Biodiesel production from yeast Cryptococcus sp. using Jerusalem artichoke

Bioresource Technology 155 (2014) 77–83

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Biodiesel production from yeast Cryptococcus sp. using Jerusalem artichoke Mina Sung, Yeong Hwan Seo, Shin Han, Jong-In Han ⇑ Department of Civil and Environmental Engineering, KAIST, 291 Daehakno, Yuseong-gu, Daejeon 305-701, Republic of Korea

h i g h l i g h t s  Various acids were used for making Jerusalem artichoke (JA) medium.  Nitric acid showed a best efficiency in this process.  Cryptococcus sp. can grow well in the JA medium.  Lipid productivity from JA medium was better than defined medium having fructose.  Cryptococcus oil was generally satisfied as a transportation fuel.

a r t i c l e

i n f o

Article history: Received 1 November 2013 Received in revised form 3 December 2013 Accepted 6 December 2013 Available online 14 December 2013 Keywords: Biodiesel Jerusalem artichoke HNO3 Pretreatment Cryptococcus sp.

a b s t r a c t Jerusalem artichoke was investigated as a cheap substrate for the heterotrophic production using a lab yeast strain Cryptococcus sp. Using Response Surface Method, 54.0% of fructose yield was achieved at 12% of dried Jerusalem artichoke powder, 0.57% of nitric acid concentration, 117 °C of reaction temperature, and 49 min of reaction time. At this optimal condition, nitric acid showed the best catalytic activity toward inulin hydrolysis and also the resulting fructose hydrolyte supported the highest microbial growth compared with other acids. In addition, lipid productivity of 1.73 g/L/d was achieved, which is higher than a defined medium using pure fructose as a substrate. Lipid quality was also found to be generally satisfactory as a feedstock for fuel, demonstrating Jerusalem artichoke could indeed be a good and cheap option for the purpose of biodiesel production. Ó 2013 Elsevier Ltd. All rights reserved.

1. Introduction The world is in great need of alternative energy sources, because of the limited availability and side effects (i.e., climate change) of the current fossil fuels (Meng et al., 2009). Biodiesel has been recognized as one renewable option of transportation fuels; in addition to the nature of renewability, it has several merits of being readily biodegradable and less toxic and containing no sulfur and/or aromatics (Li et al., 2008). The present commercial biodiesel, however, is made solely from vegetable oils which are all edible. This brings about instability of food prices and even an ethical issue. To avoid such problems and meet rapidly growing demands of bio-based fuels, ways of producing oil from non-food sources must be developed (Li et al., 2008). Recently, microbial oils have received great attention as some of potential non-food routes for biodiesel production, as they can be continuously produced and harvested all year round and yet do ⇑ Corresponding author. Tel.: +82 42 350 3629; fax: +82 42 350 3610. E-mail addresses: [email protected], [email protected] (J.-I. Han). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.12.024

not take up large arable land (Papanikolaou and Aggelis, 2002; Liu and Zhao, 2007; Angerbauer et al., 2008; Zhao et al., 2008). A variety of microorganisms, such as microalgae, fungi, and yeast, are able to accumulate oil in the form of lipid body and they are collectively called oleaginous microbes (Li et al., 2008). Among these, microalgae, especially in a photoautotrophic mode in which cells grow via photosynthesis, are the most well-known. Another way of producing microbial lipid is to grow oleaginous cells with carbon and energy sources, termed heterotrophic cultivation; oleaginous yeasts are grown this way. Heterotrophic cultivation has distinct advantages of high productivity and high final cell concentration (Ageitos et al., 2011; Seo et al., 2012). Unlike the phototrophic cultivation, however, substrates are required for the oil production, which is the prime cause of its high production cost (Meesters et al., 1996). Sufficiently cheap feedstock, therefore, must be sought to achieve the economic viability of this otherwise ideal oil production route. In this sense, Jerusalem artichoke can be a good candidate feedstock. Jerusalem artichoke has very high carbohydrate yields ranging between 5 and 14 ton per hectare (Cheng et al., 2009). This

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plant is very rugged in that it can grow vigorously even on wild land with harsh conditions. Only labor requirement is in sowing seed. What is better, Jerusalem artichoke is a non-food crop as humans do not possess an appropriate enzyme system able to digest the major carbohydrate inulin, in which fructose units are linked with the end glucose unit (Hoebregs, 1997; Cheng et al., 2009). Inulin with the relatively weak bond of ß (2?1), on the other hand, is easily hydrolyzed by microbial enzymes or acidic catalysts; the resulting fructose monomers are of course a ready food for many microbes such as oleaginous yeasts. Thus far, common acids, such as hydrochloric, sulfuric, and phosphoric acids, have been used as hydrolysis catalysts (Toran-Diaz et al., 1985; Kim and Hamdy, 1986; Zhao et al., 2010; Li et al., 2010). Nitric acid chosen in this study, though quite limited, has also been tried before. Rodr´iguez-Chong et al. (2004) found that using sugar cane bagasse nitric acid was more effective than other more common acids, in terms of short treatment time and reduced generation of inhibitors (Rodr´iguez-Chong et al., 2004). Nitrate (NO 3 ), the neutralized species of nitric acid, is known to promote cell growth and likely so in view of the typical biological way of nitrogen assimilation (Ishimoto and Yamamoto, 1977; Adda et al., 1986). In this study, therefore, we attempted to confirm it in a systematic way using our lab yeast strain, Cryptococcus sp. 2. Methods

pending the Jerusalem artichoke samples in nitric acid solutions either in the flask or the reactor, and being heated to specific temperatures for designated times. As controls, hydrochloric acid and sulfuric acid were also used; these control samples served to prove the efficacy of nitric acid as a hydrolysis catalyst and also a nitrogen source for yeast growth. After the pretreatment, pHs were adjusted to about 6 using sodium hydroxide (10 N NaOH). The undigested remains were removed by centrifugation at 12,000 rpm for 10 min and filtered with 0.2 lm PES filters (Whatman), and the clear supernatants were then used as culture media with the aforementioned amendment. All experiments were done in triplicate.

2.3. Statistical experimental design Response surface methodology (RSM) was taken to systematically design experiments and obtain the optimized yield of fructose. A three-level-three factor Box–Behnken design (BBD) was employed to obtain the interactions of three independent variables: reaction temperature (A), pretreatment time (B), and concentrations of nitric acid (C). Fructose yield was taken as the response (Y). Each of variables was varied as follows: 0.05–1.00% of nitric acid concentrations, 10–50 min of times, and 60–150 °C of temperatures. The data analysis was carried out via Design-Expert (Stat-Ease, Inc, USA) software.

2.1. Microorganism and culture medium Cryptococcus sp. was isolated from a fermented seafood obtained in Suncheon, Korea, and identified through sequencing 18S rRNA, and maintained on YM Agar Medium contained the following ingredients: 3 g of yeast extract, 3 g of malt extract, 5 g of peptone, 10 g of dextrose, in 20 g of agar in 1 L of deionized water with pH 5.5. Jerusalem artichoke medium (JAM), containing only pretreated Jerusalem artichoke and trace element (DSMZ medium 320), was mainly used to cultivate the lab yeast strain (100 mL in a 250 mL culture flask). For comparison, Cryptococcus Growth and Lipid accumulation Medium (CGLM) was used, containing 0.5 g of NH4Cl, 2.7 g of KH2PO4, 0.95 g of Na2HPO4, 1 g of MgSO47H2O, different amounts of fructose (C/N molar ratio 25, 50, 100, 150), and 10 mL of trace solution (DSMZ medium 320) in 1 L of deionized water. All YM and CGLM were sterilized by autoclave and yeast cultivation was executed at the shaking speed of 200 rpm and 30 °C. 2.2. Jerusalem artichoke preparation and pretreatment Jerusalem artichoke tubers were products of year 2012 from Gangwon province, Republic of Korea. The compositions of the Jerusalem artichoke tuber were 75.0% of moisture, 81.9% of total carbohydrate (dry basis), 0.45% of total phosphate (TP, dry basis), and 1.25% of total nitrogen (TN, dry basis). All tubers were washed, peeled, sliced into small pieces, vacuum-packed, and stored in a freezer (4 °C). For pretreatment experiments, three different types of tuber samples, i.e., slurry, powdered, and sliced tubers, were prepared. Sliced tubers were made by scissoring frozen Jerusalem artichoke. Mesh size was 1  1 cm. The slurry sample was made by mashing sliced tubers in a mortar and pestle. The dried powdered sample was prepared by crushing tubers dried at 50 °C for 1 day using the mortar and pestle. Two different pretreatment vessels were used: for under 100 °C a 200 mL flask in a water bath, and over 100 °C using a stainless steel reactor (£ 26 132 mm with 70 mL of volume) coated with Teflon on the inside in an oil bath. Pretreatment was done by sus-

2.4. Analytical method Moisture content was determined by American standard test method D2974. Total carbohydrate (TC) of Jerusalem artichoke was measured according to phenol–sulfuric acid assay (Taylor, 1995), and total phosphate (TP) and total nitrogen (TN) were measured by water test kits (Humas, HS-TP-L, HS-TN-L(CA)) with UV– Vis spectrophotometer (DR 5000, HACH). After the pretreatment, sugar (mostly fructose) contents were analyzed by high performance liquid chromatography (HPLC) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories Inc., USA). Fructose yield (%) was calculated by the following equation:

Y ¼ ðF=IÞ  100

ð1Þ

where Y is the fructose yield, F is the final fructose content produced (g/L), I is the initial carbohydrate content of Jerusalem artichoke inputted (g/L). The values of initial Jerusalem artichoke inputted were re-calculated considering its moisture content and also carbohydrate content. The growth of Cryptococcus sp. was monitored by measuring optical density at 600 nm (OD600) using a UV–Vis spectrophotometer (DR 5000, HACH). To determine dry cell weight (DCW), 1 mL of culture at each OD600 value was taken, centrifuged at 12000 rpm for 5 min, and washed with deionized water. Then, the cells were oven-dried at 50 °C until no change in weight was observed. A relationship between OD600 and DCW values was estimated via a calibration curve. Lipid contents and compositions were analyzed according to the procedure described by Folch et al. (1957) with slight modification. Briefly, cells were centrifuged at 2000 rpm for 10 min and rinsed twice with deionized water. The cell pellet was rapidly frozen in liquid nitrogen for 20 min and then lyophilized at 52 °C for 4 days. Lipid extraction and transesterification were done according to Seo et al. (2012).

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Fig. 1. Fructose concentration and yield depending on (a) various forms and (b) concentrations of Jerusalem artichoke.

Table 1 ANOVA for the quadratic model for optimizing Jerusalem artichoke pretreatment. Source

Sum of squares

df

Mean squares

F value

p-value Prob > F

Model A-Temp. B-Time C-HNO3 AB AC BC A2 B2 C2 Residual Lack of fit Pure error Cor total

7486.91 549.46 454.51 3112.60 320.41 42.90 14.82 2819.01 142.87 136.80 1009.59 1009.59 0.000 8496.50

9 1 1 1 1 1 1 1 1 1 7 3 4 16

831.88 549.46 454.51 3112.60 320.41 42.90 14.82 2819.01 142.87 136.80 144.23 336.53 0.000

5.77 3.81 3.15 21.58 2.22 0.30 0.10 19.55 0.99 0.95

0.0154 0.0919 0.1191 0.0024 0.1797 0.6024 0.7579 0.0031 0.3528 0.3625

Significant

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Table 2 The Box–Behnken Design data and corresponding fructose yield with different combinations of three independent variables when pretreated Jerusalem artichoke was used for Cryptococcus sp. growth. Run

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Coded variable level

Experimental factors

Fructose yield (%)

A

B

C

Temp. (°C)

Time (min)

HNO3 (%)

Experimental

Theoretical

0 0 1 0 0 1 0 1 1 1 0 1 0 0 1

0 1 1 1 0 1 0 0 1 0 0 0 1 0 1

0 1 0 1 0 0 0 1 0 1 0 1 1 0 0

105 105 60 105 105 150 105 150 150 150 105 60 105 105 60

30 50 50 10 30 10 30 30 50 30 30 30 50 30 10

0.53 1.00 0.53 0.05 0.53 0.53 0.53 0.05 0.53 1.00 0.53 1.00 0.05 0.53 0.53

37.6 ± 0.4 84.6 ± 3.9 6.00 ± 1.0 17.5 ± 3.1 37.6 ± 0.4 11.2 ± 2.0 37.6 ± 0.4 13.7 ± 3.8 50.6 ± 4.3 27.6 ± 3.5 37.6 ± 0.4 27.7 ± 4.4 22.3 ± 3.6 37.6 ± 0.4 2.4 ± 4.7

37.6 78.3 7.8 23.8 37.6 9.3 37.6 9.3 42.3 42.2 37.6 32.1 35.0 37.6 10.7

Fig. 2. (A) Fructose yield and (B) cultivation of Cryptococcus sp. depending on several acid solutions at the economical optimal pretreatment condition (117 °C, 49 min, same concentration of 0.57% HNO3).

M. Sung et al. / Bioresource Technology 155 (2014) 77–83

3. Results and discussion 3.1. Jerusalem artichoke preparation To determine how forms of Jerusalem artichoke tubers affected the effectiveness of acid pretreatment, sliced tubers (Slice), slurry suspension (Slurry), and powdered tubers (Powder) were prepared and tested. The pretreatment condition was fixed at the center point (a solid concentration of 12% of Jerusalem artichoke tubers (weight percent), nitric acid concentration of 1.00%, treatment time of 20 min, and temperature of 120 °C). As seen in Fig. 1(A), the powdered sample exhibited the highest fructose concentration (56.8 g/L) and yield (57.7%), which makes sense in light of its large surface area. Also in consideration of the same quantity of Jerusalem artichoke, powder itself has the highest amount of carbohydrates which can react quantitatively with the acid molecule. 12% of powder showed the best efficiency of fructose yield (Fig. 1(B)). Thus, 12% of powdered Jerusalem artichoke was employed in the following experiments.

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The theoretical values of fructose yield with different interactions of three independent variables and the experimental values are summarized in Table 2. This result clearly indicates that experimental values were distributed linearly with high correlation (R2 = 0.9126). The maximum fructose yield of 84.6% was obtained at 105 °C of reaction temperature, 50 min of reaction time, and 1.00% of nitric acid, which is the condition near to edge. Considering the overall process economics, however, another near-edge condition with lower nitric acid was selected: 117 °C of temperature, 49 min of reaction time, and 0.57% of nitric acid. Under this condition, fructose yield was found to be 54.0%. Compared with maximal conditions of fructose yield (Run 2 at Table 2), temperature was increased slightly from 105 to 117 °C, while the concentration of nitric acid reduced by 50%. By decreasing the amount of nitric acid, the ratio of carbon and nitrogen moles (C/N molar ratio) was increased, which is more favorable in terms of lipid accumulation in oleaginous microorganisms including Cryptococcus species. 3.3. Cryptococcus sp. cultivation on pretreated Jerusalem artichoke medium (JAM)

3.2. Optimization of pretreatment for Jerusalem artichoke Response surface methodology (RSM) designed by Box–Behnken was applied to discover an optimal pretreatment condition. Parameters for this included reaction time, temperature, and concentration of nitric acid. Variance for the quadratic design was analyzed to check the validity of the model, and is shown in Table 1. The model is considered significant because p-value is 0.0154. In this case, C (HNO3), A2 (Temp.2) are the most significant model terms. The second order polynomial equation of fructose yield in terms of actual factors is stated in the following equation:

Fructose yield ¼ 109:58026 þ 2:64961  Temp:  1:64742  Time þ 25:00877  HNO3 þ 0:00994444  Temp:  Time  0:15322  Temp:  HNO3 þ 0:20263  Time  HNO3  0:012778 2

 Temp:2 þ 0:014562  Time þ 25:26316  HNO23

ð2Þ

3.3.1. Benefits of HNO3 Catalytic activity of nitric acid was also compared with other acids at the selected optimal condition (i.e., 117 °C and 49 min with the same molar concentration of nitric acid). In terms of fructose yield, sulfuric acid was more efficient than nitric acid (Fig. 2(A)). In terms of the cultivability of Cryptococcus sp., however, nitric acid was the best: the yeast growth on the Jerusalem artichoke treated by nitric acid substantially exceeded that with sulfuric acid (Fig. 2(B)). It was possible that sulfuric acid was potent enough to cleave the inulin linkage and produce fructose monomers in an efficient way, but it was too strong to degrade the produced fructose and generate some growth-inhibiting byproducts like HMF, negatively effecting initial growth (or adaptability) of Cryptococcus sp. 3.3.2. Lipid production Cryptococcus sp. was cultivated in the JAM prepared using the economical pretreatment conditions (Fig. 3). Final cell concentration was almost 6.1 g/L and lipid content approximately 28.5% after 36 h, indicating that Cryptococcus sp. indeed grew and accumulated lipid with JAM, which was comparable

Fig. 3. Cryptococcus sp. growth and accumulated lipid content at pretreated Jerusalem artichoke medium.

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Table 3 Major lipid compositions of Cryptococcus sp. at Jerusalem artichoke medium and Cryptococcus Growth and Lipid accumulation Medium (CGLM), and comparison with other vegetable oil. Types of fatty acids

C C C C C

16:0 18:0 16:1 18:1 18:2

Relative amount of total fatty acids (% w/w)

(Palmitic acid) (Stearic acid) (Palmitoleic acid) (Oleic acid) (Linoleic acid)

Jatropha oil

Soybean oil

Sunflower oil

14.66 6.860 0.940 39.08 32.48

11.00 4.000  23.40 53.20

 4.500  21.10 66.20

Cryptococcus oil Jerusalem artichoke

Fructose 50

Fructose 100

Fructose 150

20.03 14.70 0.29 34.60 11.20

25.53 19.90 0.29 35.98 14.10

25.15 19.34 0.28 34.60 18.00

24.35 17.34 0.36 34.35 19.90

Table 4 An assessment of Cryptococcus oil from pretreated Jerusalem artichoke medium as a fuel. Name

C16:0

C16:1

C18:0

C18:1

C18:2

Cetane number

Oxidation stability

CFPP

Iodine value

Jatropha Soybean Sunflower Jerusalem artichoke Fructose 50 Fructose 100 Fructose 150

14.66 11.00 0.00 20.03 25.53 25.15 24.35

0.94 0.00 0.00 0.29 0.29 0.28 0.36

6.86 4.00 4.50 14.70 19.90 19.34 17.34

39.08 23.40 21.10 34.60 35.98 34.60 34.35

32.48 53.20 66.20 11.20 14.10 18.00 19.90

53.08 48.32 43.74 58.25 57.70 56.81 56.47 >51

6.22 4.81 4.37 13.12 10.95 9.14 8.52 >6

1.07 6.72 9.40 12.96 22.87 21.87 18.47 <0 (summer)<10 (winter)

93.98 113.84 132.80 55.83 61.58 66.70 69.61 <120

EN std

to CGLM. The highest amount of dry cell weight was obtained in the JAM case. Except for C/N ratio = 50, lipid content was also highest. Moreover, considering lipid productivity (dry weight  lipid content), 1.73 g/L/d of lipid was produced in a day, and this is more than C/N ratio 50 case (1.5 g/L/d). Lipid compositions of Cryptococcus sp. grown in both the pretreated JAM and CGLM were analyzed and compared with other vegetable oils such as jatropha and sunflower oil (Table 3). Cryptococcus oil was found to have greater portions of total mono-unsaturated fatty acid than the other vegetable counterparts (Cryptococcus oil (35%) vs. Soybean oil (23.4%) or Sunflower oil (21.10%)). The more mono-unsaturated fatty acids lipid contains, the better quality biodiesel can be made (Fallen, 2009). Other important properties of the Cryptococcus oil in relation to the quality as a transportation fuel were also assessed, namely, cetane number, iodine value, oxidation stability, and cold filter plugging point (CFPP) and these are list in Table 4. Cetane number (CN) is related to measure the combustion quality of diesel fuel. High CN value means short ignition delay, resulting in the efficient operation of diesel engine with reduction of nitrogen oxides (NOx) exhaust emissions (Bamgboye and Hansen, 2008). The minimum value of CN set in European unions is 51. Some vegetable oils do not meet the CN standard, whereas the Cryptococcus oil, especially cultivated in the pretreated JAM, showed the highest values, along with superb oxidation stability. Iodine value, which expresses the extent of unsaturation of the oil, also meets the European standards. Only CFPP was unsatisfactory, but even this criterion was superior in comparison with the oil from CGLM. Considering all these, oil production via heterotrophic cultivation using our yeast isolated and Jerusalem artichoke can surely be one promising way of making an alternative transportation fuel. 4. Conclusion This study proved that Jerusalem artichoke, composed of high content of inulin, could be a good low-cost feedstock of oleaginous yeasts, especially our isolated Cryptococcus sp. Nitric acid was the best acid catalyst, which efficiently hydrolyzed inulin with the lim-

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