature requirements for enzymes and the microbial Rice straw with particle size of about 1 cm was impreg- -1 strain is a major limitation in the SSF process. Thus, there nated with a nutrient solution composed of 2 g l each of is an urgent need to have thermotolerant yeast strains which yeast extract, peptone and ammonium sulfate. The biomass are able to ferment sugars effectively at higher temperatures was mixed with nutrient solution to a moisture content of than is possible with conventional S. cerevisiae strains. In 70% and exposed to ambient conditions for about 2 weeks. the recent past, there has been an upsurge in interest in Fungi were isolated by the serial dilution method using thermophilic microorganisms, mainly because of their faster sterilized Rose Bengal Chloramphenicol (RBC) agar. The reaction rates, higher product yield and higher product plates were incubated at 30°C for 4–6 days and observed resistance to degeneration at higher temperatures (Banat et regularly for fungal colonies. Different characteristic colo- al. 1998). However, we have not come across any report or nies based on their morphology, color and shape were study in which the crude enzyme produced by Aspergillus picked and purified by separate inoculation on sterilized oryzae has been used for hydrolyzing a fruit residue. RBC agar plates. The structures bearing spores and the Similarly, the use of Pichia kudriavzevii in ethanol arrangement of spores were examined microscopically. The production from any fruit residue or lignocellulosic biomass cellulose hydrolytic capability of the isolated colonies was is as yet undocumented. The aim of this study, therefore, examined by observing the clear zone on inoculated culture Ann Microbiol (2012) 62:655–666 657 plates containing potato dextrose agar (PDA) medium 50 ml sterilized yeast extract peptone dextrose (YPD) supplemented with 1% carboxymethylcellulose (CMC) broth. The pH of the medium was adjusted to 5.2, and the and congo red dye. The cellulase production capability of flasks were incubated at 40°C for 48 h and 120 rpm in an the isolates showing clear zones was further confirmed by incubator shaker. The physiological growth pattern was the filter paper cellulase (FP) assay. The isolate which studied using the culture density (OD ), cell concentration showed the highest FP activity was used for enzyme and cell biomass for the isolated yeast strain grown in YPD production. The isolate was identified through molecular broth under the incubation conditions described previously. characterization, as described in a subsequent section. The results revealed that the cells entered the log phase around 3 h and the stationary phase around 15 h, and that Enzyme production and extraction the cell concentration and cell biomass declined after 18 h (data not shown). Based on these data, 10 ml of the The isolated fungal culture which showed highest FP inoculum was aseptically transferred to 250-ml Erlenmeyer activity among those isolates showing clear zones on the flasks containing 100 ml sterilized YPD broth and the PDA medium plates containing CMC and congo red dye flasks were incubated at 40°C for 15 h at 100 rpm in an was selected for the assays of cellulase production. Spores incubator shaker. Subsequently, 50 ml inoculum from these from this isolate were harvested following inoculation on flasks was aseptically transferred to 1-l flasks containing PDA plates and incubation at 30°C for 72 h. The plates 500 ml sterilized YPD broth, and the flasks were then were removed from the incubator, and spores were incubated under the same conditions as mentioned above. harvested using sterile water. The spore count was Fresh YPD medium was prepared just before each determined with a hemocytometer, and the spore concen- fermentation experiment. The cells were concentrated to a 8 -1 9 -1 tration of 1×10 spores ml was used for enzyme level of 2×10 cells ml by centrifugation at 10,000 g, 4°C production. Solid state fermentation using rice straw and for 10 min. wheat bran at a ratio of 4:1 was employed for cellulase production. All of the enzyme production experiments were Molecular characterization of identified fungal conducted in 250-ml Erlenmeyer flasks, with each flask and yeast isolates containing 10 g substrate. The initial moisture content of 75% was made up with Mandel Weber medium, and the pH Genomic DNA was extracted from overnight cultures of the of the initial fermentation medium was set at 5.0 with 5N fungal and yeast isolates using a fungal genomic DNA NaOH before sterilization. The flasks sterilized in the isolation kit (Chromous Biotech Pvt Ltd, Bangalore, India). autoclave for 15 min, were cooled, inoculated with 1 ml For molecular characterization of the fungal strain, the D1, fungal spore suspension and incubated at 30°C for 96 h. A D2 and D3 domains of the 26S rRNA gene were amplified set of three flasks was removed at 24-h intervals from the using the primer set LROR (5′-ACCCGCTGAACT- incubator and analyzed for cellulase production. Enzyme TAAGC-3′) and LR7 (5′-TACTACCACCAAGATCT-3′). extraction from the flasks was done by the addition of a The ITS1 and ITS4 rDNA region of the yeast isolate was suitable volume of citrate buffer (0.1 M, pH 4.8) followed amplifiedbyPCR usingprimerset pITS1(5′- by vortexing, filtration and centrifugation. The supernatant TCCGTAGGTGAACCTGCGG-3′)and pITS4(5′- was collected and analyzed for FP, endogluconase TCCTCCGCTTATTGATATGC-3′) (Whiteetal. 1990). The (CMCase), pectinase (exopolygalacturonase), xylanase and PCR analysis was performed in 25 μlof reaction volume β-glucosidase activities. It was also used for enzymatic containing 50–100 ng of genomic DNA, 1× Taq buffer, -1 -1 hydrolysis and for SSF, for which it was filtered through 1.5 mmol l MgCl ,10mmol l of each dNTP, 50 ng of 0.2-μm PVDF membrane filter into a sterile bottle. Crude each primer, and 1 U of Taq DNA polymerase (Bangalore filtrate extract (CFE) was used immediately for the SSF Genei, Bangalore, India). The cycling conditions were 95°C process or stored refrigerated for 72 h prior to use. The CFE for 4.5 min, followed by 30 cycles at 95°C for 30 s, 40°C for was analyzed for concentration of sugars, such as glucose, 30 s and 72°C for 1 min, with a final extension of 10 min at fructose, galactose, xylose and arabinose, as per the method 72°C on a MiniOpticon thermal cycler (Bio-Rad, Hercules, described in the Analytical methods section. CA). Amplification was verified by electrophoresis on a -1 1.5% (w v ) agarose gel in 1× TAE buffer using a 100-bp Propagation of yeast cells ladder (Bangalore Genei) as a molecular-weight marker and -1 was visualized using ethidium bromide (1 mg ml )stain in The yeast strain used during fermentation in our study was an Alphaimager EP Geldoc system (Alpha Innotech, San isolated from freshly extracted sugarcane juice (Dhaliwal et Leandro, CA). The 918- and 485-bp amplicons thus al. 2011). The isolated yeast culture was aseptically obtained for fungal and yeast isolates, respectively, were inoculated into 150-ml Erlenmeyer flasks containing sequenced with an ABI 3130 genetic analyzer (Applied 658 Ann Microbiol (2012) 62:655–666 Biosystems, Foster City, CA), and the data obtained were Enzymatic hydrolysis using crude enzyme analyzed using BioEdit sequence analysis program (Hall 1999). A separate experiment was conducted to analyze the effect of enzymatic hydrolysis on the biomass constituents in the Effect of pretreatment and optimization of enzyme residual biomass. KP powder was suspended in distilled concentrations water in the capped polycarbonate flasks. Water was added during the pretreatment in such a way so as to maintain the -1 Kinnow peel waste powder was suspended in distilled substrate concentration at 15% (w v ) after the addition of -1 water at 15% (w v ) substrate concentration and supple- the enzyme. The flasks were sterilized in an autoclave for -1 mented with 2 g l , each of yeast extract, MgSO and 15 min, cooled and the filtered CFE was added to the flasks peptone in polycarbonate capped flasks. A substrate containing pretreated substrate. Hydrolysis was performed -1 concentration >15% (w v ) results in a solid viscous mass at 50°C for 24 h at 120 rpm in an incubator shaker. A set of not easily amenable to enzymatic hydrolysis. The flasks three flasks was removed at 6-h intervals, and the contents were sterilized in an autoclave for 15 min, removed from were filtered under vacuum using a Buchner funnel lined the autoclave while hot and opened in the laminar flow with Whatman filter paper (Whatman, New York, NY). The bench under sterile air to allow the release of volatile solid residue was dried in a hot-air oven at 70°C, and the compounds, such as D-limonene, which are considered dried biomass was analyzed for cellulose, hemicellulose, inhibitory to yeasts (Wilkins et al. 2007b). In a previous lignin and ash contents. All experiments were conducted in study, we observed a drastic reduction in D-limonene triplicate, and the statistical analysis was performed to concentration in kinnow waste obtained after the hydro- analyze the difference in treatment means. thermal pretreatment of the dried biomass (Oberoi et al. 2011). The pH of the medium was adjusted to 5.5 using Ethanol production in a laboratory batch fermentor sterilized 5 N sodium hydroxide solution. In a previous study, we had optimized the enzyme concentrations using a On the basis of the results obtained from the initial statistical design for ethanol production from kinnow waste experiments, in the batch reactor experiments, we used -1 (Oberoi et al. 2011). It is noteworthy to mention here that crude enzymes at a concentration of 3 FPU g-ds . The the composition of kinnow peel and kinnow waste is batch fermentation experiment was performed in a 2.5- different and since this study involved the use of crude l batch reactor (Minifors, Infors HT, Switzerland). A 200-g enzymes, we decided to use commercially available sample of KP (moisture content 92%) was suspended in -1 enzymes at different concentrations before finalizing the 600 ml water and supplemented with 2.0 g l yeast extract, -1 -1 crude enzyme concentration to be used during SSF. The 2.0 g l peptone and 1.0 g l MgSO ⋅H O. The fermentor 4 2 enzyme concentration range was selected on the basis of along with a calibrated pH probe, temperature probe, inlet previous studies (Oberoi et al. 2011; Wilkins et al. 2007a, air/gas provisions, condensate removal system, agitator, b). The enzyme levels selected for commercial enzymes sample collection system, NaOH (5 N) solution and were 0–100 IU per gram dry substrate (g-ds) for exhaust system was sterilized in an autoclave for 15 min. pectinase, a cellulase loading of 0–8 filter paper units The fermentor was removed from the autoclave, sparged -1 -1 (FPU) g-ds and β-glucosidase at 0–16 IU g-ds .The with sterile air to remove the volatile compounds and ratio of FPU to β-glucosidase units was maintained at 1:2 then cooled to 50°C by immersion in cold water. The pH as no cellobiose was detected during the hydrolysis of of the medium was adjusted to 5.0 with the sterilized 5 kinnow waste using cellulase and β-glucosidase in the N NaOH solution, and 567 ml of filtered CFE was added ratio 1:2 (Oberoi et al. 2011). Commercial enzymes were to the fermentor, which accounted for an FP activity of 3 -1 appropriately diluted with citrate buffer (0.1 M, pH 4.8), FPU g-ds . Hydrolysis of KP was performed at 50°C for filtered through 0.45-μm PVDF membrane (Millipore 3 h at 120 rpm and a pH of 5.0. After a 3-h pre-hydrolysis, India Pvt Ltd, Bangalore, India) and added at the various the temperature of the medium was brought down to 40°C, -1 selected concentrations to the flasks containing KP and the and the medium was inoculated with 10% (v v )yeast 9 -1 other nutrients mentioned previously. The flasks were cells at a cell concentration of 2×10 cells ml in the subsequently inoculated with 10% yeast inoculum having inoculum. Agitation, pH and temperature were maintained 9 -1 a cell concentration of 2×10 cells ml and incubated at at 120 rpm, 5.0 and 40°C, respectively, throughout the 40°C, 100 rpm for 15 h in an incubator shaker. The fermentation process. The temperature was selected to experiment was conducted in triplicate, and the superna- accommodate the enzymes as well as the fermenting tant was separated after centrifugation. Ethanol was microbial strain in the same vessel at the same time. determined in the supernatant using ethanol membranes Samples were drawn at 6-h intervals up to 24 h and described elsewhere in the paper. analyzed for sugars, ethanol and GA concentrations. The Ann Microbiol (2012) 62:655–666 659 experiment was conducted three times in the same Results and discussion fermentor, and the results were statistically analyzed. Isolation and screening of cellulolytic microbial strains Analytical methods Among the 12 fungal isolates which showed characteristic Arabinose, fructose, glucose, sucrose, xylose and oligosac- diversity in terms of colony morphology, spore color and charides, such as cellobiose, cellotriose, xylobiose and microscopic spore characteristics, only four isolates showed xylotriose, were analyzed with HPLC (Dionex Corp, clear zones on the PDA plates containing CMC and congo Sunnyvale, CA) using a Shodex SP-0810 column (300× red. These four isolates were identified on the basis of 7.8 mm) fitted with a SP-G guard column (Waters , Milford, morphological characterization and microscopic examina- MA). Degassed HPLC grade water was used as a mobile tions. All four isolates belong to the genus Aspergillus and -1 phase at a flow rate of 1.0 ml min . The column oven and were screened for FP activity. Filter paper cellulase activity refractive index (RI) detector were maintained at 80°C and is a relative measure of the overall cellulose-hydrolyzing 50°C, respectively. Samples were diluted, centrifuged, and capacity of microbial cellulase preparations (Urbanszki et filtered through 0.45-μm RC membranes (Phenomenex al. 2000). Isolate I showed the highest FP activity among Corp, Santa Clara, CA). Peaks were detected by the RI the four strains and it also produced a higher enzyme detector and quantified on the basis of area and retention concentration in a shorter time (Fig. 1). Thus, this isolate time of the standards. Galacturonic acid concentration was was used for the assays on enzyme production. Isolate I determined using the modified dinitrosalicylic acid method was identified and characterized on the basis of sequencing described previously (Wang et al. 1997). Ethanol was of the 26S rDNA region, which are described in detail later determined with YSI 2786 ethanol membrane kits using in this paper. YSI 2700 Select biochemical analyzer (YSI Inc, Buffalo, FP activity and CMCase, β-glucosidase and xylanase NY). The instrument was calibrated using the ethanol production of the selected isolate increased until 72 h, -1 -1 standards of 2.0 g l and 3.2 g l provided by the levelling off thereafter, whereas exopolygalacturonase ac- manufacturers. Samples were adequately diluted, centri- tivity levelled off after 96 h (Fig. 2). Fungi initially fuged and injected into the instrument for analysis consumed the readily available sugars and produced following the manufacturers’ specified procedure. hydrolytic enzymes; following depletion of the sugar Cellulose content was analyzed using a gravimetric concentration, particularly when the glucose concentration method employing acetic nitrate reagent (Pereira et al. was low, the fungi began to use these hydrolytic enzymes 1998), while hemicellulose was determined by subtracting for the production of sugars, resulting in a decrease in enzyme activity. In a previous study, we observed a similar acid detergent fiber (ADF) from neutral detergent fiber (NDF) using the method previously described by Goering trend in enzyme production using mixed-culture solid-state and Vansoest (1970). Moisture and ash contents in the fermentation (Oberoi et al. 2010a). The major factors which samples were determined by the AOAC method (2000). determine the enzyme production capability of a culture are Sugars were extracted with ethanol and analyzed by the the C:N ratio, amount of available sugars in the fermenta- method described previously by Oberoi et al. (2011). tion medium, concentration of insoluble polysaccharides, Enzyme assays for FP activity, CMCase and xylanase were such as cellulose, hemicellulose and pectin, and cultural performed using previously described methods (Bailey et al. 1992;Ghose 1987; Wood and Bhat 1988). The concentration of reducing sugars (RS) was determined using the DNS reagent with glucose as a standard (Miller 1959). β- Glucosidase activity was estimated using p-nitrophenyl-β-D- glucopyranoside (pNPG) as a substrate (Oberoi et al. 2010b). Pectinase was determined in terms of exopolygalacturonase activity as per the previously described procedure of Oberoi et al. (2010a). Statistical analysis All experiments were carried out in triplicate, and the mean and standard deviation (SD) values were calculated using the MS Excel program. The significance for the treatment Fig. 1 Screening of four different isolates for filter paper cellulase (FP) activity means was determined with JMP software (SAS, Cary, NC) 660 Ann Microbiol (2012) 62:655–666 are synonyms for the same organism. We have recently reported ethanol production from sugarcane juice using P. kudriavzevii (Dhaliwal et al. 2011). Therefore, the literature reporting use of I. orientalis for ethanol production is cited elsewhere in this paper. Effect of pretreatment on the composition of kinnow mandarin peel Kinnow mandarin peel waste contains sugars, cellulose, hemicellulose and pectin (Table 1). Cellulose, hemicellulose and pectin can be enzymatically hydrolyzed to yield fermentable sugars. The presence of lignin in low concen- trations (Table 1) renders KP an ideal substrate for ethanol Fig. 2 Effect of incubation time on enzyme production by the newly production. Lignin in the lignocellulosic biomass binds to isolated strain of Aspergillus oryzae the cellulose and hemicellulose fractions, thereby reducing conditions. On the basis of our results, we decided to their accessibility to enzymes. Because of its composition, harvest the enzyme after 72 h for use during SSF. KP can potentially serve as a good substrate for ethanol production, especially for use as a biofuel. The increase in Identification and characterization of fungal and yeast sugar concentration in ethanol extracts in pretreated KP strains accounted for the increase in total content when compared with KP (Table 1). The sterilization pretreatment facilitated Sequencing and analysis of the 26S rDNA region of the the solubilization and subsequent extraction with ethanol of isolated fungal strain and of the ITS rDNA region of the some of the sugars in KP that were strongly bonded to the isolated yeast strain revealed that these regions had the insoluble polysaccharide fractions (Oberoi et al. 2011). The highest identity with Aspergillus oryzae and Pichia observed significant increase in cellulose concentration in kudriavzevii (Issatchenkia orientalis), respectively. Phylo- pretreated KP (Table 1) indicates that the sterilization genetic relationships were inferred through the alignment pretreatment did not hydrolyze cellulose. and cladistic analysis of homologous nucleotide sequences Because of the sterilization hydrothermal pretreatment, of known microorganisms. The isolated fungal and yeast sugars bound to the polysaccharide fractions solubilized, strains and A. oryzae (NCBI accession no: AP007172) and I. leading to a higher concentration of cellulose, hemicellu- orientalis (NCBI accession nos EF568018 and EF568014), lose and lignin in the pretreated KP (Oberoi et al. 2011). respectively, belonged to the same branch. I. orientalis is a However, pretreatment led to partial solubilization of pectin National Center for Biotechnology Information (NCBI) (Table 1). Cellulose forms intra-molecular hydrogen bonds synonym for P. kudriavzevii. As per the molecular phyloge- between adjacent glucose molecules. Pectin forms a matrix netic work of Kurtzman et al. (2008), the species ascribed to in which the cellulose microfibrils are embedded and bind the genus Issatchenkia has been clustered within Pichia,and adjacent cell walls together making it a fairly resistant thus Issatchenkia orientalis has been replaced by Pichia kudriavzevii as the taxonomically valid entity. Based on Table 1 Compositional analysis of Kinnow mandarin peels morphology and the comparison of 26S rRNA and ITS rDNA gene sequences, the isolated fungal and yeast strains Composition (%) Kinnow peel Pretreated kinnow peel were identified as strains of A. oryzae and P. kudriavzevii, Cellulose 10.72±0.36 12.85±0.41 respectively. The 26S rRNA gene sequences and ITS Hemicellulose 3.88±0.27 4.36±0.23 sequences for the newly isolated strains of A. oryzae and P. Pectin 22.88±1.24 16.45±0.95 kudriavzevii were submitted to GenBank under accession Sugars in ethanol extract 29.66±1.48 37.12±1.74 numbers HQ 122940 and HQ 122942, respectively. The Ash 3.52±0.19 3.89±0.20 isolates of A. oryzae and P. kudriavzevii have been deposited Lignin 1.91±0.15 2.5±0.17 with the National Bureau of Agriculturally Important Micro- Protein 5.65±0.34 6.12±0.40 organisms (NBAIM), Mau Nath Bhanjan, India. There are a number of published reports on ethanol production using I. Data are presented as the mean ± standard deviation (SD) of n =3 orientalis, whereas we have not come across any report on trials ethanol production using P. kudriavzevii from any fruit Phenolic compounds and fat account for the remainder of the residue or cellulosic biomass, although both designations composition Ann Microbiol (2012) 62:655–666 661 complex. We observed a similar trend in the profile of treated KP with CFE resulted in the solubilization of constituents after sterilization pretreatment of kinnow waste cellulose and hemicellulose (Table 3), which in turn led to (Oberoi et al. 2011). Steric hindrance of cellulose and an increase in ash and lignin concentrations. Cellulose, hemicellulose hydrolysis by pectin is supported by the cell hemicellulose or residual biomass concentrations did not wall model for flowering plants which states that a pectin show a significant decline after 18 h (Table 3). The matrix surrounds cellulose fibers coated with xyloglucan solubilization of cellulose and hemicellulose by the hydro- (Carpita and Gibeaut 1993). Mild pretreatment is thus lytic enzymes led to an increase in ash and lignin contents. essential for partial solubilization of hemicellulose and The observed decline in hydrolysis rate after 12 h (Table 3) pectin, which otherwise are strongly bonded to each other. may be mainly due to the increased ash and lignin contents, Pretreatment increases the surface area allowing the which may envelope the cellulose and hemicellulose hydrolyzing enzymes to work more efficiently on pectin fractions, thereby reducing their accessibility to enzymes. and cellulose (Wilkins et al. 2007b). The limited availability of cellulose and hemicellulose after 18 h could also have led to a decline in the hydrolytic Simultaneous saccharification and fermentation using ability of the enzymes. It is possible that some of the commercial enzymes catalytic sites in the crude enzyme were blocked by salts or small-molecular-weight polypeptides. No cellobiose, cello- Ethanol concentration increased by about 80% with the use triose, xylobiose or xylotriose were formed during hydro- of cellulolytic and pectinolytic enzymes relative to the lysis of the pretreated KP with crude enzymes, indicating control for which no enzymes were used (Table 2). The that the enzymes, such as β-xylosidase and xylan esterase, absence of either cellulase or pectinase during SSF were present in the CFE. However, enzyme assays for β- adversely affected ethanol yield (Table 2). Ethanol produc- xylosidase, α-L-arabinofuranosidase and xylan esterase -1 tion with cellulase (3 FPU g-ds ) and pectinase (50 IU g- were not performed in our study. In a previous study, we -1 ds ) did not differ significantly with higher cellulase and reported that the sodium dodecyl sulfate–polyacrylamide pectinase concentrations (Table 2). Wilkins et al. (2007b) gel electrophoresis (SDS–PAGE) of CFE produced by made similar observations during hydrolysis studies of mixed-cultures of Trichoderma reesei and Aspergillus citrus peel waste with cellulolytic and pectinolytic enzymes. oryzae produced many characteristic bands (Brijwani et Therefore, we decided to use the crude enzyme at 3 FPU g- al. 2010). The significant drop in pH during the enzymatic -1 ds during SSF for ethanol production, which means that hydrolysis of KP is caused by the increase in GA the concentrations of β-glucosidase, endoglucanase, exo- concentration. Pectin in the cell walls of fruit residues is polygalacturonase and xylanase in the CFE were 261, 210, composed of GA units linked to the sugar moiety composed -1 69, and 1560 IU g-ds , respectively. of rhamnose, galactose, arabinose and glucose. Several weak organic acids, such as acetic, malic, malonic, lactic Hydrolysis with the crude enzyme consortium and citric acid, are known to be present in citrus peels (Grohmann et al. 1999), and these inhibit S. cerevisiae No sugars were detected in the CFE used for the hydrolysis growth at a lower pH due to an increase in undissociated and ethanol production experiments. Hydrolysis of pre- acids (Wilkins et al. 2007b). A continuous decline in pH Table 2 Ethanol production by -1 -1 -1 -1 Cellulase (FPU g-ds ) β-glucosidase (IU g-ds ) Pectinase (IU g-ds ) Ethanol (g l ) simultaneous saccharification and fermentation using different 0 0 0 18.92±0.37 concentrations of commercial enzymes 5 10 20 30.12±0.28 5 10 50 32.11±0.35 5 10 80 32.54±0.46 5 10 100 32.23±0.30 5 10 0 24.56±0.39 3 6 50 31.89±0.28 8 16 50 31.69±0.26 0 0 50 21.13±0.17 3 6 100 32.01±0.22 8 16 100 31.77±0.41 Data are given as the mean ± SD 0 0 100 22.16±0.21 of n=3 trials 662 Ann Microbiol (2012) 62:655–666 Table 3 Compositional changes during enzymatic hydrolysis of kinnow peel waste with the crude filtrate extract Time (h) Cellulose (%) Hemicellulose (%) Ash (%) Lignin (%) pH 0 12.85±0.41 a 4.36±0.23 a 3.89±0.20 a 2.50±0.1 7 a 5.0 6 6.90±0.37 b 2.77±0.24 b 5.15±0.34 b 2.76±0.80 a 4.2 12 4.24±0.28 c 1.70±0.27 b 5.35±0.19 b 2.95±0.11 b 3.9 18 2.86±0.20 d 0.89±0.06 c 5.89±0.18 c 3.25±0.08 c 3.7 24 1.83±0.09 e 0.88±0.16 c 6.25±0.19 c 4.02±0.08 d 3.5 Least significant difference (p<0.05) 0.57 0.38 0.41 0.69 Data are given as the mean ± SD for n =3 trials. The SD for pH values was within 5% of the mean value Mean values followed by the same lowercase letter do not differ significantly. The calculations for constituents were made on the basis of the initial substrate concentration might adversely affect the hydrolytic ability of the enzymes Fermentation beyond 12 h led to a significant reduction in present in the CFE. volumetric productivity because of the drop in fermentation rate. Ethanol concentration and productivity obtained in our Ethanol production in a batch fermentor study are comparable with or higher than those reported in previous studies (Table 5). Although, higher ethanol Hydrolysis of pretreated KP with the CFE at concentrations concentrations were reported in some of the previous described previously resulted in a significant increase in studies (Table 5), it should be noted that crude enzymes glucose and fructose concentrations during the 3-h pre- were used in the present study for the hydrolysis of KP. hydrolysis. Arabinose, xylose and GA together with the Glucose and fructose obtained at high concentrations hexose sugars were also formed during the 3-h pre- after a 3-h pre-hydrolysis of KP in the laboratory fermenter hydrolysis (Table 4). No glucose, fructose or sucrose was are the preferred substrates for yeasts and are consumed detected after 12 h SSF, and their concentrations even at 6 h before other substrates (Gancedo 1998). A high concentra- were low compared to their initial concentrations (Table 4), tion of inoculum, the harvesting of cells during the log indicating rapid fermentation of these sugars by yeast cells. phase and the availability of fresh medium may have Sucrose is converted to glucose and fructose by the reduced the lag phase for the yeast cells. An increase in invertase present in the yeast cells, and such monomers xylose, arabinose and GA concentrations indicates that the are subsequently fermented to ethanol. Some of the sugars, P. kudriavzevii strain was not able to metabolize and such as glucose, fructose and galactose, produced by ferment these compounds. Issatchenkia orientalis strains enzymatic hydrolysis of insoluble polysaccharides during are not capable of metabolizing pentose sugars, such as SSF were fermented to ethanol. Ethanol concentration xylose and arabinose (Kurtzman et al. 1980). Arabinose is levelled off after 12 h (Fig. 3), which could largely be due found in pectin side chains, whereas polymers of GA form to the non-availability of hexose sugars for fermentation. the backbone of pectin molecules (Carpita and Gibeaut Table 4 Sugar consumption and galacturonic acid production during simultaneous saccharification and fermentation in a batch fermentor Time (h) Glucose Fructose Galactose Arabinose Sucrose Xylose Galacturonic acid -1 -1 -1 -1 -1 -1 -1 (g l ) (g l ) (g l ) (g l ) (g l ) (g l ) (g l ) 0 24.87±0.75 a 21.98 ±0.53 a 1.25 ±0.12 a 1.48±0.14 a 10.86±0.34 a 0.23±0.03 a 6.56±0.29 a 6 3.33±0.15 b 4.9±0.29 b 1.04±0.09 a 2.56±0.13 a 2.5±0.17 b 0.25±0.05 b 7.12±0.25 b 12 ND ND 1.14 ±0.10 a 2.68±0.19 b ND 0.36±0.06 b 8.25±0.27 c 18 ND ND 1.04±0.15 a,b 3.25±0.09 b ND 0.39±0.31 c 9.01±0.52 c,d 24 ND ND 0.85±0.09 b 3.57±0.16 c ND 0.48±0.08 d 9.84±0.34 d Least significant 0.63 0.50 0.21 0.26 0.30 0..27 0.63 difference (p<0.05) After a 3-h hydrolysis Data are given as the mean ± SD for n=3 trials Mean values followed by the same lowercase letter do not differ significantly Ann Microbiol (2012) 62:655–666 663 1993). Arabinose and GA might have solubilized because production, such as S. cerevisiae, cannotbeusedattemper- of the hydrolysis of KP by the pectinase present in the CFE. atures in the vicinity of 45–50°C that are optimum for An increase in xylose concentration during hydrolysis may cellulases (Oberoi et al. 2011).The traditional yeasts used in be due to the solubilization of hemicellulose by the industrial fermentations perform well within the temperature xylanase and β-xylosidase present in the CFE. No glucose range 30–35°C, and their fermentative ability is compro- was left after 6 h during SSF, whereas the RS concentration mised at temperatures >35°C (Sa-Correia and van Uden increased after 12 h. This result could primarily be due to 1982; Wilkins et al. 2007b). Abdel-Banat et al. (2009) the increase in the concentrations of xylose, arabinose and reported that a 5°C increase in fermentation temperature GA, which are not fermented by P. kudriavzevii cells, as greatly affects the cost of fuel ethanol production. mentioned previously (Fig. 3). Grohmann et al. (1994) To maximize ethanol production, it is thus important to reported that ethanol production from enzymatically hydro- conduct SSF at temperatures close to optimum for cellulase, lyzed orange peel using S. cerevisiae at 35°C and pH 5.0 did use thermotolerant yeasts for faster cellulose hydrolysis and not increase beyond 12 h. As noted earlier, commercial a shorter fermentation time. Although the temperature enzymes are expensive and enzymes such as cellulase, β- during SSF was maintained at 40°C, we are now attempting glucosidase, xylanase and pectinase are added separately to to partially purify the crude enzyme obtained from the the fermentation medium, thereby further increasing the strain of A. oryzae used in this study and conduct SSF at production cost. temperatures ranging from 45 to 50°C with thermotolerant The concentration of ethanol produced in the batch yeast strains in an attempt to produce ethanol from fermenter was higher than that produced in the shake flask, lignocellulosic biomass. A higher ethanol concentration mainly because the pH was controlled during the entire has been reported from citrus peel waste by previous process in the batch fermentor, which means that the researchers using a combination of commercial enzymes, enzyme and yeast cells did not have to adapt to continu- such as cellulase, β-glucosidase and pectinase, but the use ously decreasing pH conditions. In addition, the batch of a CFE obtained from a strain of A. oryzae for ethanol fermentor provided better operational conditions, such as production from any fruit residue is unprecedented. In agitation and contact between the cells and medium (Oberoi addition, the use of a thermotolerant strain of newly et al. 2011). We also believe that the 3-h pre-hydrolysis at isolated yeast strain of P. kudriavzevii holds promise for 50°C helped to effectively release soluble sugars bound to further evaluations. our results in terms of product yield and the insoluble polysaccharide matrix and also to have volumetric productivity are encouraging for future scale-up partially hydrolyzed the insoluble fractions, as 50°C is studies. considered to be the optimal temperature for effective hydrolysis by cellulase and pectinase. The residual biomass at the end of the SSF process could be exploited for use as cattle Conclusions feed because of impregnation of biomass with yeast cells. In our study, we also used a newly isolated thermotolerant strain Hydrothermal pretreatment helped in partial solubilization of P. kudriavzevii, which to the best of our knowledge has not of cellulose and pectin and also helped release sugars been used for ethanol production from any lignocellulosic bonded to the insoluble fractions. A 3-h pre-hydrolysis with biomass or fruit residue. Traditional yeasts used for ethanol the crude enzyme produced by a newly isolated strain of Aspergillus oryzae prior to SSF resulted in production of glucose, fructose, galactose, arabinose, xylose, sucrose and galacturonic acid. The newly isolated thermotolerant strain of Pichia kudriavzevii fermented glucose, fructose, sucrose and galactose to ethanol whereas it could not metabolize arabinose, xylose and galacturonic acid produced by enzymatic hydrolysis during SSF. This study demonstrated that SSF with crude enzyme consortium obtained from A. oryzae and fermentation with P. kudriavzevii strain resulted -1 in 33.87 g l ethanol from kinnow peel in 12 h in a laboratory fermenter. Ethanol concentration during SSF leveled off after 12 h, suggesting high ethanol productivity for the process which means that a large number of batches could be completed in a short time. Hydrolysis with crude Fig. 3 Effect of fermentation time on sugar consumption and ethanol -1 -1 enzyme and ethanol productivity of 2.8 g l h in a SSF production during the simultaneous saccharification and fermentation (SSF) process in a laboratory fermentor process indicates a good potential for scale-up studies. 664 Ann Microbiol (2012) 62:655–666 Table 5 Comparative study of results obtained in the present study with those of previous investigations Substrate Pretreatment process Microorganism used Fermentation Ethanol Volumetric productivity Reference -1 -1 -1 temperature (°C) concentration (g l ) (g l h ) Primary Secondary Kinnow waste Autoclave–sterilization SSF using commercial Saccharomyces 37 43.0 3.50 Oberoi et al. at 15 psi for 15 min enzymes and galactose cerevisiae 2011 adapted cells Orange peel Separate hydrolysis and S. cerevisiae and 35 37-40 0.50–0.55 Wilkins et al. hydrolysate fermentation (SHF) Kluveromyces 2007a marxianus Citrus peel waste Live steam 150–160°C SSF with S. cerevisiae 37 42 1.75 Wilkins et al. for 2–4 min commercial enzymes 2007b Orange peel 0.5% (w/v) H SO Dilute acid hydrolysis S. cerevisiae 30 30.3 3.37 Oberoi et al. 2 4 at 121°C for 15 min 2010c Kinnow:banana Autoclave–sterilization Enzymatic hydrolysis Pachysolen 30 26.84 0.74 Sharma et al. peel (4:6) at 15 psi for 1 h with crude enzymes tannophillus 2007 and S. cerevisiae Citrus peel waste High pressure steam Enzymatic hydrolysis with S. cerevisiae 38 32.4 1.8 Zhou et al. at 70 psi commercial enzymes 2008 Kinnow peel Autoclave– sterilization Enzymatic hydrolysis Pichia kudriavzevii 40 33.87 2.82 This study at 15 psi for 15 min with crude enzymes SSF Simultaneous saccharification and fermentation Ann Microbiol (2012) 62:655–666 665 Acknowledgments The authors gratefully acknowledge the finan- Hall TA (1999) BioEdit: a user-friendly biological sequence alignment cial assistance received under AMAAS project of Indian Council of editor and analysis program for Windows 95/98/NT. Nucleic Agricultural Research (ICAR), Government of India for conducting Acids Symp Ser 41:95–98 this study. Hill J, Nelson E, Tilman D, Polasky S, Tiffany D (2006) Environ- mental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci USA 103:11206–11210 Kadam KL (1996) Cellulase production. In: Wyman CE (ed) Handbook on bioethanol: production and utilization. Taylor and References Francis, Washington D.C., pp 213–252, Chapter 11 Kalra KL, Grewal HS, Kahlon SS (1989) Bioconversion of kinnow- Abdel-Banat BMA, Hoshida H, Ano A, Nonklang S, Akada R (2009) mandarin waste into single-cell protein. World J Microbiol High-temperature fermentation: how can processes for ethanol Biotech 5:321–326 production at high temperatures become superior to the tradi- Kurtzman CP, Smiley MJ, Johnson CJ (1980) Emendation of the tional process using mesophilic yeast. Appl Microbiol Biotechnol genus Issatchenkia kudriavzev and comparison of species by 85:861–867 deoxyribonucleic acid reassociation, mating reaction, and asco- AOAC (2000) Official methods of analysis, vol. 2, 17th edn. spore ultrastructure. Int J Sys Bacteriol 30:503–513 Association of Official Analytical Chemists, Gaithersburg Kurtzman CP, Robnett CJ, Basehoar-Powers E (2008) Phylogenetic Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of relationships among species of Pichia, Issatchenkia and Williop- methods for assay of xylanase activity. J Biotechnol 23:257– sis determined from multigene sequence analysis, and the 270 proposal of Barnettozyma gen.nov., Lindnera gen.nov. and Banat IM, Nigam P, Singh D, Marchant R, McHale AP (1998) Wickerhamomyces gen.nov. FEMS Yeast Res 8:939–954 Review: ethanol production at elevated temperatures and alcohol Limtong S, Sringiew C, Yonganitchai W (2007) Production of fuel concentrations: Part I—yeasts in general. World J Microbiol ethanol at high temperature from sugarcane juice by new isolated Biotechnol 14:809–821 Kluyeromyces marxianus. Bioresour Technol 98:3367–3374 Brijwani K, Oberoi HS, Vadlani PV (2010) Production of a cellulolytic Miller GL (1959) Dinitro salicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428 enzyme system in mixed-culture solid state fermentation of soybean hulls supplemented with wheat bran. Process Biochem Oberoi HS, Babbar N, Dhaliwal SS, Kaur S, Vadlani PV, Bhargav VK, 45:120–128 Patil RT (2010a) Enhanced oil recovery by pre-treatment of mustard Carpita NC, Gibeaut DM (1993) Structural models of primary cell seeds using crude enzyme extract obtained from mixed-culture walls in flowering plants: consistency of molecular structure solid-state fermentation of Kinnow (Citrus reticulata) waste and with the physical properties of the walls during growth. Plant wheat bran. Food and Bioprocess Technol. doi:10.1007/s11947- J3:1–30 010-03830-y Dhaliwal SS, Oberoi HS, Sandhu SK, Nanda D, Kumar D, Uppal Oberoi HS, Vadlani PV, Brijwani K, Bhargav VK, Patil RT (2010b) SK (2011) Enhanced ethanol production from sugarcane juice Enhanced ethanol production via fermentation of rice straw with by galactose adaptation of a newly isolated thermotolerant hydrolysate-adapted Candida tropicalis ATCC 13803. Process strain of Pichia kudriavzevii. Bioresour Technol 102:5968– Biochem 45:1299–1306 5975 Oberoi HS, Vadlani PV, Madl RL, Saida L, Abeykoon JP (2010c) Dhillon SS, Gill RK, Gill SS, Singh M (2004) Studies on utilization of Ethanol production from orange peels: Two stage hydrolysis and citrus peel for pectinase production using fungus Aspergillus fermentation studies using optimized parameters through exper- niger. Int J Environ Stud 61:199–210 imental design. J Agric Food Chem 58:3422–3429 Faga BA, Wilkins MR, Banat IM (2010) Ethanol production Oberoi HS, Vadlani PV, Nanjundaswamy A, Bansal S, Singh S, Kaur through simultaneous saccharification and fermentation of S, Babbar N (2011) Enhanced ethanol production from Kinnow switchgrass using Saccharomyces cerevisiae D A and thermoto- mandarin (Citrus reticulata) waste via a statistically optimized lerant Kluyveromyces marxianus IMB strains. Bioresour Technol simultaneous saccharification and fermentation process. Biore- 101:2273–2279 sour Technol 102:1593–1601 Farrell AE, Pelvin RJ, Turner BT, Jones AD, O’Hare M, Kammen DM Pereira AN, Mobedshahi M, Ladisch MR (1998) Preparation of (2006) Ethanol can contribute to energy and environmental goals. cellodextrins. Methods Enzymol 160:26–43 Science 311:506–508 Sa-Correia I, van Uden N (1982) Temperature profiles of ethanol Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol tolerance: effects of ethanol on the minimum and the maximum Biol Rev 62:334–361 for growth of the yeasts Saccharomyces cerevisiae and Kluyver- Ghose TK (1987) Measurement of cellulose activities. Pure Appl omyces fralilis. Biotechnol Bioeng 25:1665–1667 Chem 59:257–268 Sharma N, Kalra KL, Oberoi HS, Bansal S (2007) Optimization of Goering HK, Vansoest PJ (1970) Forage fibre analysis. Agricultural fermentation parameters for production of ethanol from Research Services, United States Department of Agriculture, kinnow waste and banana peels by simultaneous saccharifica- Agricultural Handbook, No 379. ARS, USDA, Washington D. tion and fermentation. Indian J Microbiol 47:310–316 C. Shen Y, Zhang Y, Bao X, Du F, Zhuang G, Qu Y (2008) Simultaneous Grohmann K, Elizabeth AB, Busling BS (1994) Production of ethanol saccharification and fermentation of acid-pretreated corncobs from enzymatically hydrolyzed orange peel by the yeast with a recombinant Saccharomyces cerevisiae expressing β- Saccharomyes cerevisiae. Appl Biochem Biotechnol 45/46:315– glucosidase. Bioresour Technol 99:5099–5103 327 Urbanszki K, Szakacs G, Tengerdy RP (2000) Standardization of filter Grohmann K, Cameron RG, Buslig BS (1995) Fractionation and paper activity assay for solid substrate fermentation. Biotechnol pretreatment of orange peel by dilute acid hydrolysis. Bioresour Lett 22:65–69 Technol 54:129–141 Wang G, Michailides TJ, Bostock RM (1997) Improved detection of Grohmann K, Manthey JA, Cameron RG, Buslig BS (1999) polygalacturonase activity due to Mucor piriformis with a Purification of citrus peel juice and molasses. J Agric Food modified dinitrosalicylic acid reagent. Phytopathology 87:161– Chem 47:4859–4867 163 666 Ann Microbiol (2012) 62:655–666 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Wilkins MR, Widmer W, Grohmann K (2007b) Simultaneous sacchar- sequencing of fungal ribosomal RNA genes for phylogenetics. ification and fermentation of citrus peel waste by Saccharomyces In: Innis MA, Gelfand DH Sninsky JJ, White TJ (eds) PCR cerevisiae to produce ethanol. Process Biochem 42(12):1614–1619 protocols: a guide to methods and applications. Academic Press, Wood TM, Bhat KM (1988) Methods for measuring cellulase San Diego, pp 315–322 activities. Methods Enzymol Biomass Part A Cellulose Hemicel- Wilkins MR, Suryawati L, Maness NO, Churz D (2007a) Ethanol lulose 160:87–112 production by Saccharomyces cerevisiae and Kluyveromyces Zhou W, Widmer W, Grohmann K (2008) Developments in ethanol marxianus in the presence of orange-peel oil. World J Microbiol production from citrus peel waste. Proc Fla State Hortic Soc Biotech 23(8):1161–1168 121:307–310 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Ethanol production from Kinnow mandarin (Citrus reticulata) peels via simultaneous saccharification and fermentation using crude enzyme produced by Aspergillus oryzae and the thermotolerant Pichia kudriavzevii strain

Download PDF
12 pages

Loading next page...
 
/lp/springer-journals/ethanol-production-from-kinnow-mandarin-citrus-reticulata-peels-via-hgjmvog75j

References (35)

Publisher
Springer Journals
Copyright
Copyright © 2011 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-011-0302-x
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2012) 62:655–666 DOI 10.1007/s13213-011-0302-x ORIGINAL ARTICLE Ethanol production from Kinnow mandarin (Citrus reticulata) peels via simultaneous saccharification and fermentation using crude enzyme produced by Aspergillus oryzae and the thermotolerant Pichia kudriavzevii strain Simanjeet Kaur Sandhu & Harinder Singh Oberoi & Sandeep Singh Dhaliwal & Neha Babbar & Ujjal Kaur & Dhiraj Nanda & Dinesh Kumar Received: 12 March 2011 /Accepted: 16 June 2011 /Published online: 8 July 2011 # Springer-Verlag and the University of Milan 2011 . . Abstract The aim of this study was to assess the potential Keywords Aspergillus oryzae Ethanol . . of using the crude filtrate extract (CFE) produced by a Pichia kudriavzevii Kinnow peels newly isolated strain of Aspergillus oryzae and fermentation Simultaneous saccharification and fermentation with a novel thermotolerant strain of Pichia kudriavzevii for the production of ethanol from kinnow peel waste (KP) via Introduction simultaneous saccharification and fermentation (SSF). High-performance liquid chromatography determination Demand for fuels produced from renewable sources has showed that pre-hydrolysis of KP with CFE at 3 cellulase increased in recent years due to increased oil prices, filter paper units/g dry substrate (FPU/g-ds) at 50°C -1 -1 concerns about greenhouse gas production and increasing resulted in 24.87±0.75 g l glucose, 21.98±0.53 g l -1 -1 dependence on foreign sources of energy (Hill et al. 2006). fructose, 10.86±0.34 g l sucrose and 6.56±0.29 g l The development of energy from renewable sources has the galacturonic acid (GA) along with insignificant amounts of potential to provide domestic energy supplies, while arabinose, galactose and xylose. Simultaneous saccharifi- reducing net greenhouse gas emission and developing a cation and fermentation of hydrothermally pretreated KP at -1 more favorable energy balance than traditional petroleum a substrate concentration of 15% (w v ) was conducted in a production (Farrell et al. 2006). Unlike fossil fuels, ethanol 2.5-l laboratory fermentor with P. kudriavzevii at 40°C after is a renewable energy source produced through the a 3-h pre-hydrolysis. Oligosaccharides were not produced fermentation of sugars. It is currently used as a partial during the SSF process. Ethanol production leveled off after gasoline replacement in a number of countries in the world. 12 h, resulting in an ethanol concentration and productivity -1 -1 -1 Kinnow mandarin (Citrus reticulata) belongs to the citrus of 33.87 g l and 2.82 g l h , respectively. These results family of fruits and is an economically important fruit demonstrate the potentiality of SSF using crude enzymes grown in India and Pakistan. According to Food and and P. kudriavzevii for the scale-up production of ethanol Agriculture Organization of the United Nations statistics, from KP. India produces around 7.13 million tonnes of citrus fruits annually, of which kinnow mandarin accounts for about 15%. Because of its high juice content and sweet taste, : : : S. Kaur Sandhu H. Singh Oberoi (*) S. Singh Dhaliwal there has been an upsurge in kinnow production in India N. Babbar U. Kaur during the past few years. Kalra et al. (1989) reported that Central Institute of Post Harvest Engineering and Technology, P.O. PAU, Ludhiana 141 004 Punjab, India kinnow mandarin processing residues, composed of peels, e-mail: hari_manu@yahoo.com pulp and seeds, are rich in carbohydrates and account for about 55–60% of the weight of the raw fruit. Dhillon et al. D. Nanda D. Kumar (2004) proposed that citrus peel waste could be used to National Bureau of Animal Genetic Resources, Karnal, India produce important commercial products, such as enzymes, 656 Ann Microbiol (2012) 62:655–666 ethanol, microbial biomass, volatile flavoring compounds, was to assess the potential of using crude enzymes obtained organic acids and antioxidants. The residues obtained from from a newly isolated A. oryzae strain and a novel citrus fruits are rich in fermentable sugars, such as glucose, thermotolerant P. kudriavzevii isolate in a SSF process for fructose, sucrose and galactose, as well as in insoluble ethanol production using kinnow peel waste (KP) in a 2.5- polysaccharides, such as cellulose, hemicellulose and pectin l laboratory fermentor. (Grohmann et al. 1995; Oberoi et al. 2011). Despite this richness in sugars, proteins and minerals, fruit residues do not have any significant commercial use or value in India Materials and methods and are disposed off in municipal bins, leading to environmental pollution problems. Materials The low lignin content in citrus fruit residues relative to other lignocellulosic substrates renders the former attractive Kinnow mandarin (Citrus reticulata) residues composed of substrates for ethanol production as there is no need for peels, pulp and seeds were procured from a fruit processing expensive and energy-intensive pretreatment. Fruit residues unit located in Ludhiana, Punjab, India. The peels were may also offer substrate flexibility in the biomass-to- manually separated and washed with distilled water to ethanol conversion process for use as biofuel in countries remove any extraneous material, cut into small pieces and producing substantial quantities of fruits. The successful dried at 70°C in a hot-air oven until completely dry. The hydrolysis of citrus peel waste into sugars and their dried peels were ground to a fine powder using an electric subsequent conversion into ethanol have been reported mill. Celluclast (C-2730), Novozyme 188 (C-6105), and (Grohmann et al. 1994; Oberoi et al. 2011; Wilkins et al. pectinase (P-2611) were procured from Sigma-Aldrich (St. 2007a, b). Previous studies conducted on the production of Louis, MO). The fungal isolate used for cellulase produc- ethanol from citrus fruit residues used commercial enzymes tion was isolated from rice straw, while the yeast strain used and wild-type strains of Saccharomyces cerevisiae or for fermentation was isolated from sugarcane juice. Details recombinant Escherichia coli (Grohmann et al. 1994; on the isolation of fungal and yeast strains are presented in Wilkins et al. 2007a). The total cost of cellulosic ethanol the following subsections. The dehydrated media and media production is very high, but the cost can be drastically ingredients and analytical grade chemicals were purchased reduced if in-house produced enzymes are used for from Hi-Media Laboratories Pvt Ltd (Mumbai, India) and saccharification (Kadam 1996). According to Limtong et Fisher Scientific, India, respectively. The standards used for al. (2007), an ideal microorganism used for ethanol high-performance liquid chromatography (HPLC) determi- production must have rapid fermentation potential, appre- nations, such as those for glucose, fructose, sucrose, xylose, ciable thermotolerance, ethanol tolerance and high osmo- arabinose, galactose and galacturonic acid (GA), were tolerance. Simultaneous saccharification and fermentation procured from Sigma-Aldrich, and those for oligosacchar- (SSF) increases the hydrolysis rate by reducing the product ides, such as cellobiose, cellotriose, xylobiose and xylo- inhibition of enzymes, reduces tank usage by combining the triose, were procured from Megazyme International (Bray, saccharification and fermentation tanks into one tank, County Wicklow, Ireland). simplifies the operational process and decreases the processing time, thereby improving process economics Isolation of cellulase-producing fungal strains (Faga et al. 2010; Shen et al. 2008). Different optimum temperature requirements for enzymes and the microbial Rice straw with particle size of about 1 cm was impreg- -1 strain is a major limitation in the SSF process. Thus, there nated with a nutrient solution composed of 2 g l each of is an urgent need to have thermotolerant yeast strains which yeast extract, peptone and ammonium sulfate. The biomass are able to ferment sugars effectively at higher temperatures was mixed with nutrient solution to a moisture content of than is possible with conventional S. cerevisiae strains. In 70% and exposed to ambient conditions for about 2 weeks. the recent past, there has been an upsurge in interest in Fungi were isolated by the serial dilution method using thermophilic microorganisms, mainly because of their faster sterilized Rose Bengal Chloramphenicol (RBC) agar. The reaction rates, higher product yield and higher product plates were incubated at 30°C for 4–6 days and observed resistance to degeneration at higher temperatures (Banat et regularly for fungal colonies. Different characteristic colo- al. 1998). However, we have not come across any report or nies based on their morphology, color and shape were study in which the crude enzyme produced by Aspergillus picked and purified by separate inoculation on sterilized oryzae has been used for hydrolyzing a fruit residue. RBC agar plates. The structures bearing spores and the Similarly, the use of Pichia kudriavzevii in ethanol arrangement of spores were examined microscopically. The production from any fruit residue or lignocellulosic biomass cellulose hydrolytic capability of the isolated colonies was is as yet undocumented. The aim of this study, therefore, examined by observing the clear zone on inoculated culture Ann Microbiol (2012) 62:655–666 657 plates containing potato dextrose agar (PDA) medium 50 ml sterilized yeast extract peptone dextrose (YPD) supplemented with 1% carboxymethylcellulose (CMC) broth. The pH of the medium was adjusted to 5.2, and the and congo red dye. The cellulase production capability of flasks were incubated at 40°C for 48 h and 120 rpm in an the isolates showing clear zones was further confirmed by incubator shaker. The physiological growth pattern was the filter paper cellulase (FP) assay. The isolate which studied using the culture density (OD ), cell concentration showed the highest FP activity was used for enzyme and cell biomass for the isolated yeast strain grown in YPD production. The isolate was identified through molecular broth under the incubation conditions described previously. characterization, as described in a subsequent section. The results revealed that the cells entered the log phase around 3 h and the stationary phase around 15 h, and that Enzyme production and extraction the cell concentration and cell biomass declined after 18 h (data not shown). Based on these data, 10 ml of the The isolated fungal culture which showed highest FP inoculum was aseptically transferred to 250-ml Erlenmeyer activity among those isolates showing clear zones on the flasks containing 100 ml sterilized YPD broth and the PDA medium plates containing CMC and congo red dye flasks were incubated at 40°C for 15 h at 100 rpm in an was selected for the assays of cellulase production. Spores incubator shaker. Subsequently, 50 ml inoculum from these from this isolate were harvested following inoculation on flasks was aseptically transferred to 1-l flasks containing PDA plates and incubation at 30°C for 72 h. The plates 500 ml sterilized YPD broth, and the flasks were then were removed from the incubator, and spores were incubated under the same conditions as mentioned above. harvested using sterile water. The spore count was Fresh YPD medium was prepared just before each determined with a hemocytometer, and the spore concen- fermentation experiment. The cells were concentrated to a 8 -1 9 -1 tration of 1×10 spores ml was used for enzyme level of 2×10 cells ml by centrifugation at 10,000 g, 4°C production. Solid state fermentation using rice straw and for 10 min. wheat bran at a ratio of 4:1 was employed for cellulase production. All of the enzyme production experiments were Molecular characterization of identified fungal conducted in 250-ml Erlenmeyer flasks, with each flask and yeast isolates containing 10 g substrate. The initial moisture content of 75% was made up with Mandel Weber medium, and the pH Genomic DNA was extracted from overnight cultures of the of the initial fermentation medium was set at 5.0 with 5N fungal and yeast isolates using a fungal genomic DNA NaOH before sterilization. The flasks sterilized in the isolation kit (Chromous Biotech Pvt Ltd, Bangalore, India). autoclave for 15 min, were cooled, inoculated with 1 ml For molecular characterization of the fungal strain, the D1, fungal spore suspension and incubated at 30°C for 96 h. A D2 and D3 domains of the 26S rRNA gene were amplified set of three flasks was removed at 24-h intervals from the using the primer set LROR (5′-ACCCGCTGAACT- incubator and analyzed for cellulase production. Enzyme TAAGC-3′) and LR7 (5′-TACTACCACCAAGATCT-3′). extraction from the flasks was done by the addition of a The ITS1 and ITS4 rDNA region of the yeast isolate was suitable volume of citrate buffer (0.1 M, pH 4.8) followed amplifiedbyPCR usingprimerset pITS1(5′- by vortexing, filtration and centrifugation. The supernatant TCCGTAGGTGAACCTGCGG-3′)and pITS4(5′- was collected and analyzed for FP, endogluconase TCCTCCGCTTATTGATATGC-3′) (Whiteetal. 1990). The (CMCase), pectinase (exopolygalacturonase), xylanase and PCR analysis was performed in 25 μlof reaction volume β-glucosidase activities. It was also used for enzymatic containing 50–100 ng of genomic DNA, 1× Taq buffer, -1 -1 hydrolysis and for SSF, for which it was filtered through 1.5 mmol l MgCl ,10mmol l of each dNTP, 50 ng of 0.2-μm PVDF membrane filter into a sterile bottle. Crude each primer, and 1 U of Taq DNA polymerase (Bangalore filtrate extract (CFE) was used immediately for the SSF Genei, Bangalore, India). The cycling conditions were 95°C process or stored refrigerated for 72 h prior to use. The CFE for 4.5 min, followed by 30 cycles at 95°C for 30 s, 40°C for was analyzed for concentration of sugars, such as glucose, 30 s and 72°C for 1 min, with a final extension of 10 min at fructose, galactose, xylose and arabinose, as per the method 72°C on a MiniOpticon thermal cycler (Bio-Rad, Hercules, described in the Analytical methods section. CA). Amplification was verified by electrophoresis on a -1 1.5% (w v ) agarose gel in 1× TAE buffer using a 100-bp Propagation of yeast cells ladder (Bangalore Genei) as a molecular-weight marker and -1 was visualized using ethidium bromide (1 mg ml )stain in The yeast strain used during fermentation in our study was an Alphaimager EP Geldoc system (Alpha Innotech, San isolated from freshly extracted sugarcane juice (Dhaliwal et Leandro, CA). The 918- and 485-bp amplicons thus al. 2011). The isolated yeast culture was aseptically obtained for fungal and yeast isolates, respectively, were inoculated into 150-ml Erlenmeyer flasks containing sequenced with an ABI 3130 genetic analyzer (Applied 658 Ann Microbiol (2012) 62:655–666 Biosystems, Foster City, CA), and the data obtained were Enzymatic hydrolysis using crude enzyme analyzed using BioEdit sequence analysis program (Hall 1999). A separate experiment was conducted to analyze the effect of enzymatic hydrolysis on the biomass constituents in the Effect of pretreatment and optimization of enzyme residual biomass. KP powder was suspended in distilled concentrations water in the capped polycarbonate flasks. Water was added during the pretreatment in such a way so as to maintain the -1 Kinnow peel waste powder was suspended in distilled substrate concentration at 15% (w v ) after the addition of -1 water at 15% (w v ) substrate concentration and supple- the enzyme. The flasks were sterilized in an autoclave for -1 mented with 2 g l , each of yeast extract, MgSO and 15 min, cooled and the filtered CFE was added to the flasks peptone in polycarbonate capped flasks. A substrate containing pretreated substrate. Hydrolysis was performed -1 concentration >15% (w v ) results in a solid viscous mass at 50°C for 24 h at 120 rpm in an incubator shaker. A set of not easily amenable to enzymatic hydrolysis. The flasks three flasks was removed at 6-h intervals, and the contents were sterilized in an autoclave for 15 min, removed from were filtered under vacuum using a Buchner funnel lined the autoclave while hot and opened in the laminar flow with Whatman filter paper (Whatman, New York, NY). The bench under sterile air to allow the release of volatile solid residue was dried in a hot-air oven at 70°C, and the compounds, such as D-limonene, which are considered dried biomass was analyzed for cellulose, hemicellulose, inhibitory to yeasts (Wilkins et al. 2007b). In a previous lignin and ash contents. All experiments were conducted in study, we observed a drastic reduction in D-limonene triplicate, and the statistical analysis was performed to concentration in kinnow waste obtained after the hydro- analyze the difference in treatment means. thermal pretreatment of the dried biomass (Oberoi et al. 2011). The pH of the medium was adjusted to 5.5 using Ethanol production in a laboratory batch fermentor sterilized 5 N sodium hydroxide solution. In a previous study, we had optimized the enzyme concentrations using a On the basis of the results obtained from the initial statistical design for ethanol production from kinnow waste experiments, in the batch reactor experiments, we used -1 (Oberoi et al. 2011). It is noteworthy to mention here that crude enzymes at a concentration of 3 FPU g-ds . The the composition of kinnow peel and kinnow waste is batch fermentation experiment was performed in a 2.5- different and since this study involved the use of crude l batch reactor (Minifors, Infors HT, Switzerland). A 200-g enzymes, we decided to use commercially available sample of KP (moisture content 92%) was suspended in -1 enzymes at different concentrations before finalizing the 600 ml water and supplemented with 2.0 g l yeast extract, -1 -1 crude enzyme concentration to be used during SSF. The 2.0 g l peptone and 1.0 g l MgSO ⋅H O. The fermentor 4 2 enzyme concentration range was selected on the basis of along with a calibrated pH probe, temperature probe, inlet previous studies (Oberoi et al. 2011; Wilkins et al. 2007a, air/gas provisions, condensate removal system, agitator, b). The enzyme levels selected for commercial enzymes sample collection system, NaOH (5 N) solution and were 0–100 IU per gram dry substrate (g-ds) for exhaust system was sterilized in an autoclave for 15 min. pectinase, a cellulase loading of 0–8 filter paper units The fermentor was removed from the autoclave, sparged -1 -1 (FPU) g-ds and β-glucosidase at 0–16 IU g-ds .The with sterile air to remove the volatile compounds and ratio of FPU to β-glucosidase units was maintained at 1:2 then cooled to 50°C by immersion in cold water. The pH as no cellobiose was detected during the hydrolysis of of the medium was adjusted to 5.0 with the sterilized 5 kinnow waste using cellulase and β-glucosidase in the N NaOH solution, and 567 ml of filtered CFE was added ratio 1:2 (Oberoi et al. 2011). Commercial enzymes were to the fermentor, which accounted for an FP activity of 3 -1 appropriately diluted with citrate buffer (0.1 M, pH 4.8), FPU g-ds . Hydrolysis of KP was performed at 50°C for filtered through 0.45-μm PVDF membrane (Millipore 3 h at 120 rpm and a pH of 5.0. After a 3-h pre-hydrolysis, India Pvt Ltd, Bangalore, India) and added at the various the temperature of the medium was brought down to 40°C, -1 selected concentrations to the flasks containing KP and the and the medium was inoculated with 10% (v v )yeast 9 -1 other nutrients mentioned previously. The flasks were cells at a cell concentration of 2×10 cells ml in the subsequently inoculated with 10% yeast inoculum having inoculum. Agitation, pH and temperature were maintained 9 -1 a cell concentration of 2×10 cells ml and incubated at at 120 rpm, 5.0 and 40°C, respectively, throughout the 40°C, 100 rpm for 15 h in an incubator shaker. The fermentation process. The temperature was selected to experiment was conducted in triplicate, and the superna- accommodate the enzymes as well as the fermenting tant was separated after centrifugation. Ethanol was microbial strain in the same vessel at the same time. determined in the supernatant using ethanol membranes Samples were drawn at 6-h intervals up to 24 h and described elsewhere in the paper. analyzed for sugars, ethanol and GA concentrations. The Ann Microbiol (2012) 62:655–666 659 experiment was conducted three times in the same Results and discussion fermentor, and the results were statistically analyzed. Isolation and screening of cellulolytic microbial strains Analytical methods Among the 12 fungal isolates which showed characteristic Arabinose, fructose, glucose, sucrose, xylose and oligosac- diversity in terms of colony morphology, spore color and charides, such as cellobiose, cellotriose, xylobiose and microscopic spore characteristics, only four isolates showed xylotriose, were analyzed with HPLC (Dionex Corp, clear zones on the PDA plates containing CMC and congo Sunnyvale, CA) using a Shodex SP-0810 column (300× red. These four isolates were identified on the basis of 7.8 mm) fitted with a SP-G guard column (Waters , Milford, morphological characterization and microscopic examina- MA). Degassed HPLC grade water was used as a mobile tions. All four isolates belong to the genus Aspergillus and -1 phase at a flow rate of 1.0 ml min . The column oven and were screened for FP activity. Filter paper cellulase activity refractive index (RI) detector were maintained at 80°C and is a relative measure of the overall cellulose-hydrolyzing 50°C, respectively. Samples were diluted, centrifuged, and capacity of microbial cellulase preparations (Urbanszki et filtered through 0.45-μm RC membranes (Phenomenex al. 2000). Isolate I showed the highest FP activity among Corp, Santa Clara, CA). Peaks were detected by the RI the four strains and it also produced a higher enzyme detector and quantified on the basis of area and retention concentration in a shorter time (Fig. 1). Thus, this isolate time of the standards. Galacturonic acid concentration was was used for the assays on enzyme production. Isolate I determined using the modified dinitrosalicylic acid method was identified and characterized on the basis of sequencing described previously (Wang et al. 1997). Ethanol was of the 26S rDNA region, which are described in detail later determined with YSI 2786 ethanol membrane kits using in this paper. YSI 2700 Select biochemical analyzer (YSI Inc, Buffalo, FP activity and CMCase, β-glucosidase and xylanase NY). The instrument was calibrated using the ethanol production of the selected isolate increased until 72 h, -1 -1 standards of 2.0 g l and 3.2 g l provided by the levelling off thereafter, whereas exopolygalacturonase ac- manufacturers. Samples were adequately diluted, centri- tivity levelled off after 96 h (Fig. 2). Fungi initially fuged and injected into the instrument for analysis consumed the readily available sugars and produced following the manufacturers’ specified procedure. hydrolytic enzymes; following depletion of the sugar Cellulose content was analyzed using a gravimetric concentration, particularly when the glucose concentration method employing acetic nitrate reagent (Pereira et al. was low, the fungi began to use these hydrolytic enzymes 1998), while hemicellulose was determined by subtracting for the production of sugars, resulting in a decrease in enzyme activity. In a previous study, we observed a similar acid detergent fiber (ADF) from neutral detergent fiber (NDF) using the method previously described by Goering trend in enzyme production using mixed-culture solid-state and Vansoest (1970). Moisture and ash contents in the fermentation (Oberoi et al. 2010a). The major factors which samples were determined by the AOAC method (2000). determine the enzyme production capability of a culture are Sugars were extracted with ethanol and analyzed by the the C:N ratio, amount of available sugars in the fermenta- method described previously by Oberoi et al. (2011). tion medium, concentration of insoluble polysaccharides, Enzyme assays for FP activity, CMCase and xylanase were such as cellulose, hemicellulose and pectin, and cultural performed using previously described methods (Bailey et al. 1992;Ghose 1987; Wood and Bhat 1988). The concentration of reducing sugars (RS) was determined using the DNS reagent with glucose as a standard (Miller 1959). β- Glucosidase activity was estimated using p-nitrophenyl-β-D- glucopyranoside (pNPG) as a substrate (Oberoi et al. 2010b). Pectinase was determined in terms of exopolygalacturonase activity as per the previously described procedure of Oberoi et al. (2010a). Statistical analysis All experiments were carried out in triplicate, and the mean and standard deviation (SD) values were calculated using the MS Excel program. The significance for the treatment Fig. 1 Screening of four different isolates for filter paper cellulase (FP) activity means was determined with JMP software (SAS, Cary, NC) 660 Ann Microbiol (2012) 62:655–666 are synonyms for the same organism. We have recently reported ethanol production from sugarcane juice using P. kudriavzevii (Dhaliwal et al. 2011). Therefore, the literature reporting use of I. orientalis for ethanol production is cited elsewhere in this paper. Effect of pretreatment on the composition of kinnow mandarin peel Kinnow mandarin peel waste contains sugars, cellulose, hemicellulose and pectin (Table 1). Cellulose, hemicellulose and pectin can be enzymatically hydrolyzed to yield fermentable sugars. The presence of lignin in low concen- trations (Table 1) renders KP an ideal substrate for ethanol Fig. 2 Effect of incubation time on enzyme production by the newly production. Lignin in the lignocellulosic biomass binds to isolated strain of Aspergillus oryzae the cellulose and hemicellulose fractions, thereby reducing conditions. On the basis of our results, we decided to their accessibility to enzymes. Because of its composition, harvest the enzyme after 72 h for use during SSF. KP can potentially serve as a good substrate for ethanol production, especially for use as a biofuel. The increase in Identification and characterization of fungal and yeast sugar concentration in ethanol extracts in pretreated KP strains accounted for the increase in total content when compared with KP (Table 1). The sterilization pretreatment facilitated Sequencing and analysis of the 26S rDNA region of the the solubilization and subsequent extraction with ethanol of isolated fungal strain and of the ITS rDNA region of the some of the sugars in KP that were strongly bonded to the isolated yeast strain revealed that these regions had the insoluble polysaccharide fractions (Oberoi et al. 2011). The highest identity with Aspergillus oryzae and Pichia observed significant increase in cellulose concentration in kudriavzevii (Issatchenkia orientalis), respectively. Phylo- pretreated KP (Table 1) indicates that the sterilization genetic relationships were inferred through the alignment pretreatment did not hydrolyze cellulose. and cladistic analysis of homologous nucleotide sequences Because of the sterilization hydrothermal pretreatment, of known microorganisms. The isolated fungal and yeast sugars bound to the polysaccharide fractions solubilized, strains and A. oryzae (NCBI accession no: AP007172) and I. leading to a higher concentration of cellulose, hemicellu- orientalis (NCBI accession nos EF568018 and EF568014), lose and lignin in the pretreated KP (Oberoi et al. 2011). respectively, belonged to the same branch. I. orientalis is a However, pretreatment led to partial solubilization of pectin National Center for Biotechnology Information (NCBI) (Table 1). Cellulose forms intra-molecular hydrogen bonds synonym for P. kudriavzevii. As per the molecular phyloge- between adjacent glucose molecules. Pectin forms a matrix netic work of Kurtzman et al. (2008), the species ascribed to in which the cellulose microfibrils are embedded and bind the genus Issatchenkia has been clustered within Pichia,and adjacent cell walls together making it a fairly resistant thus Issatchenkia orientalis has been replaced by Pichia kudriavzevii as the taxonomically valid entity. Based on Table 1 Compositional analysis of Kinnow mandarin peels morphology and the comparison of 26S rRNA and ITS rDNA gene sequences, the isolated fungal and yeast strains Composition (%) Kinnow peel Pretreated kinnow peel were identified as strains of A. oryzae and P. kudriavzevii, Cellulose 10.72±0.36 12.85±0.41 respectively. The 26S rRNA gene sequences and ITS Hemicellulose 3.88±0.27 4.36±0.23 sequences for the newly isolated strains of A. oryzae and P. Pectin 22.88±1.24 16.45±0.95 kudriavzevii were submitted to GenBank under accession Sugars in ethanol extract 29.66±1.48 37.12±1.74 numbers HQ 122940 and HQ 122942, respectively. The Ash 3.52±0.19 3.89±0.20 isolates of A. oryzae and P. kudriavzevii have been deposited Lignin 1.91±0.15 2.5±0.17 with the National Bureau of Agriculturally Important Micro- Protein 5.65±0.34 6.12±0.40 organisms (NBAIM), Mau Nath Bhanjan, India. There are a number of published reports on ethanol production using I. Data are presented as the mean ± standard deviation (SD) of n =3 orientalis, whereas we have not come across any report on trials ethanol production using P. kudriavzevii from any fruit Phenolic compounds and fat account for the remainder of the residue or cellulosic biomass, although both designations composition Ann Microbiol (2012) 62:655–666 661 complex. We observed a similar trend in the profile of treated KP with CFE resulted in the solubilization of constituents after sterilization pretreatment of kinnow waste cellulose and hemicellulose (Table 3), which in turn led to (Oberoi et al. 2011). Steric hindrance of cellulose and an increase in ash and lignin concentrations. Cellulose, hemicellulose hydrolysis by pectin is supported by the cell hemicellulose or residual biomass concentrations did not wall model for flowering plants which states that a pectin show a significant decline after 18 h (Table 3). The matrix surrounds cellulose fibers coated with xyloglucan solubilization of cellulose and hemicellulose by the hydro- (Carpita and Gibeaut 1993). Mild pretreatment is thus lytic enzymes led to an increase in ash and lignin contents. essential for partial solubilization of hemicellulose and The observed decline in hydrolysis rate after 12 h (Table 3) pectin, which otherwise are strongly bonded to each other. may be mainly due to the increased ash and lignin contents, Pretreatment increases the surface area allowing the which may envelope the cellulose and hemicellulose hydrolyzing enzymes to work more efficiently on pectin fractions, thereby reducing their accessibility to enzymes. and cellulose (Wilkins et al. 2007b). The limited availability of cellulose and hemicellulose after 18 h could also have led to a decline in the hydrolytic Simultaneous saccharification and fermentation using ability of the enzymes. It is possible that some of the commercial enzymes catalytic sites in the crude enzyme were blocked by salts or small-molecular-weight polypeptides. No cellobiose, cello- Ethanol concentration increased by about 80% with the use triose, xylobiose or xylotriose were formed during hydro- of cellulolytic and pectinolytic enzymes relative to the lysis of the pretreated KP with crude enzymes, indicating control for which no enzymes were used (Table 2). The that the enzymes, such as β-xylosidase and xylan esterase, absence of either cellulase or pectinase during SSF were present in the CFE. However, enzyme assays for β- adversely affected ethanol yield (Table 2). Ethanol produc- xylosidase, α-L-arabinofuranosidase and xylan esterase -1 tion with cellulase (3 FPU g-ds ) and pectinase (50 IU g- were not performed in our study. In a previous study, we -1 ds ) did not differ significantly with higher cellulase and reported that the sodium dodecyl sulfate–polyacrylamide pectinase concentrations (Table 2). Wilkins et al. (2007b) gel electrophoresis (SDS–PAGE) of CFE produced by made similar observations during hydrolysis studies of mixed-cultures of Trichoderma reesei and Aspergillus citrus peel waste with cellulolytic and pectinolytic enzymes. oryzae produced many characteristic bands (Brijwani et Therefore, we decided to use the crude enzyme at 3 FPU g- al. 2010). The significant drop in pH during the enzymatic -1 ds during SSF for ethanol production, which means that hydrolysis of KP is caused by the increase in GA the concentrations of β-glucosidase, endoglucanase, exo- concentration. Pectin in the cell walls of fruit residues is polygalacturonase and xylanase in the CFE were 261, 210, composed of GA units linked to the sugar moiety composed -1 69, and 1560 IU g-ds , respectively. of rhamnose, galactose, arabinose and glucose. Several weak organic acids, such as acetic, malic, malonic, lactic Hydrolysis with the crude enzyme consortium and citric acid, are known to be present in citrus peels (Grohmann et al. 1999), and these inhibit S. cerevisiae No sugars were detected in the CFE used for the hydrolysis growth at a lower pH due to an increase in undissociated and ethanol production experiments. Hydrolysis of pre- acids (Wilkins et al. 2007b). A continuous decline in pH Table 2 Ethanol production by -1 -1 -1 -1 Cellulase (FPU g-ds ) β-glucosidase (IU g-ds ) Pectinase (IU g-ds ) Ethanol (g l ) simultaneous saccharification and fermentation using different 0 0 0 18.92±0.37 concentrations of commercial enzymes 5 10 20 30.12±0.28 5 10 50 32.11±0.35 5 10 80 32.54±0.46 5 10 100 32.23±0.30 5 10 0 24.56±0.39 3 6 50 31.89±0.28 8 16 50 31.69±0.26 0 0 50 21.13±0.17 3 6 100 32.01±0.22 8 16 100 31.77±0.41 Data are given as the mean ± SD 0 0 100 22.16±0.21 of n=3 trials 662 Ann Microbiol (2012) 62:655–666 Table 3 Compositional changes during enzymatic hydrolysis of kinnow peel waste with the crude filtrate extract Time (h) Cellulose (%) Hemicellulose (%) Ash (%) Lignin (%) pH 0 12.85±0.41 a 4.36±0.23 a 3.89±0.20 a 2.50±0.1 7 a 5.0 6 6.90±0.37 b 2.77±0.24 b 5.15±0.34 b 2.76±0.80 a 4.2 12 4.24±0.28 c 1.70±0.27 b 5.35±0.19 b 2.95±0.11 b 3.9 18 2.86±0.20 d 0.89±0.06 c 5.89±0.18 c 3.25±0.08 c 3.7 24 1.83±0.09 e 0.88±0.16 c 6.25±0.19 c 4.02±0.08 d 3.5 Least significant difference (p<0.05) 0.57 0.38 0.41 0.69 Data are given as the mean ± SD for n =3 trials. The SD for pH values was within 5% of the mean value Mean values followed by the same lowercase letter do not differ significantly. The calculations for constituents were made on the basis of the initial substrate concentration might adversely affect the hydrolytic ability of the enzymes Fermentation beyond 12 h led to a significant reduction in present in the CFE. volumetric productivity because of the drop in fermentation rate. Ethanol concentration and productivity obtained in our Ethanol production in a batch fermentor study are comparable with or higher than those reported in previous studies (Table 5). Although, higher ethanol Hydrolysis of pretreated KP with the CFE at concentrations concentrations were reported in some of the previous described previously resulted in a significant increase in studies (Table 5), it should be noted that crude enzymes glucose and fructose concentrations during the 3-h pre- were used in the present study for the hydrolysis of KP. hydrolysis. Arabinose, xylose and GA together with the Glucose and fructose obtained at high concentrations hexose sugars were also formed during the 3-h pre- after a 3-h pre-hydrolysis of KP in the laboratory fermenter hydrolysis (Table 4). No glucose, fructose or sucrose was are the preferred substrates for yeasts and are consumed detected after 12 h SSF, and their concentrations even at 6 h before other substrates (Gancedo 1998). A high concentra- were low compared to their initial concentrations (Table 4), tion of inoculum, the harvesting of cells during the log indicating rapid fermentation of these sugars by yeast cells. phase and the availability of fresh medium may have Sucrose is converted to glucose and fructose by the reduced the lag phase for the yeast cells. An increase in invertase present in the yeast cells, and such monomers xylose, arabinose and GA concentrations indicates that the are subsequently fermented to ethanol. Some of the sugars, P. kudriavzevii strain was not able to metabolize and such as glucose, fructose and galactose, produced by ferment these compounds. Issatchenkia orientalis strains enzymatic hydrolysis of insoluble polysaccharides during are not capable of metabolizing pentose sugars, such as SSF were fermented to ethanol. Ethanol concentration xylose and arabinose (Kurtzman et al. 1980). Arabinose is levelled off after 12 h (Fig. 3), which could largely be due found in pectin side chains, whereas polymers of GA form to the non-availability of hexose sugars for fermentation. the backbone of pectin molecules (Carpita and Gibeaut Table 4 Sugar consumption and galacturonic acid production during simultaneous saccharification and fermentation in a batch fermentor Time (h) Glucose Fructose Galactose Arabinose Sucrose Xylose Galacturonic acid -1 -1 -1 -1 -1 -1 -1 (g l ) (g l ) (g l ) (g l ) (g l ) (g l ) (g l ) 0 24.87±0.75 a 21.98 ±0.53 a 1.25 ±0.12 a 1.48±0.14 a 10.86±0.34 a 0.23±0.03 a 6.56±0.29 a 6 3.33±0.15 b 4.9±0.29 b 1.04±0.09 a 2.56±0.13 a 2.5±0.17 b 0.25±0.05 b 7.12±0.25 b 12 ND ND 1.14 ±0.10 a 2.68±0.19 b ND 0.36±0.06 b 8.25±0.27 c 18 ND ND 1.04±0.15 a,b 3.25±0.09 b ND 0.39±0.31 c 9.01±0.52 c,d 24 ND ND 0.85±0.09 b 3.57±0.16 c ND 0.48±0.08 d 9.84±0.34 d Least significant 0.63 0.50 0.21 0.26 0.30 0..27 0.63 difference (p<0.05) After a 3-h hydrolysis Data are given as the mean ± SD for n=3 trials Mean values followed by the same lowercase letter do not differ significantly Ann Microbiol (2012) 62:655–666 663 1993). Arabinose and GA might have solubilized because production, such as S. cerevisiae, cannotbeusedattemper- of the hydrolysis of KP by the pectinase present in the CFE. atures in the vicinity of 45–50°C that are optimum for An increase in xylose concentration during hydrolysis may cellulases (Oberoi et al. 2011).The traditional yeasts used in be due to the solubilization of hemicellulose by the industrial fermentations perform well within the temperature xylanase and β-xylosidase present in the CFE. No glucose range 30–35°C, and their fermentative ability is compro- was left after 6 h during SSF, whereas the RS concentration mised at temperatures >35°C (Sa-Correia and van Uden increased after 12 h. This result could primarily be due to 1982; Wilkins et al. 2007b). Abdel-Banat et al. (2009) the increase in the concentrations of xylose, arabinose and reported that a 5°C increase in fermentation temperature GA, which are not fermented by P. kudriavzevii cells, as greatly affects the cost of fuel ethanol production. mentioned previously (Fig. 3). Grohmann et al. (1994) To maximize ethanol production, it is thus important to reported that ethanol production from enzymatically hydro- conduct SSF at temperatures close to optimum for cellulase, lyzed orange peel using S. cerevisiae at 35°C and pH 5.0 did use thermotolerant yeasts for faster cellulose hydrolysis and not increase beyond 12 h. As noted earlier, commercial a shorter fermentation time. Although the temperature enzymes are expensive and enzymes such as cellulase, β- during SSF was maintained at 40°C, we are now attempting glucosidase, xylanase and pectinase are added separately to to partially purify the crude enzyme obtained from the the fermentation medium, thereby further increasing the strain of A. oryzae used in this study and conduct SSF at production cost. temperatures ranging from 45 to 50°C with thermotolerant The concentration of ethanol produced in the batch yeast strains in an attempt to produce ethanol from fermenter was higher than that produced in the shake flask, lignocellulosic biomass. A higher ethanol concentration mainly because the pH was controlled during the entire has been reported from citrus peel waste by previous process in the batch fermentor, which means that the researchers using a combination of commercial enzymes, enzyme and yeast cells did not have to adapt to continu- such as cellulase, β-glucosidase and pectinase, but the use ously decreasing pH conditions. In addition, the batch of a CFE obtained from a strain of A. oryzae for ethanol fermentor provided better operational conditions, such as production from any fruit residue is unprecedented. In agitation and contact between the cells and medium (Oberoi addition, the use of a thermotolerant strain of newly et al. 2011). We also believe that the 3-h pre-hydrolysis at isolated yeast strain of P. kudriavzevii holds promise for 50°C helped to effectively release soluble sugars bound to further evaluations. our results in terms of product yield and the insoluble polysaccharide matrix and also to have volumetric productivity are encouraging for future scale-up partially hydrolyzed the insoluble fractions, as 50°C is studies. considered to be the optimal temperature for effective hydrolysis by cellulase and pectinase. The residual biomass at the end of the SSF process could be exploited for use as cattle Conclusions feed because of impregnation of biomass with yeast cells. In our study, we also used a newly isolated thermotolerant strain Hydrothermal pretreatment helped in partial solubilization of P. kudriavzevii, which to the best of our knowledge has not of cellulose and pectin and also helped release sugars been used for ethanol production from any lignocellulosic bonded to the insoluble fractions. A 3-h pre-hydrolysis with biomass or fruit residue. Traditional yeasts used for ethanol the crude enzyme produced by a newly isolated strain of Aspergillus oryzae prior to SSF resulted in production of glucose, fructose, galactose, arabinose, xylose, sucrose and galacturonic acid. The newly isolated thermotolerant strain of Pichia kudriavzevii fermented glucose, fructose, sucrose and galactose to ethanol whereas it could not metabolize arabinose, xylose and galacturonic acid produced by enzymatic hydrolysis during SSF. This study demonstrated that SSF with crude enzyme consortium obtained from A. oryzae and fermentation with P. kudriavzevii strain resulted -1 in 33.87 g l ethanol from kinnow peel in 12 h in a laboratory fermenter. Ethanol concentration during SSF leveled off after 12 h, suggesting high ethanol productivity for the process which means that a large number of batches could be completed in a short time. Hydrolysis with crude Fig. 3 Effect of fermentation time on sugar consumption and ethanol -1 -1 enzyme and ethanol productivity of 2.8 g l h in a SSF production during the simultaneous saccharification and fermentation (SSF) process in a laboratory fermentor process indicates a good potential for scale-up studies. 664 Ann Microbiol (2012) 62:655–666 Table 5 Comparative study of results obtained in the present study with those of previous investigations Substrate Pretreatment process Microorganism used Fermentation Ethanol Volumetric productivity Reference -1 -1 -1 temperature (°C) concentration (g l ) (g l h ) Primary Secondary Kinnow waste Autoclave–sterilization SSF using commercial Saccharomyces 37 43.0 3.50 Oberoi et al. at 15 psi for 15 min enzymes and galactose cerevisiae 2011 adapted cells Orange peel Separate hydrolysis and S. cerevisiae and 35 37-40 0.50–0.55 Wilkins et al. hydrolysate fermentation (SHF) Kluveromyces 2007a marxianus Citrus peel waste Live steam 150–160°C SSF with S. cerevisiae 37 42 1.75 Wilkins et al. for 2–4 min commercial enzymes 2007b Orange peel 0.5% (w/v) H SO Dilute acid hydrolysis S. cerevisiae 30 30.3 3.37 Oberoi et al. 2 4 at 121°C for 15 min 2010c Kinnow:banana Autoclave–sterilization Enzymatic hydrolysis Pachysolen 30 26.84 0.74 Sharma et al. peel (4:6) at 15 psi for 1 h with crude enzymes tannophillus 2007 and S. cerevisiae Citrus peel waste High pressure steam Enzymatic hydrolysis with S. cerevisiae 38 32.4 1.8 Zhou et al. at 70 psi commercial enzymes 2008 Kinnow peel Autoclave– sterilization Enzymatic hydrolysis Pichia kudriavzevii 40 33.87 2.82 This study at 15 psi for 15 min with crude enzymes SSF Simultaneous saccharification and fermentation Ann Microbiol (2012) 62:655–666 665 Acknowledgments The authors gratefully acknowledge the finan- Hall TA (1999) BioEdit: a user-friendly biological sequence alignment cial assistance received under AMAAS project of Indian Council of editor and analysis program for Windows 95/98/NT. Nucleic Agricultural Research (ICAR), Government of India for conducting Acids Symp Ser 41:95–98 this study. Hill J, Nelson E, Tilman D, Polasky S, Tiffany D (2006) Environ- mental, economic, and energetic costs and benefits of biodiesel and ethanol biofuels. Proc Natl Acad Sci USA 103:11206–11210 Kadam KL (1996) Cellulase production. In: Wyman CE (ed) Handbook on bioethanol: production and utilization. Taylor and References Francis, Washington D.C., pp 213–252, Chapter 11 Kalra KL, Grewal HS, Kahlon SS (1989) Bioconversion of kinnow- Abdel-Banat BMA, Hoshida H, Ano A, Nonklang S, Akada R (2009) mandarin waste into single-cell protein. World J Microbiol High-temperature fermentation: how can processes for ethanol Biotech 5:321–326 production at high temperatures become superior to the tradi- Kurtzman CP, Smiley MJ, Johnson CJ (1980) Emendation of the tional process using mesophilic yeast. Appl Microbiol Biotechnol genus Issatchenkia kudriavzev and comparison of species by 85:861–867 deoxyribonucleic acid reassociation, mating reaction, and asco- AOAC (2000) Official methods of analysis, vol. 2, 17th edn. spore ultrastructure. Int J Sys Bacteriol 30:503–513 Association of Official Analytical Chemists, Gaithersburg Kurtzman CP, Robnett CJ, Basehoar-Powers E (2008) Phylogenetic Bailey MJ, Biely P, Poutanen K (1992) Interlaboratory testing of relationships among species of Pichia, Issatchenkia and Williop- methods for assay of xylanase activity. J Biotechnol 23:257– sis determined from multigene sequence analysis, and the 270 proposal of Barnettozyma gen.nov., Lindnera gen.nov. and Banat IM, Nigam P, Singh D, Marchant R, McHale AP (1998) Wickerhamomyces gen.nov. FEMS Yeast Res 8:939–954 Review: ethanol production at elevated temperatures and alcohol Limtong S, Sringiew C, Yonganitchai W (2007) Production of fuel concentrations: Part I—yeasts in general. World J Microbiol ethanol at high temperature from sugarcane juice by new isolated Biotechnol 14:809–821 Kluyeromyces marxianus. Bioresour Technol 98:3367–3374 Brijwani K, Oberoi HS, Vadlani PV (2010) Production of a cellulolytic Miller GL (1959) Dinitro salicylic acid reagent for determination of reducing sugars. Anal Chem 31:426–428 enzyme system in mixed-culture solid state fermentation of soybean hulls supplemented with wheat bran. Process Biochem Oberoi HS, Babbar N, Dhaliwal SS, Kaur S, Vadlani PV, Bhargav VK, 45:120–128 Patil RT (2010a) Enhanced oil recovery by pre-treatment of mustard Carpita NC, Gibeaut DM (1993) Structural models of primary cell seeds using crude enzyme extract obtained from mixed-culture walls in flowering plants: consistency of molecular structure solid-state fermentation of Kinnow (Citrus reticulata) waste and with the physical properties of the walls during growth. Plant wheat bran. Food and Bioprocess Technol. doi:10.1007/s11947- J3:1–30 010-03830-y Dhaliwal SS, Oberoi HS, Sandhu SK, Nanda D, Kumar D, Uppal Oberoi HS, Vadlani PV, Brijwani K, Bhargav VK, Patil RT (2010b) SK (2011) Enhanced ethanol production from sugarcane juice Enhanced ethanol production via fermentation of rice straw with by galactose adaptation of a newly isolated thermotolerant hydrolysate-adapted Candida tropicalis ATCC 13803. Process strain of Pichia kudriavzevii. Bioresour Technol 102:5968– Biochem 45:1299–1306 5975 Oberoi HS, Vadlani PV, Madl RL, Saida L, Abeykoon JP (2010c) Dhillon SS, Gill RK, Gill SS, Singh M (2004) Studies on utilization of Ethanol production from orange peels: Two stage hydrolysis and citrus peel for pectinase production using fungus Aspergillus fermentation studies using optimized parameters through exper- niger. Int J Environ Stud 61:199–210 imental design. J Agric Food Chem 58:3422–3429 Faga BA, Wilkins MR, Banat IM (2010) Ethanol production Oberoi HS, Vadlani PV, Nanjundaswamy A, Bansal S, Singh S, Kaur through simultaneous saccharification and fermentation of S, Babbar N (2011) Enhanced ethanol production from Kinnow switchgrass using Saccharomyces cerevisiae D A and thermoto- mandarin (Citrus reticulata) waste via a statistically optimized lerant Kluyveromyces marxianus IMB strains. Bioresour Technol simultaneous saccharification and fermentation process. Biore- 101:2273–2279 sour Technol 102:1593–1601 Farrell AE, Pelvin RJ, Turner BT, Jones AD, O’Hare M, Kammen DM Pereira AN, Mobedshahi M, Ladisch MR (1998) Preparation of (2006) Ethanol can contribute to energy and environmental goals. cellodextrins. Methods Enzymol 160:26–43 Science 311:506–508 Sa-Correia I, van Uden N (1982) Temperature profiles of ethanol Gancedo JM (1998) Yeast carbon catabolite repression. Microbiol Mol tolerance: effects of ethanol on the minimum and the maximum Biol Rev 62:334–361 for growth of the yeasts Saccharomyces cerevisiae and Kluyver- Ghose TK (1987) Measurement of cellulose activities. Pure Appl omyces fralilis. Biotechnol Bioeng 25:1665–1667 Chem 59:257–268 Sharma N, Kalra KL, Oberoi HS, Bansal S (2007) Optimization of Goering HK, Vansoest PJ (1970) Forage fibre analysis. Agricultural fermentation parameters for production of ethanol from Research Services, United States Department of Agriculture, kinnow waste and banana peels by simultaneous saccharifica- Agricultural Handbook, No 379. ARS, USDA, Washington D. tion and fermentation. Indian J Microbiol 47:310–316 C. Shen Y, Zhang Y, Bao X, Du F, Zhuang G, Qu Y (2008) Simultaneous Grohmann K, Elizabeth AB, Busling BS (1994) Production of ethanol saccharification and fermentation of acid-pretreated corncobs from enzymatically hydrolyzed orange peel by the yeast with a recombinant Saccharomyces cerevisiae expressing β- Saccharomyes cerevisiae. Appl Biochem Biotechnol 45/46:315– glucosidase. Bioresour Technol 99:5099–5103 327 Urbanszki K, Szakacs G, Tengerdy RP (2000) Standardization of filter Grohmann K, Cameron RG, Buslig BS (1995) Fractionation and paper activity assay for solid substrate fermentation. Biotechnol pretreatment of orange peel by dilute acid hydrolysis. Bioresour Lett 22:65–69 Technol 54:129–141 Wang G, Michailides TJ, Bostock RM (1997) Improved detection of Grohmann K, Manthey JA, Cameron RG, Buslig BS (1999) polygalacturonase activity due to Mucor piriformis with a Purification of citrus peel juice and molasses. J Agric Food modified dinitrosalicylic acid reagent. Phytopathology 87:161– Chem 47:4859–4867 163 666 Ann Microbiol (2012) 62:655–666 White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct Wilkins MR, Widmer W, Grohmann K (2007b) Simultaneous sacchar- sequencing of fungal ribosomal RNA genes for phylogenetics. ification and fermentation of citrus peel waste by Saccharomyces In: Innis MA, Gelfand DH Sninsky JJ, White TJ (eds) PCR cerevisiae to produce ethanol. Process Biochem 42(12):1614–1619 protocols: a guide to methods and applications. Academic Press, Wood TM, Bhat KM (1988) Methods for measuring cellulase San Diego, pp 315–322 activities. Methods Enzymol Biomass Part A Cellulose Hemicel- Wilkins MR, Suryawati L, Maness NO, Churz D (2007a) Ethanol lulose 160:87–112 production by Saccharomyces cerevisiae and Kluyveromyces Zhou W, Widmer W, Grohmann K (2008) Developments in ethanol marxianus in the presence of orange-peel oil. World J Microbiol production from citrus peel waste. Proc Fla State Hortic Soc Biotech 23(8):1161–1168 121:307–310

Journal

Annals of Microbiology – Springer Journals

Published: Jul 8, 2011

There are no references for this article.