Notable mixed substrate fermentation by native Kodamaea ohmeri strains isolated from Lagenaria siceraria flowers and ethanol production on paddy straw hydrolysates
© The Author(s) 2018
Received: 22 December 2016
Accepted: 20 January 2018
Published: 5 February 2018
Bioethanol obtained by fermenting cellulosic fraction of biomass holds promise for blending in petroleum. Cellulose hydrolysis yields glucose while hemicellulose hydrolysis predominantly yields xylose. Economic feasibility of bioethanol depends on complete utilization of biomass carbohydrates and an efficient co-fermenting organism is a prerequisite. While hexose fermentation capability of Saccharomyces cerevisiae is a boon, however, its inability to ferment pentose is a setback.
Two xylose fermenting Kodamaea ohmeri strains were isolated from Lagenaria siceraria flowers through enrichment on xylose. They showed 61% glucose fermentation efficiency in fortified medium. Medium engineering with 0.1% yeast extract and peptone, stimulated co-fermentation potential of both strains yielding maximum ethanol 0.25 g g−1 on mixed sugars with ~ 50% fermentation efficiency. Strains were tolerant to inhibitors like 5-hydroxymethyl furfural, furfural and acetic acid. Both K. ohmeri strains grew well on biologically pretreated rice straw hydrolysates and produced ethanol.
Recent environmental disturbances, fluctuating prices, and uncertainties associated with the use of conventional fuels, have led to paradigm shift to displace conventional fuels with sustainable, renewable, and environmentally friendly/clean energy sources, among which biomass-derived energy appears to be the most promising option . Of various alternative energy sources, bioenergy derived from lignocellulosic biomass has attracted significant attention as one of the routes to address energy crisis, especially bioethanol in transport sector . Second generation bioethanol, produced by fermenting sugar slurries obtained from enzymatic hydrolysis of cellulose present in lignocellulosic biomass, has the potential of being a major contributor to meet the global energy demand, as biomass is the most abundant, sustainable, and renewable resource on earth. However, unfavorable economics is the foremost impediment in successful deployment of this process on industrial scale. An efficient pretreatment with lower inhibitor generation followed by enzymatic hydrolysis for maximum sugar recovery, and complete utilization and fermentation of all the sugars present in hydrolysates will aid in making the process cost effective . In addition to cellulose, biomass also has hemicellulose, which is the second major polysaccharide, consisting of hexoses and pentoses, with xylose as the major pentose sugar.
Thus, complete conversion of lignocellulosic biomass entails a co-fermenting yeast, capable of fermenting both glucose and xylose yielding high ethanol titers. Development of strains for use in industrial-scale facilities is continuously being carried out in parallel with the process optimization. Commercial strains of S. cerevisiae, the most widely used organisms for ethanol production are exclusively involved in glucose fermentation, thus completely utilizing cellulosic fraction while xylose is left unfermented. To overcome this drawback of S. cerevisiae, recombinant strains capable of utilizing xylose have been developed since 1980s but ethanol yield was found to be low . Since then, several genetic engineering approaches have been adopted for developing a recombinant strain capable of mixed substrate fermentation but with limited success . This is due to the constraints associated with co-fermentation, like aerobic process of xylose fermentation, co-factor (NADH) imbalance  and glucose repression [7, 8]. In addition, inhibitors present in biomass hydrolysates  and medium constituents  have been observed to affect yeast physiology and fermentation efficiency [11, 12]. All these issues need to be addressed earnestly.
On the other hand, native pentose fermenting yeasts are well known [4, 13]. First report of ethanol production from xylose by yeast came in 1958 when Karczewska  observed ethanol production from Candida tropicalis. Pichia and Scheffersomyces are the most interesting pentose fermenting yeasts but their co-fermenting abilities on mixed substrates are yet to be established to the extent suitable for commercial application . Numerous native yeasts are known for xylose assimilation but very few are reported for efficient fermentation of xylose to ethanol. Such yeast include Pichia, Candida, Pachysolen, Clavispora, Debaromyces, Kluyveromyces, Cryptococcus, Rhodotorula etc. Researchers have demonstrated low to high ethanol production from xylose in rich medium, by different yeasts isolated from natural habitats like tree bark, decaying wood samples and insect gut [16–18]. Mixed substrate utilization and co-fermentation is still a challenge. Thus, rational bio prospecting for native pentose assimilating and fermenting yeasts is the contemporary approach and increasing efforts have recently been put into evaluating natural xylose fermenting potential of yeasts [19, 20].
A yeast genus Kodamaea, earlier placed under Pichia genus has been reported for pentose utilization including xylose and arabinose but fermentation of pentoses to ethanol has not been reported. A novel sp. of Kodamaea, K. kakuduensis, isolated from Australian Hibiscus flower, was reported to be a good glucose fermenter with weak xylose assimilation properties . Kodamaea ohmeri has been explored for its food fermentation properties especially for pickling and cocoa beans but ethanol production has not been reported yet . Zhu et al.  described d-arabitol as the main product from glucose by K. ohmeri. This study illustrates mixed sugar utilization, ethanol fermentation potential, and inhibitor tolerance of two native K. ohmeri strains isolated from the flowers of L. siceraria plant for their possible exploitation in bioethanol production.
Isolation of yeast strains
Lagenaria siceraria flowers were collected, washed with distilled water and crushed in pestle mortar with 0.8% saline under aseptic conditions. 1 mL of this suspension was inoculated into 50 mL MXYP broth (0.5% malt extract, 1% xylose, 0.5% yeast extract and 0.3% peptone, pH 5) in 100 mL flasks with 0.25% sodium propionate, for enrichment of xylose utilizing yeasts. After 48 h incubation at 30 °C, culture samples were plated on MXYP agar with chloramphenicol (50 µg mL−1) antibiotic. Plates were incubated for 24 h at 30 °C and colonies were selected based on their morphology. Selected colonies were purified and grown on same medium and glycerol stocks were prepared.
Identification and characterization of selected yeast strains
Two potent xylose assimilating strains were selected, strain 5 and strain 6. Both the strains were characterized on morphological, biochemical as well as on molecular level. Phenotypic characterization was done on the basis of their colony and cell morphology using phase contrast microscopy and scanning electron microscopy. Molecular characterization included sequencing of the ITS region of the yeast strains.
Studying cell morphology using phase contrast microscopy and scanning electron microscopy
To study morphology, overnight grown cultures were observed under phase contrast microscope (Olympus America Inc.) at magnification 10× and 40×. Cell morphology was also studied using scanning electron microscope (Zeiss EVOMA10). Overnight incubated cultures on xylose (1 mL) were centrifuged at 8000g for 10 min, 2.5% glutaraldehyde fixative was added to the pellet and kept for 2–4 h to arrest growth. Cultures were then washed with 0.1 M phosphate buffer thrice at an interval of 15 min. Samples were dehydrated with a graded series of acetone (30, 50, 70, 80, 90, 95 and 100%), fixed on cover slips placed over stuff grids. A drop of hexamethyl disilazone was added over the cover slips and then allowed to dry in a fume hood. Cells were observed with scanning electron microscope at an acceleration voltage of 20 kV and images recorded.
Molecular identification through ITS sequencing
Further confirmation was done by PCR amplification of ITS region. PCR procedures involved denaturation at 95 °C for 5 min, followed by 35 cycles of 94 °C for 5 min, 55 °C for 30 s and extension at 72 °C for 45 s, with final extension for 10 min at 72 °C. Amplified products were run over 1% agarose gel to confirm their molecular size. ITS sequencing of the amplified products was completed by Xcelris, India and further analyzed using Basic Local Alignment Search Tool (BLAST) . Partial sequencing of the strains was done using ITS 1 and ITS 4 degenerate primers i.e., ITS1-forward primer (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4-reverse primer (5′-TCCTCCGCTTATTGATATGC-3′) .
Ability of Kodamaea ohmeri strains to assimilate different sugars was tested using biochemical strips (Hi Media) for yeast. Overnight cultures were inoculated on the strips (100 µL each) and incubated at 28 °C. Results were observed for 72 h.
Determining enzyme activities
K. ohmeri strains were grown for 48 h on 2% xylose, and mixed sugars (2% xylose + 2% glucose) in minimal medium with shaking at 150 rpm at 30 °C. After 48 h, cultures were centrifuged at 8000 rpm for 10 min and supernatant was discarded. Pellet was processed for xylose reductase (XR) and xylitol dehydrogenase (XDH) activities were measured and expressed as specific activities. Protein concentration in crude extracts was measured using BSA as standard.
Reaction cocktail for xylose reductase activity
Reaction cocktail for xylitol dehydrogenase activity
500 mM tris–HCl
Fermentation abilities of K. ohmeri strains
Both strains were grown in minimal medium (1.36 mg L−1 KH2PO4, 0.2 g L−1 MgSO4·7H2O, 2.0 g L−1 NaCl, 1.0 g L−1 (NH4)2SO4, 10 mg L−1 FeSO4, pH 5) with 5% xylose/10% glucose or both as carbon source for 72 h at 30 °C to check their ability to grow and ferment xylose. Effect of salts like NaCl and FeSO4 was studied. Medium (50 mL) in 100 mL Erlenmeyer flasks was inoculated (10% inoculum) and incubated for 72 h at 30 °C. Inoculum was prepared in MXYP broth (pH 7.0) by incubating it at 30 °C for 48 h and shaking (150 rpm). Aliquots were aseptically withdrawn at regular intervals and the absorbance read at 660 nm (Specord 200) to measure growth. These aliquots were then centrifuged at 10,000 rpm for 10 min and supernatants were used for estimation of sugar consumption and ethanol production by HPLC as described later.
Fermentation of mixed sugars
Stimulation of fermentation ability upon medium supplementation
Effect of medium supplementation with yeast extract and peptone on ethanol production was studied. Treatments with combinations of yeast extract (0.1–1%) and peptone (0.1 and 1%) with pure or mixed sugars (10% glucose or 10% glucose + 5% xylose) were applied. Incubation was carried out as described earlier and samples were analyzed for growth and fermentation.
Ethanol levels were estimated using chromatographic techniques, such as HPLC and GC.
High performance liquid chromatography
Cultures were harvested at regular intervals, centrifuged at 8000 rpm for 10 min, filtered using 0.22 µ syringe filters and subjected to analysis by HPLC. Samples were run on Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 65 °C using 5 mM H2SO4 as mobile phase at 0.5 mL min−1 and measured with a Shodex RI-101 refraction index detector (Shoko Scientific Co. Ltd., Yokohama, Japan). Ethanol concentration and sugar consumption were determined.
Inhibitor tolerance of K. ohmeri strains
For exploitation of K. ohmeri strains for fermentation of biomass hydrolysates, it is important to check their capability to grow in presence of HMF, furfural, formic acid and acetic acid, the predominant by-products of biomass pretreatment which are present in hydrolysates and reported to inhibit growth.
Cultures were grown in presence of HMF (0.5–5.0 g L−1) and furfural (0.25–0.65 g L−1) in minimal medium with 5% glucose + 2.5% xylose and 0.1% yeast extract for 96 h. Growth was checked every 24 h by reading absorbance at 660 nm. Appropriate controls were maintained and growth was compared. Similar experiment was carried out using acetic acid (5–15 g L−1) and formic acid (3–11 g L−1) under similar conditions. All the experiments were carried out in triplicates.
Growth and fermentation on biologically pretreated paddy straw hydrolysates
Rice straw of the aromatic rice (Pusa 2511) was pretreated under solid state fermentation using Trametes hirsute, for 7 days and cellulose content was analysed in pretreated solids . Enzymatic hydrolysis of biologically pretreated solids was carried out using accellerase®1500 (Genencor) loading corresponding to 0.5 mL (~ 15 FPU) per g glucan . Total sugars in hydrolysates were estimated using DNS .
Both strains were grown in hydrolysates  and culture samples were periodically withdrawn. Samples were processed. Growth and sugar consumption were observed. Ethanol production was detected by HPLC. Defined medium with 1.3% glucose served as control.
Statistical analyses of the results was done using SPSS (Version 21.0. Armonk, NY: IBM Corp).
Results and discussion
Growth and characterization
Attributes pertaining xylose metabolism
Xylose reductase and xylitol dehydrogenase enzyme activities pertaining to xylose metabolism [36, 37] were exhibited by both the strains but levels were low. The activities suggested the presence of xylose metabolizing pathway in these strains but levels were too low and their ratio predicted the flow of the pathway towards ethanol production. Specific activities (U mg−1 protein) of the strains were found to be 0.024, 0.2 (XR) for strain 5 and 6 respectively, while 0.011 and 0.015 (XDH) for strain 5 and strain 6 respectively.
Fermentation and co-fermentation capabilities and effect of supplementation
Glucose utilization and ethanol yield of strain 5 and strain 6
Glucose (g L−1)
Ethanol yield (g g−1)
0.1% yeast extract + 0.1% peptone
90.40 ± 16.6
97.95 ± 1.8
88.90 ± 17.2
0.16 ± 0.04
0.28 ± 0.05
0.20 ± 0.09
0.5% yeast extract
99.97 ± 0.06
0.16 ± 0.09
0.25 ± 0.06
0.28 ± 0.12
1% yeast extract + 1% peptone
97.74 ± 3.71
99.91 ± 0.16
0.13 ± 0.02
0.12 ± 0.02
0.12 ± 0.02
0.1% yeast extract + 0.1% peptone
99.9 ± 0.17
0.14 ± 0.01
0.12 ± 0.02
0.5% yeast extract
99.18 ± 1.42
0.22 ± 0.05
0.24 ± 0.12
1% yeast extract + 1% peptone
99.83 ± 0.21
100 ± 0.01
0.24 ± 0.14
0.31 ± 0.10
0.20 ± 0.10
Effect of supplementation on sugar utilization and ethanol yield of K. ohmeri strains
Xylose consumed (g L−1)
Glucose consumed (g L−1)
Ethanol yield (g g−1)
Fermentation efficiency (%)
0.1% (YE + P)
20.47 ± 1.9
20.4 ± 3.2
16.2 ± 3.05
0.21 ± 0.017
0.22 ± 0.033
44 ± 3.3
40.8 ± 6.5
43.6 ± 5.9
20.3 ± 2.2
18.8 ± 0.57
14.3 ± 1.9
0.16 ± 0.02
0.17 ± 0.01
0.18 ± 0.01
32 ± 4.5
32.5 ± 1.5
34.9 ± 2.4
1% (YE + P)
17.7 ± 2.8
15 ± 7.4
13.3 ± 0.02
0.21 ± 0.03
0.19 ± 0.07
0.2 ± 0.001
41.3 ± 5.4
37.1 ± 14.6
39.7 ± 0.18
0.1% (YE + P)
11.7 ± 1.7
13 ± 4.3
14 ± 0.45
49.3 ± 0.19
0.25 ± 0.02
0.19 ± 0.05
0.2 ± 0.001
48.6 ± 3.13
38 ± 8.8
39.7 ± 0.25
15.1 ± 9.13
18 ± 6.15
14.2 ± 3.8
0.2 ± 0.01
0.19 ± 0.06
0.2 ± 0.03
38.7 ± 18.1
38 ± 12.1
39.6 ± 6.2
1% (YE + P)
12.2 ± 0.67
66.6 ± 2.4
14.9 ± 0.12
0.25 ± .007
0.18 ± 0.03
0.18 ± 0.001
49.5 ± 1.5
36 ± 5.3
35.1 ± 0.28
Ethanol yields of pentose fermenting strains
Lignocellulosic biomass is pretreated to facilitate higher conversion of biomass polysaccharides to fermentable sugars such as glucose, xylose, arabinose etc. This process generates by-products which inhibit growth of microbes and obstruct fermentation process. In general, these inhibitors are classified into four groups including lignin degradation by-products (phenolics), sugar degradation by-products (HMF and furfural), and products derived from the structure of the biomass and heavy metal ions (chromium and nickel) . Effect of most commonly found inhibitors like HMF, furfural, acetic acid and formic acid was determined on growth of K. ohmeri strains.
Growth and ethanol production by K. ohmeri strains from biomass hydrolysates
Ethanol yields of K. ohmeri strains from rice straw biomass hydrolysates
Ethanol produced (g L−1)
K. ohmeri strain 5 (control)
0.98 ± 0.01
0.065 ± 0.003
0.059 ± 0.0005
0.015 ± 0.0025
K. ohmeri strain 5 (hydrolysate)
0.3 ± 0.001
0.35 ± 0.01
1.92 ± 0.04
0.001 ± 0.0015
K. ohmeri strain 6 (control)
1.07 ± 0.01
1.15 ± 0.05
0.71 ± 0.02
0.26 ± 0.02
K. ohmeri strain 6 (hydrolysate)
0.12 ± 0.03
0.78 ± 0.02
1.28 ± 0.01
0.04 ± 0.008
Screening for microbes capable of co-fermentation is necessary for efficient conversion of lignocellulosic biomass into ethanol with enhanced productivity. There is a significant advancement in developing a robust microbial strain with co-fermentation potential as well as tolerance to inhibitors. K. ohmeri strains, studied here showed promising mixed sugar fermentation potential with enhanced xylose utilization. Strains were also tolerant to HMF, furfural, formic acid and could grow well in presence of acetic acid on prolonged incubation. The study emphasizes that this genus could provide robust native yeast strains with co-fermentation properties which can be evolved further. Lignocellulosic hydrolysates often generate unexpected results due to the presence of inhibitors, as they vary widely in nature . These strains displayed efficient growth and ethanol production from biologically pretreated rice straw hydrolysates.
SS and PS carried out the experimental work. AA conceptualized the study, designing experiments and helped in the finalization of manuscript. Dr. SS performed HPLC of all the samples. Dr. LN and Dr. DP contributed for the saccharification and fermentation experimental work. All authors read and approved the final manuscript.
This work was supported by AMAAS (Grant No. 12-124), ICAR, India. Scanning electron microscopy was carried out in the Division of Entomology, ICAR-IARI, India.
The authors declare that they have no competing interests.
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