Detoxification of Jatropha curcas kernel cake by a novel Streptomyces fimicarius strain

Journal of Hazardous Materials 260 (2013) 238–246

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Detoxification of Jatropha curcas kernel cake by a novel Streptomyces fimicarius strain Xing-Hong Wang a,1 , Lingcheng Ou b,1 , Liang-Liang Fu a , Shui Zheng a , Ji-Dong Lou c , José Gomes-Laranjo d , Jiao Li a , Changhe Zhang d,∗ a

Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, China School of Development Studies, Yunnan University, Kunming 650091, China c College of Life Science, China Jiliang University, Hangzhou 310018, China d Center for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Department of Biology and Environment, Universidade de Trás-os-Montes e Alto Douro (UTAD), Apartado 1013, Vila Real 5001-801, Portugal b

h i g h l i g h t s • • • • •

The kernel cake was highly toxic even though the phorbol esters were undetectable. An animal model was established to quantify the general toxicity of the kernel cake. A new Streptomyces fimicarius strain was able to degrade the toxins by 97%. The detoxified kernel cake was nontoxic to carp fingerling and improved plant growth. Strain profile essential for the kernel cake detoxification was discussed.

a r t i c l e

i n f o

Article history: Received 1 March 2013 Received in revised form 6 May 2013 Accepted 8 May 2013 Available online 16 May 2013 Keywords: Jatropha curcas Biodiesel Kernel cake detoxification Solid state fermentation Streptomyces fimicarius YUCM 310038

a b s t r a c t A huge amount of kernel cake, which contains a variety of toxins including phorbol esters (tumor promoters), is projected to be generated yearly in the near future by the Jatropha biodiesel industry. We showed that the kernel cake strongly inhibited plant seed germination and root growth and was highly toxic to carp fingerlings, even though phorbol esters were undetectable by HPLC. Therefore it must be detoxified before disposal to the environment. A mathematic model was established to estimate the general toxicity of the kernel cake by determining the survival time of carp fingerling. A new strain (Streptomyces fimicarius YUCM 310038) capable of degrading the total toxicity by more than 97% in a 9-day solid state fermentation was screened out from 578 strains including 198 known strains and 380 strains isolated from air and soil. The kernel cake fermented by YUCM 310038 was nontoxic to plants and carp fingerlings and significantly promoted tobacco plant growth, indicating its potential to transform the toxic kernel cake to bio-safe animal feed or organic fertilizer to remove the environmental concern and to reduce the cost of the Jatropha biodiesel industry. Microbial strain profile essential for the kernel cake detoxification was discussed. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Jatropha curcas L., a tropical and sub-tropical shrub/tree, has been emerging as the most promising biodiesel crop of the second generation, because of its high oil content (43–61% in the seed kernel) and endurance to grow in wasteland and marginal land even polluted soils without using arable land [1–8]. Recently, the Jatropha based biodiesel industry has been developing very quickly.

∗ Corresponding author. Tel.: +351 259350222; fax: +351 259350480. E-mail addresses: [email protected], changhe [email protected] (C.H. Zhang). 1 These authors contributed equally to this work. 0304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jhazmat.2013.05.012

For instance, by 2007, China has built up more than 2000 Jatropha biodiesel production plants, with a present J. curcas planting area about 200,000 ha [4]. Currently J. curcas oil is mainly used for the production of aviation fuel aiming to reduce the emission of CO2 in China. A production base with a capacity of 60,000 tons of Jatropha aviation biofuel has been established in Nanchong, Sichuan, by PetroChina. Nevertheless, Jatropha biodiesel industry is still at its infancy stage due to high cost, the lack of full use of the toxic by-products [9–12], and lack of policy support in some countries, such as India [13]. The kernel cake is the major by-product of the Jatropha biodiesel industry. The seeds and the kernel cake of J. curcas have been proven to be toxic to mammals and human being, and its use

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as animal feed has been restricted [14]. The seed cake contains various toxins and anti-nutritional factors such as trypsin inhibitors, saponins, phytate, curcin and lectin [15]. Phorbol esters, a group of tetracyclic tigliane diterpenes, are recognized as the main toxins of J. curcas because of their tumor promotion effect [16,17]. The most common and only commercially available phorbol ester is phorbol 12-myristate-13-acetate (PMA). In addition to PMA, at least 6 intramolecular diesters derived from 12-deoxy-16hydroxyphorbol have been identified from J. curcas [17]. Recently, a dozen of other kinds of toxins have been purified and identified from J. curcas seeds [14,18]. These toxins are detrimental to bacteria, fungi, invertebrates, vertebrates as well as humans. Therefore, rational use and save disposal of the seed cake are a main challenge to reduce the cost of Jatropha biodiesel industry and to avoid environmental concern it may cause. To detoxify the kernel cake with complex and unknown toxic constituents is still a great challenge to the Jatropha biodiesel industry. Previous efforts mainly focused on the degradation of phorbol esters [12,19,20]. No encouraging progress has been made from various chemical and physical methods to fully degrade/inactivate phorbol esters from J. curcas seed cake to convert it to animal feed [21,22]. It is possibly to completely remove (not degrade) the phorbol esters from the kernel cake by methanol or ethanol extraction [23,24]. However, no reports on how to handle the toxins extracted; the disposal of the toxins also raises environmental and health concern. In addition, the use of organic solvent is expensive [25] and could have a residual effect on the animals and human beings consuming the feed. The current investigation showed that even though phorbol esters were undetectable, the kernel cake was highly toxic to plant and animal. Therefore, a mathematic model was established to estimate the general toxicity of the kernel cake by the survival time of carp fingerlings. Microorganisms capable of simultaneously degrading all kinds of the toxins of the kernel cake would be useful to solve the above-mentioned problems. We hypothesize there are such microorganisms in nature. We aimed to obtain such a strain by a high throughput strain isolation and screening strategy from soil and air as well as from known strains, and to detoxify the kernel cake by an environmentally friendly solid state fermentation (SSF) by the newly isolated strain. 2. Experimental 2.1. Kernel cake The seeds of J. curcas were produced in Chuxiong (101◦ 63 E, 24◦ 70 N), Yunnan, China. Clean mature seeds were de-hulled to isolate the kernels. The oil of the kernels was extracted by mechanical press to obtain the kernel cake. 2.2. Extraction of the toxins and analysis of the phorbol esters The toxins in the kernel cake were extracted with absolute methanol. In brief, 36 ml methanol was added to a flask containing 5 g kernel cake and the mixture was incubated in an ultrasound bath for 3 × 15 min. The extraction process was repeated for another two times and the extract fractions were pooled together. The methanol was removed at a rotary evaporator under vacuum at 60 ◦ C; the extract was then fully dried in an air flow oven at 40 ◦ C. For HPLC analysis, the extract powder was dissolved in methanol at 2 mg/ml and then filtered using Whatman filter paper no. 2. A Waters 515 HPLC system equipped with a 2996 Photodiode Array Detector (Waters, USA) and a Zorbax SB-C18 reverse phase C18 column (5 ␮m, 4.6 × 250 mm i.d., Agilent, USA) was used. The separation was carried out at 30 ◦ C with a flow rate at 1.0 ml/min starting with

239

60% water (A) and 40% acetonitrile (B) for 30 min, and decreasing A to 25%, increasing B to 75% for 20 min, then B at 100% for the last 15 min. The detector wavelength was set at 254 nm. Standard PMA and other chemical reagents were obtained from Sigma (U.K.), except for specified elsewhere. 2.3. Establishment of a toxicity model The methanol extract of the unfermented kernel cake was dissolved in DMSO (1:3, W/V) and added to 200 ml of edible tap water in a 500-ml beaker drop by drop with agitation, respectively, forming a serial concentration gradient. The beakers were sonicated in an ultrasound bath for 3 × 10 min to totally dissolve the toxins, or, to make the toxins reach saturation in the water when added at higher concentrations. Three 2-cm-long carp fingerlings (Cirrhinus chinensis) purchased from the local market were fostered in an individual beaker without feeding. The survival time of the carp fingerlings was recorded. The toxin concentration gradient test was repeated two times. The mean value was used to establish the model on the relationship between the concentration of the total toxins in the methanol extract and the survival time. Two sets of controls using tap water alone and tap water with 3 ml DMSO (the maximal volume added to the water with the methanol extracts) were included, respectively, in all the tests. 2.4. Strain isolation In principle, J. curcas kernel cake was used in the medium as carbon and energy source for strain isolation and screening. Strain isolation was performed on autoclaved (121 ◦ C, 30 min) kernel cake–agar plate containing 100 g/l kernel cake powder and 20 g/l agar. Sixty different locations were chosen in Kunming (102◦ 72 E, 25◦ 05 N), Dali (99◦ 53 E, 25◦ 47 N) and Weixi (99◦ 28 E, 27◦ 18 N), Yunnan Province, for sampling from air. Two plates were exposed in the air at each location for 5 min and sealed. The plates were then incubated at 28 ◦ C for 1–5 d. All the colonies with different characters from one location were picked out and purified by single-cell or single-spore colony culture on the kernel cake–agar plates. In addition, 40 soil samples in the root zone of J. curcas trees was collected from Shuangbai (101◦ 63 E, 24◦ 70 N) and Yongren (101◦ 67 E, 26◦ 07 N), Yunnan, as well. Soil samples were collected 5 cm below the land surface in sterile containers. One gram of each soil sample was dispersed in 9 ml of 0.85% NaCl in sterile test tubes. Thereafter, a series of dilution from 10−2 to 10−8 were prepared in 0.85% NaCl. A 0.2 ml aliquot of the appropriate dilution was spread aseptically onto the plates. 2.5. Toxicity evaluation and strain screening The evaluation of the kernel cake toxicity to microbe, plant and animal was integrated in the strain screening process. The strains isolated from the air and soils as well as the 198 known strains from SCMGB (Southwest China Microbial Germplasm Bank)–YUMRC (Yunnan University Microbial Resource Center) were used for the screening. The scheme of the strain isolation and screening was summarized in Fig. 1 and detailed as follows. 2.5.1. Microbial toxicity – kernel cake fermentation and the primary screening The isolated strains as well as the known strains were inoculated to the autoclaved moistened kernel cake (10 g + 20 ml water, 121 ◦ C for 60 min) and incubated at 28 ◦ C to perform the fermentation. A high growth rate in the kernel cake is a prerequisite for a microbial strain to detoxify the cake. Therefore, at the first step of the strain

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filter papers moistened with 10 ml of tap water. The seeds were then incubated at 28 ◦ C in the dark for germination. Three days later the germination percentage was scored. The strains that conferred the fermented seed cake did not significantly inhibit the seed germination, as compared with the control, were screen out. 2.5.3. Animal toxicity – carp fingerling lethality test and the 3rd step strain screening Two hundred milligram methanol extract of the kernel cake was dissolved in 0.6 ml DMSO and added to a 500-ml beaker containing 200 ml edible tap water, drop by drop. Three 2-cm-long carp fingerlings were incubated in each flask. The average survival time of the carp fingerlings in the tap water that contained the methanol extract of the kernel cake fermented by a strain, which passed the 2nd step screening, being close to that in the tap water alone was marked; the corresponded strain was screened out. 2.6. Time course of SSF and detoxification Ten grams of kernel cake and 20 ml of tap water were added to a flask and autoclaved at 121 ◦ C for 1 h. One ml of S. fimicarius YUCM 310038 liquid culture at the late exponential phase (OD405 = 1.5) was inoculated to one flask and incubated at 28 ◦ C in the dark. During the SSF process, samples were taken at 24 h intervals. Three flasks were sampled at each time point. One gram of the kernel cake was dispersed in 9 ml of 0.85% NaCl and serially diluted from 10−2 to 10−9 in 0.85% NaCl solution in sterile test tubes for determining the microbial concentration by colony formation on potato dextrose agar. The toxins of the sampled kernel cake were extracted by methanol and the survival time of the carp fingerlings in the water that contained 1000 mg of the methanol extract at different time points was determined as described previously. 2.7. Analysis of the main components of the kernel cake

Fig. 1. The scheme of the strain isolation and screening.

screening we selected those, whose mycelia/colonies were able to occupy the whole surface of the kernel cake within one week.

Organic matter, protein, lipid and ash were analyzed using the AOAC (1980) procedure. The organic carbon and crude fiber were analyzed by potassium dichromate method (Agriculture Industry Standard of PR China, NY 481-2002). The content of N, P, K, Ca, Mg, Fe, Zn and Na was analyzed by inductively coupled plasma atomic emission spectrometry (ICPS-1000II, Shimadzu, Japan). Sample preparation: 5.0 mg kernel cake was put in a 200 ml beaker, 20 ml GR HNO3 was added to the beaker and stayed overnight. The sample was heated until completely digested and the volume was adjusted to 25 ml by carefully dripping Milli-Q water. Analysis condition: high-frequency power, 1.2 kW; observation height, 15 mm; cooling gas flow rate: 15 L/min; auxiliary gas flow rate: 1.0 L/min; nebulizer gas flow rate: 3.5 L/min; plasma gas flow rate: 1.2 L/min; sample flow rate: 0.8 ml/min. 2.8. Effect of the kernel cake on plant growth

2.5.2. Phytotoxicity – seed germination test and the 2nd step screening The aim of the germination tests was to screen out strains conferring the fermented seed cake to be nontoxic to plants. Seeds of three plant species, pumpkin [Cucurbita moschata (Duch.) Poir], Chinese cabbage (Brassica chinensis L. Gent.), and garden radish (Raphanus sativus L.) were used for the germination test. The raw unfermented kernel cake and those fermented by the microbial strains, which passed the primary screening, were extracted with 10 times of water (w/v) for 6 h with agitation. Eight milliliters of the extract was transferred to each Petri dish ( = 10.5 cm). Two pieces of filter paper were placed in a Petri dish. Four replicates of 10 seeds of C. moschata, R. sativus or B. chinensis were arranged on the filter paper in each Petri dish. Controls were maintained on

One kilogram moist forest surface soil was filled to a pot ( = 15 cm). Three tobacco (Nicotiana tabacum) seedlings (ca. 7 cm high) were randomly planted in each pot. The fermented or unfermented J. curcas kernel cake was applied to the soil at 10 g per pot. The control pots contained the soil alone. The plants were incubated at 24 ◦ C with a 16 h/8 h irradiation/dark cycle at 1000 ␮mol/mm2 /s. They were watered regularly and the growth was measured 14 d later. 2.9. Statistical analyses Statistical analyses were carried out by Student’s t-test. Results were expressed as mean ± standard deviation (n ≥ 3), and

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Table 1 Components of the Jatropha curcas kernel cake (g/kg, for the organic matters; mg/kg for the elements). ND: not detected. Protein

Crude fiber

Oil

Ash

Organic carbon

C/N

P

K

Ca

Mg

Fe

Zn

Na

423

245

20

88

411.2

12.8:1

12,900

8520

3600

5840

167

62.57

ND

differences were considered significant at P < 0.05, except for specified elsewhere. 3. Results and discussion 3.1. Phorbol ester and nutrient contents in the kernel cake Due to the existence of a variety of toxins and the unknown toxins, it is impossible to evaluate the toxicity of the kernel cake by the determination of all the individual toxins. Phorbol ester content is usually used as an indicator of the toxicity of the kernel cake [20,26]. Owing to that PMA is the only commercially available compound as a standard for the analysis of phorbol esters using HPLC, the compounds with retention time “close to”, e.g., from 1–5 min to 5–13 min less than, that of PMA are arbitrarily considered as phorbol esters [20,27–30]. Obviously, the determination of phorbol esters in this way is not accurate and the contents from different authors are not comparable. Purification and identification of the individual toxic compounds are essential to solve the problem. It is worthy to be mentioned that all of the J. curcas samples did not contain detectable PMA in the reports mentioned above. As shown in Fig. 2 the peak of pure PMA appeared at 49.18 min in the HPLC chromatogram; there were a few small peaks with retention times much less than 49.18 min in the extract of J. curcas kernel cake; there was no detectable PMA in the kernel cake. With the facility to detect the toxicity by the carp fingerling lethality test, as described in Sections 2.5.3 and 3.3, we have separated and purified a few toxic compounds from the liquid chromatographic effluents of the kernel cake extract and obtained their 1 H NMR spectra. The purified compounds were highly unstable. When performed the 13 C NMR to elucidate their molecular structures after the 1 H NMR analyses, we found that they had all decayed. Anyway, the 1 H NMR spectra showed that they were none of the known toxic compounds presently identified from J. curcas. In brief, there were no detectable phorbol esters in the kernel cake. However, as shown in the following sections the kernel cake was highly toxic. Therefore, the content of phorbol esters could not indicate the toxicity of the kernel cake. Heating and the fermentation by some microbial strains greatly increased the toxicity of the kernel cake (data not shown). Based on these observations combined with that the toxic compounds were highly degradable, we infer that some intermediates degraded were more toxic than the toxic ingredients themselves and that partial of the toxicity of the kernel cake came from the degradation of the unknown toxic compounds. Work to elucidate the chemical identities of the toxic compounds and their major intermediates degraded, as well as, their toxicity is under way in our lab. From animal feed point of view, the main components of the kernel cake were protein (42.3%), crude fiber (24.5%), ash (8.8%), oil (2%) and water (3.8%) (Table 1). From fertilizer point of view, it contained 41.1% organic matter and rich N, P, K and other plant essential elements (Table 1). 3.2. Toxicity of the kernel cake, microbial strain isolation and screening Three hundred and eighty microbial strains including bacteria, actinobacteria, fungi and yeasts were isolated in total (120 strains from air, 260 strains from the soils). Upon inoculation of the 380

strains and the 198 known strains to the kernel cake, 10% of them could not survive at all; 59% of them grew very slow; 31% of them grew well (the mycelia/colonies covered the total surface of the medium within one week). The growth inhibition by the kernel cake might be due to its antimicrobial components [31,32] and its high protein content, especially the toxic proteins [33]. Based on the growth rate on the kernel cake, 181 strains were screened out. The water extract of the unfermented kernel cake significantly inhibited the seed germination percentage of C. moschata, R. sativus and B. chinensis by 20%, 45% and 30%, respectively. The extract of the kernel cake fermented by most of the strains did not improve the seed germination percentage as compared with that of the unfermented kernel cake. By the plant seed germination test, 69 strains that conferred the fermented kernel cake did not significantly inhibited the seed germination of all the three plant species were screened out from the 181 strains obtained previously. The unfermented kernel cake was highly toxic to the carp fingerlings. The addition of the extract of the unfermented kernel cake to the water significantly reduced the survival time from 60–80 h to 12–18 h. The survival time in the water with the addition of DMSO at 1.5% (v/v), the maximal dosage used in this work, was also 12–18 h, indicating that DMSO at the used dosage did not affect the survival time of the carp fingerlings. After the carp fingerling lethality test strains YUCM 310038, A003 and E290 were screened out from the 69 strains. Strains YUCM 310038, A003 and E290 were identified as Streptomyces fimicarius, Aspergillus versicolor and Scopulariopsis brevicaulis, respectively. Our research indicated that A. versicolor A003 produced sterigmatocystin (data not shown), a very toxic and highly carcinogenic mycotoxin [34], by itself. S. brevicaulis is a human pathogen, causing onychomycosis [35] and invasive disease as well [36,37]. We confirmed that S. brevicaulis E290 grew on human detached nails (data not shown). To date, no report shows that S. fimicarius infects or does harm to human, animal or plant. Therefore, we had the most interest in S. fimicarius YUCM 310038. Strain S. fimicarius YUCM 310038 has been deposited in SCMGB–YUMRC, with an accession no. YUCM 310038, and in China Center for Type Culture Collection (CCTCC) as well (accession no. M2011248). Its mycelium and spore morphology and 16S rDNA (GenBank accession number JQ696990) phylogenesis were shown in Figs. 3 and 4, respectively. Similar to our results, the phorbol esters in some J. curcas genotypes in Mexico [21] and Thailand [38] were also undetectable. These varieties were classified as “nontoxic”. However, as demonstrated in this work, even though phorbol esters were undetectable, the kernel cake was severely toxic to carp fingerlings and strongly inhibited plant seed germination and the growth of some microorganisms. Seed germination and seedling establishment are critical stages in plant life cycle. Germination test is commonly used for the assessment of phytotoxicity [39,40]. The kernel cake severely inhibited the seed germination of pumpkin, Chinese cabbage and garden radish, indicating its phytotoxicity. The toxic agents might be the derivatives of phorbol esters and/or other toxins, in addition to the undetectable phorbol esters. For instance, phorbol compounds without any side chain also have tumor promoting activity [41]; Jatropherol-I, a phorbol-type compound, is the main toxin of J. curcas to silkworm [42]. Consequently, using the content of

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A 0.40

AU

0.30

0.20

0.10

0.00 5.00

10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

B 3.00

2.50

AU

2.00

1.50

1.00

0.50

0.00 10.00

20.00

30.00

40.00

50.00

60.00

Retention time (min) Fig. 2. HPLC chromatograms of the methanol extract of the fresh Jatropha curcas kernel cake (A) and standard phorbol 12-myristate-13-acetate (PMA) (B).

phorbol esters as the sole criterion for evaluating the toxicity of J. curcas kernel cake is not correct. Toxicity evaluation by animal and plant tests is essential. The toxicity evaluation on animal and plant has the advantage to reveal the general toxicity and does not need to quantify the individual components of the toxins. This kind of evaluation is particularly essential for future development of the fermented kernel cake as animal feed or bio-safe fertilizer. In fact, as mentioned previously, due to the large variety of the toxins and the existence of unknown toxins it is impossible to evaluate the detoxification effect by determining all the individual toxins of the fermented kernel cake in the strain screening program. In previous reports, by the determination of phorbol esters the authors only evaluated the degradation effects of one [20] or a few microbial strains [26,43]. We only obtained three strains capable of effectively detoxifying the kernel cake from the 578 strains. This indicates that a large strain resource is the prerequisite for the strain screening, which makes a strain screening process a huge project. The efficient step by step target oriented strain screening strategy especially the novel detoxification evaluation methods by plant seed germination and carp fingerling lethality tests facilitated the large-scale screening process.

3.3. A mathematic model to estimate the general toxicity of the kernel cake The relationship between the concentration of the methanol extract of the unfermented kernel cake in the water (c) and the corresponded survival time of the carp fingerlings (t) is shown in Fig. 5. When the concentration of the methanol extract below a threshold value (cTmin ) in the water, the toxins did not affect the viability of the carp fingerlings, so the survival time reaches the maximum (Tmax ), i.e., the same as in tap water. That is If

t(c) = Tmax

c ≤ cTmin

(1)

(2)

When the concentration of the methanol extract above a threshold value (cTmax ) in the water, the toxins reached saturation in the water or reached the concentration to the maximal toxicity, manifesting the maximal lethality and resulting in the minimal survival

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243

Fig. 3. Morphological characteristics of strain Streptomyces fimicarius YUCM 310038. Colonial morphology on potato dextrose agar (A, surface; B, reverse), mycelium and spore under scanning electronic microscopy (C, magnification 12,000×; D, magnification 60,000×).

time (Tmin ); a higher concentration of the methanol extract did not reduce the survival time anymore. That is If

t(c) = Tmin

c ≥ cTmax If

Tmin < t(c) < Tmax

t reduced exponentially against c t(c) = aekc

(6)

(4)

t = ekc a

(7)

(5)

kc = ln

(3)

t a

= ln t − ln a

(8)

Fig. 4. Neighbor-joining phylogenetic tree constructed with the 16S rRNA gene sequence of strain YUCM 310038 and those of other related Streptomyces strains retrieved from GenBank database. GenBank accession numbers are given in the parentheses for reference sequences. Numbers at nodes were bootstrap values based on 1000 resamplings. Bar, substitutions per nucleotide position.

X.-H. Wang et al. / Journal of Hazardous Materials 260 (2013) 238–246 1.E+10

90

Num ber of m icrobial cells/g kernel cake

Numbe r of mic rob ia l c e lls

80

S urviva l time (h)

1.E+09

70 60

1.E+08 50 40 1.E+07 30 20

1.E+06

Carp fingerling survival time (h)

244

10 1.E+05 0

24

48

72

96

120

144

168

192

216

240

0 264

Fermentatioin time (h)

Fig. 5. The relationship between the carp fingerling survival time and the concentration of the methanol extract of the Jatropha curcas kernel cake in the water. The survival time was the average of the fish (3 × 3, triplicates). Within the tested concentrations, at each concentration point the differences of the survival time of three independent tests were not significant at P < 0.01 level.

c(t) =

ln t − ln a k

that the kernel cake fermented for 9 d was not detrimental to the carp fingerlings any more. After 9 d fermentation (9)

where a = 71.863, k = −0.0085 (R2 = 0.991), calculated by an exponential regression. For fish from the same batch or with the same viability, Tmax , Tmin , cTmin and cTmax were constants. In the case shown in Fig. 5, Tmax = 60 h, Tmin = 8 h; cTmin = 25 mg/l, cTmax = 350 mg/l. In brief, in this model, if t was defined, the c could be estimated or calculated by formula (2), (4) or (9). In other words, the general toxin concentration in the kernel cake was determined by the survival time of the carp fingerlings. The determination of the t was very simple, quick and repeatable. The percentage (p) of the toxins degraded by an SSF process can be calculated by p=

cf − cunf cf

× 100%

Fig. 6. The relationship between the fermentation time of Jatropha curcas kernel cake by strain Streptomyces fimicarius YUCM 310038 and the average survival time of carp fingerlings living in the 200 ml water that contained 1000 mg of the methanol extract of the fermented kernel cake. Values were mean of three replicates.

(10)

where cf is the concentration of the methanol extract of the kernel cake fermented by a strain used in the toxicity test; cunf is that of the unfermented kernel cake from the same batch, which results in the same survival time as that of cf .

3.4. Time course of the SSF and detoxification As shown in Fig. 6, during the SSF process, there was no obvious lag phase; the maximal microbial cell density appeared at 168 h; the cell density declined sharply afterwards. The survival time of the carp fingerlings incubated in the water that contained the methanol extract of the unfermented kernel cake (Tmin ) was 12 h, while the survival time of those in the water that contained the methanol extract of the kernel cake fermented for 24 h extended to 24 h. This indicated that the microbial detoxification started as soon as the initiation of the fermentation. The survival time in the water that contained the extract of the kernel cake fermented for 192 h, i.e., one day after the peak density of the bacteria in the kernel cake, was the same as that in the tap water (Tmax = 80 h). This indicated

∵ t(c) = Tmax , ∴ cunf ≤ cTmin = 30 mg/l of the methanol extract of the unfermented kernel cake. In other words, the toxin concentration (cf ) in the water containing 1000 mg/l of the methanol extract of the kernel cake fermented by S. fimicarius YUCM 310038 for 9 d was equivalent to that in the water containing ≤30 mg/l of the methanol extract of the unfermented kernel cake. Therefore the total toxins were degraded by ≥97% by the fermentation as calculated by formula (10): p ≥ 100% (1000 − 30)/1000. If we want to have a more accurate calculation, we can increase the cf . For instance, in this case, if we increase cf to 2000 mg/l and still get t(c) = Tmax , then cunf ≤ cTmin = 30 mg/l, p ≥ 100% (2000 − 30)/2000 = 98.5%. The present acute toxicity test showed that the fermented kernel was not toxic to the carp fingerlings. Whether the fermented kernel cake is safe as animal feed may need further chronic toxicity test on domestic animals and birds, such as pigs and chickens. 3.5. Improved tobacco plant growth by the application of the fermented kernel cake The kernel cake contained rich N, P, K and other essential elements for plant growth (Table 1). However, as shown in Table 2, the application of the unfermented kernel cake to the soil did not significantly improve tobacco seedling growth but inhibited the root growth by 48% as compared with the control. This indicates that the phytotoxicity of the unfermented kernel cake also included root growth inhibition, in addition to germination inhibition mentioned previously. Interestingly, the application of the kernel cake fermented by S. fimicarius YUCM 310038 significantly improved tobacco seedling growth by 80% based on biomass in 14 days (Table 2), indicating the potential of the fermented kernel cake as safe organic fertilizer. Collectively, the kernel cake was highly toxic to both animal and plant even though the phorbol esters were undetectable. Consequently, any uncontrolled disposal or spread of J. curcas seed cake either in aquatic or terrestrial environment may cause

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Table 2 Comparison of tobacco seedling growth in pots applied with unfermented Jatropha curcas kernel cake (unfermented) and with that after 9 day fermentation by Streptomyces fimicarius YUCM 310038 (fermented) at 10 g/kg soil. Control, soil alone without kernel cake. FW: fresh weight, DW: dry weight. Different letters (a, b and c) in the same column mean significantly different at P < 0.05 level. Treatments

FW (g/plant)

DW (g/plant)

Leaf FW (g/plant)

Leaf length (cm)

Root FW (g/plant)

Control Unfermented Fermented

14.4 ± 2.8b 15.2 ± 2.6b 25.8 ± 2.9a

2.4 ± 0.3b 2.5 ± 0.4b 4.3 ± 0.5a

8.6 ± 1.1c 11.3 ± 1.6b 15.6 ± 1.9a

5.8 ± 0.7b 6.8 ± 0.7b 8.2 ± 0.9a

5.8 ± 0.8b 3.9 ± 0.6c 10.2 ± 1.2a

ecological concern. Presently, most Jatropha industries directly dispose the kernel cake to the soils as fertilizer without any detoxification treatment because of not knowing that doing so will pose a great risk to the environment and human health. The present work not only greatly improved our understanding of the toxicity of the kernel cake but also provided strategies to detoxify it. The toxicity model and the strain screening strategy may be significant for other microbial detoxification/decomposition systems for substrate with complex and/or unknown toxic ingredients. The ultimate goal of the detoxification of J. curcas kernel cake is effectively converting it from toxic waste to bio-safe animal feed or organic fertilizer to reduce the cost of Jatropha industry. To fulfill this goal via an efficient and environmentally friendly microbial fermentation, the microbial strain should have the following characters: (1) the effective detoxification capacity, (2) a rapid growth rate in the kernel cake, (3) not producing any toxins by itself, in particular, (4) not being an animal (especially human) or plant pathogen. Phorbol esters could be degraded by SSF through Pseudomonas aeruginosa [20]. Nevertheless, no animal or plant test shows that the fermented kernel cake was nontoxic. In addition, P. aeruginosa is a notorious human and animal (both vertebrate and invertebrate) opportunistic pathogen [44], as well as plant pathogen [45], causing diseases fatal to the hosts. S. fimicarius YUCM 310038 is the only known and available strain that has all these 4 characters. It is able to directly and efficiently detoxify the moistened kernel cake under benign condition without any additional reagent. The application of the kernel cake fermentation by YUCM 310038 has the potential to transform the kernel cake with a variety of toxins to bio-safe animal feed or organic fertilizer, not only to remove the environmental concern but to reduce the cost of the Jatropha industry as well. 4. Conclusions The J. curcas kernel cake strongly inhibited plant seed germination and root growth and was highly toxic to carp fingerlings even though the phorbol esters were undetectable. Therefore, it should be detoxified before release to the environment. The mathematic model enabled us to estimate the concentration of the total toxins in the kernel cake by determining the survival time of the carp fingerlings without the determination of the (unknown) individual toxins. The effective strain screening strategy facilitated the screening process. Strain S. fimicarius YUCM 310038 was able to degrade the total toxicity by more than 97% in a single fermentation process, conferring the fermented kernel cake nontoxic to carp fingerlings and plants, and promoting tobacco plant growth. Therefore, the present work represents a novel method for effectively simultaneous detoxification of multiple toxins in plant biomass. Acknowledgements This work was financed by the Ministry of Science and Technology for the China National Science and Technology Pillar Program under the project 2011BAD30B00, and by European Union Funds (FEDER/COMPETE–Operational Competitiveness Programme) and Portugal national funds (FCT – Portuguese Foundation for Science

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