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Journal of Hazardous Materials 260 (2013) 238– 246

Contents lists available at SciVerse ScienceDirect

Journal of Hazardous Materials

jou rn al hom epage: www.elsev ier .com/ locate / jhazmat

etoxification of Jatropha curcas kernel cake by a novel Streptomycesmicarius strain

ing-Hong Wanga,1, Lingcheng Oub,1, Liang-Liang Fua, Shui Zhenga, Ji-Dong Louc,osé Gomes-Laranjod, Jiao Lia, Changhe Zhangd,∗

Yunnan Institute of Microbiology, Yunnan University, Kunming 650091, ChinaSchool of Development Studies, Yunnan University, Kunming 650091, ChinaCollege of Life Science, China Jiliang University, Hangzhou 310018, ChinaCenter for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Department of Biology and Environment, Universidade derás-os-Montes e Alto Douro (UTAD), Apartado 1013, Vila Real 5001-801, Portugal

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

rticle history:eceived 1 March 2013eceived in revised form 6 May 2013ccepted 8 May 2013vailable online 16 May 2013

eywords:atropha curcas

a b s t r a c t

A huge amount of kernel cake, which contains a variety of toxins including phorbol esters (tumor promo-ters), is projected to be generated yearly in the near future by the Jatropha biodiesel industry. We showedthat the kernel cake strongly inhibited plant seed germination and root growth and was highly toxic tocarp fingerlings, even though phorbol esters were undetectable by HPLC. Therefore it must be detoxifiedbefore disposal to the environment. A mathematic model was established to estimate the general tox-icity of the kernel cake by determining the survival time of carp fingerling. A new strain (Streptomycesfimicarius YUCM 310038) capable of degrading the total toxicity by more than 97% in a 9-day solid state

iodieselernel cake detoxificationolid state fermentationtreptomyces fimicarius YUCM 310038

fermentation was screened out from 578 strains including 198 known strains and 380 strains isolatedfrom air and soil. The kernel cake fermented by YUCM 310038 was nontoxic to plants and carp fingerlingsand significantly promoted tobacco plant growth, indicating its potential to transform the toxic kernelcake to bio-safe animal feed or organic fertilizer to remove the environmental concern and to reduce thecost of the Jatropha biodiesel industry. Microbial strain profile essential for the kernel cake detoxificationwas discussed.

. Introduction

Jatropha curcas L., a tropical and sub-tropical shrub/tree, haseen emerging as the most promising biodiesel crop of the secondeneration, because of its high oil content (43–61% in the seed ker-

el) and endurance to grow in wasteland and marginal land evenolluted soils without using arable land [1–8]. Recently, the Jat-opha based biodiesel industry has been developing very quickly.

∗ Corresponding author. Tel.: +351 259350222; fax: +351 259350480.E-mail addresses: czhang@utad.pt, changhe zhang9@hotmail.com (C.H. Zhang).

1 These authors contributed equally to this work.

304-3894/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jhazmat.2013.05.012

© 2013 Elsevier B.V. All rights reserved.

For instance, by 2007, China has built up more than 2000 Jatrophabiodiesel production plants, with a present J. curcas planting areaabout 200,000 ha [4]. Currently J. curcas oil is mainly used for theproduction of aviation fuel aiming to reduce the emission of CO2in China. A production base with a capacity of 60,000 tons of Jat-ropha aviation biofuel has been established in Nanchong, Sichuan,by PetroChina. Nevertheless, Jatropha biodiesel industry is still atits infancy stage due to high cost, the lack of full use of the toxicby-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 biodieselindustry. The seeds and the kernel cake of J. curcas have beenproven to be toxic to mammals and human being, and its use

ardous

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X.-H. Wang et al. / Journal of Haz

s animal feed has been restricted [14]. The seed cake con-ains various toxins and anti-nutritional factors such as trypsinnhibitors, saponins, phytate, curcin and lectin [15]. Phorbol esters,

group of tetracyclic tigliane diterpenes, are recognized as theain toxins of J. curcas because of their tumor promotion effect

16,17]. The most common and only commercially available phor-ol ester is phorbol 12-myristate-13-acetate (PMA). In addition toMA, at least 6 intramolecular diesters derived from 12-deoxy-16-ydroxyphorbol have been identified from J. curcas [17]. Recently,

dozen of other kinds of toxins have been purified and identifiedrom J. curcas seeds [14,18]. These toxins are detrimental to bacte-ia, fungi, invertebrates, vertebrates as well as humans. Therefore,ational use and save disposal of the seed cake are a main chal-enge to reduce the cost of Jatropha biodiesel industry and to avoidnvironmental concern it may cause.

To detoxify the kernel cake with complex and unknown toxiconstituents is still a great challenge to the Jatropha biodiesel indus-ry. Previous efforts mainly focused on the degradation of phorbolsters [12,19,20]. No encouraging progress has been made fromarious chemical and physical methods to fully degrade/inactivatehorbol esters from J. curcas seed cake to convert it to animal feed21,22]. It is possibly to completely remove (not degrade) the phor-ol esters from the kernel cake by methanol or ethanol extraction23,24]. However, no reports on how to handle the toxins extracted;he disposal of the toxins also raises environmental and healthoncern. In addition, the use of organic solvent is expensive [25]nd could have a residual effect on the animals and human beingsonsuming the feed.

The current investigation showed that even though phorbolsters were undetectable, the kernel cake was highly toxic to plantnd animal. Therefore, a mathematic model was established tostimate the general toxicity of the kernel cake by the survivalime of carp fingerlings. Microorganisms capable of simultaneouslyegrading all kinds of the toxins of the kernel cake would be usefulo solve the above-mentioned problems. We hypothesize there areuch microorganisms in nature. We aimed to obtain such a strainy a high throughput strain isolation and screening strategy fromoil and air as well as from known strains, and to detoxify the kernelake by an environmentally friendly solid state fermentation (SSF)y the newly isolated strain.

. Experimental

.1. Kernel cake

The seeds of J. curcas were produced in Chuxiong (101◦63′ E,4◦70′ N), Yunnan, China. Clean mature seeds were de-hulled to iso-

ate the kernels. The oil of the kernels was extracted by mechanicalress to obtain the kernel cake.

.2. Extraction of the toxins and analysis of the phorbol esters

The toxins in the kernel cake were extracted with absoluteethanol. In brief, 36 ml methanol was added to a flask containing

g kernel cake and the mixture was incubated in an ultrasound bathor 3 × 15 min. The extraction process was repeated for another twoimes and the extract fractions were pooled together. The methanolas removed at a rotary evaporator under vacuum at 60 ◦C; the

xtract was then fully dried in an air flow oven at 40 ◦C. For HPLCnalysis, the extract powder was dissolved in methanol at 2 mg/mlnd then filtered using Whatman filter paper no. 2. A Waters 515

PLC system equipped with a 2996 Photodiode Array Detector

Waters, USA) and a Zorbax SB-C18 reverse phase C18 column5 �m, 4.6 × 250 mm i.d., Agilent, USA) was used. The separationas carried out at 30 ◦C with a flow rate at 1.0 ml/min starting with

Materials 260 (2013) 238– 246 239

60% water (A) and 40% acetonitrile (B) for 30 min, and decreasingA to 25%, increasing B to 75% for 20 min, then B at 100% for thelast 15 min. The detector wavelength was set at 254 nm. StandardPMA 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 dis-solved in DMSO (1:3, W/V) and added to 200 ml of edible tap waterin a 500-ml beaker drop by drop with agitation, respectively, form-ing a serial concentration gradient. The beakers were sonicated inan ultrasound bath for 3 × 10 min to totally dissolve the toxins, or, tomake the toxins reach saturation in the water when added at higherconcentrations. Three 2-cm-long carp fingerlings (Cirrhinus chinen-sis) purchased from the local market were fostered in an individualbeaker without feeding. The survival time of the carp fingerlingswas recorded. The toxin concentration gradient test was repeatedtwo times. The mean value was used to establish the model on therelationship between the concentration of the total toxins in themethanol extract and the survival time. Two sets of controls usingtap water alone and tap water with 3 ml DMSO (the maximal vol-ume 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 ascarbon and energy source for strain isolation and screening. Strainisolation was performed on autoclaved (121 ◦C, 30 min) kernelcake–agar plate containing 100 g/l kernel cake powder and 20 g/lagar. 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 exposedin the air at each location for 5 min and sealed. The plates were thenincubated at 28 ◦C for 1–5 d. All the colonies with different charac-ters from one location were picked out and purified by single-cellor single-spore colony culture on the kernel cake–agar plates. Inaddition, 40 soil samples in the root zone of J. curcas trees was col-lected from Shuangbai (101◦63′ E, 24◦70′ N) and Yongren (101◦67′

E, 26◦07′ N), Yunnan, as well. Soil samples were collected 5 cmbelow the land surface in sterile containers. One gram of each soilsample 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 in0.85% NaCl. A 0.2 ml aliquot of the appropriate dilution was spreadaseptically onto the plates.

2.5. Toxicity evaluation and strain screening

The evaluation of the kernel cake toxicity to microbe, plant andanimal was integrated in the strain screening process. The strainsisolated from the air and soils as well as the 198 known strains fromSCMGB (Southwest China Microbial Germplasm Bank)–YUMRC(Yunnan University Microbial Resource Center) were used for thescreening. The scheme of the strain isolation and screening wassummarized in Fig. 1 and detailed as follows.

2.5.1. Microbial toxicity – kernel cake fermentation and theprimary screening

The isolated strains as well as the known strains were inoculated

to the autoclaved moistened kernel cake (10 g + 20 ml water, 121 ◦Cfor 60 min) and incubated at 28 ◦C to perform the fermentation. Ahigh growth rate in the kernel cake is a prerequisite for a microbialstrain to detoxify the cake. Therefore, at the first step of the strain

240 X.-H. Wang et al. / Journal of Hazardous

so

2s

foC(us1tpoo

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

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

.5.2. Phytotoxicity – seed germination test and the 2nd stepcreening

The aim of the germination tests was to screen out strains con-erring the fermented seed cake to be nontoxic to plants. Seedsf three plant species, pumpkin [Cucurbita moschata (Duch.) Poir],hinese cabbage (Brassica chinensis L. Gent.), and garden radishRaphanus sativus L.) were used for the germination test. The rawnfermented kernel cake and those fermented by the microbialtrains, which passed the primary screening, were extracted with0 times of water (w/v) for 6 h with agitation. Eight milliliters of

he extract was transferred to each Petri dish (� = 10.5 cm). Twoieces of filter paper were placed in a Petri dish. Four replicatesf 10 seeds of C. moschata, R. sativus or B. chinensis were arrangedn the filter paper in each Petri dish. Controls were maintained on

Materials 260 (2013) 238– 246

filter papers moistened with 10 ml of tap water. The seeds werethen incubated at 28 ◦C in the dark for germination. Three dayslater the germination percentage was scored. The strains that con-ferred the fermented seed cake did not significantly inhibit the seedgermination, as compared with the control, were screen out.

2.5.3. Animal toxicity – carp fingerling lethality test and the 3rdstep strain screening

Two hundred milligram methanol extract of the kernel cake wasdissolved in 0.6 ml DMSO and added to a 500-ml beaker containing200 ml edible tap water, drop by drop. Three 2-cm-long carp fin-gerlings were incubated in each flask. The average survival time ofthe carp fingerlings in the tap water that contained the methanolextract of the kernel cake fermented by a strain, which passed the2nd step screening, being close to that in the tap water alone wasmarked; 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 aflask and autoclaved at 121 ◦C for 1 h. One ml of S. fimicarius YUCM310038 liquid culture at the late exponential phase (OD405 = 1.5)was inoculated to one flask and incubated at 28 ◦C in the dark. Dur-ing the SSF process, samples were taken at 24 h intervals. Threeflasks were sampled at each time point. One gram of the kernel cakewas 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 determiningthe microbial concentration by colony formation on potato dex-trose agar. The toxins of the sampled kernel cake were extractedby methanol and the survival time of the carp fingerlings in thewater that contained 1000 mg of the methanol extract at differenttime points was determined as described previously.

2.7. Analysis of the main components of the kernel cake

Organic matter, protein, lipid and ash were analyzed using theAOAC (1980) procedure. The organic carbon and crude fiber wereanalyzed by potassium dichromate method (Agriculture IndustryStandard of PR China, NY 481-2002). The content of N, P, K, Ca, Mg,Fe, Zn and Na was analyzed by inductively coupled plasma atomicemission spectrometry (ICPS-1000II, Shimadzu, Japan). Samplepreparation: 5.0 mg kernel cake was put in a 200 ml beaker, 20 mlGR HNO3 was added to the beaker and stayed overnight. The samplewas heated until completely digested and the volume was adjustedto 25 ml by carefully dripping Milli-Q water. Analysis condition:high-frequency power, 1.2 kW; observation height, 15 mm; coolinggas flow rate: 15 L/min; auxiliary gas flow rate: 1.0 L/min; nebulizergas flow rate: 3.5 L/min; plasma gas flow rate: 1.2 L/min; sampleflow rate: 0.8 ml/min.

2.8. Effect of the kernel cake on plant growth

One kilogram moist forest surface soil was filled to a pot(� = 15 cm). Three tobacco (Nicotiana tabacum) seedlings (ca. 7 cmhigh) were randomly planted in each pot. The fermented or unfer-mented J. curcas kernel cake was applied to the soil at 10 g per pot.The control pots contained the soil alone. The plants were incubatedat 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 dlater.

2.9. Statistical analyses

Statistical analyses were carried out by Student’s t-test.Results were expressed as mean ± standard deviation (n ≥ 3), and

X.-H. Wang et al. / Journal of Hazardous Materials 260 (2013) 238– 246 241

Table 1Components 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

1

di

3

3

itt[pttpeaiicicmtdicnctwtpplcngBpdadtio

koie

3s

af

423 245 20 88 411.2 12.8:

ifferences were considered significant at P < 0.05, except for spec-fied elsewhere.

. Results and discussion

.1. Phorbol ester and nutrient contents in the kernel cake

Due to the existence of a variety of toxins and the unknown tox-ns, it is impossible to evaluate the toxicity of the kernel cake byhe determination of all the individual toxins. Phorbol ester con-ent is usually used as an indicator of the toxicity of the kernel cake20,26]. Owing to that PMA is the only commercially available com-ound as a standard for the analysis of phorbol esters using HPLC,he compounds with retention time “close to”, e.g., from 1–5 mino 5–13 min less than, that of PMA are arbitrarily considered ashorbol esters [20,27–30]. Obviously, the determination of phorbolsters in this way is not accurate and the contents from differentuthors are not comparable. Purification and identification of thendividual toxic compounds are essential to solve the problem. Its worthy to be mentioned that all of the J. curcas samples did notontain detectable PMA in the reports mentioned above. As shownn Fig. 2 the peak of pure PMA appeared at 49.18 min in the HPLChromatogram; there were a few small peaks with retention timesuch less than 49.18 min in the extract of J. curcas kernel cake;

here was no detectable PMA in the kernel cake. With the facility toetect the toxicity by the carp fingerling lethality test, as described

n Sections 2.5.3 and 3.3, we have separated and purified a few toxicompounds from the liquid chromatographic effluents of the ker-el cake extract and obtained their 1H NMR spectra. The purifiedompounds were highly unstable. When performed the 13C NMRo elucidate their molecular structures after the 1H NMR analyses,e found that they had all decayed. Anyway, the 1H NMR spec-

ra showed that they were none of the known toxic compoundsresently identified from J. curcas. In brief, there were no detectablehorbol esters in the kernel cake. However, as shown in the fol-

owing sections the kernel cake was highly toxic. Therefore, theontent of phorbol esters could not indicate the toxicity of the ker-el cake. Heating and the fermentation by some microbial strainsreatly increased the toxicity of the kernel cake (data not shown).ased on these observations combined with that the toxic com-ounds were highly degradable, we infer that some intermediatesegraded were more toxic than the toxic ingredients themselvesnd that partial of the toxicity of the kernel cake came from theegradation of the unknown toxic compounds. Work to elucidatehe chemical identities of the toxic compounds and their majorntermediates degraded, as well as, their toxicity is under way inur lab.

From animal feed point of view, the main components of theernel cake were protein (42.3%), crude fiber (24.5%), ash (8.8%),il (2%) and water (3.8%) (Table 1). From fertilizer point of view,t contained 41.1% organic matter and rich N, P, K and other plantssential elements (Table 1).

.2. Toxicity of the kernel cake, microbial strain isolation andcreening

Three hundred and eighty microbial strains including bacteria,ctinobacteria, fungi and yeasts were isolated in total (120 strainsrom air, 260 strains from the soils). Upon inoculation of the 380

12,900 8520 3600 5840 167 62.57 ND

strains and the 198 known strains to the kernel cake, 10% of themcould not survive at all; 59% of them grew very slow; 31% of themgrew well (the mycelia/colonies covered the total surface of themedium within one week). The growth inhibition by the kernelcake might be due to its antimicrobial components [31,32] andits high protein content, especially the toxic proteins [33]. Basedon the growth rate on the kernel cake, 181 strains were screenedout.

The water extract of the unfermented kernel cake significantlyinhibited the seed germination percentage of C. moschata, R. sativusand B. chinensis by 20%, 45% and 30%, respectively. The extract ofthe kernel cake fermented by most of the strains did not improvethe seed germination percentage as compared with that of theunfermented kernel cake. By the plant seed germination test,69 strains that conferred the fermented kernel cake did not sig-nificantly inhibited the seed germination of all the three plantspecies were screened out from the 181 strains obtained previ-ously.

The unfermented kernel cake was highly toxic to the carp fin-gerlings. The addition of the extract of the unfermented kernel caketo the water significantly reduced the survival time from 60–80 hto 12–18 h. The survival time in the water with the addition ofDMSO at 1.5% (v/v), the maximal dosage used in this work, wasalso 12–18 h, indicating that DMSO at the used dosage did notaffect the survival time of the carp fingerlings. After the carp fin-gerling lethality test strains YUCM 310038, A003 and E290 werescreened out from the 69 strains. Strains YUCM 310038, A003and E290 were identified as Streptomyces fimicarius, Aspergillusversicolor and Scopulariopsis brevicaulis, respectively. Our researchindicated that A. versicolor A003 produced sterigmatocystin (datanot shown), a very toxic and highly carcinogenic mycotoxin [34],by itself. S. brevicaulis is a human pathogen, causing onychomy-cosis [35] and invasive disease as well [36,37]. We confirmedthat S. brevicaulis E290 grew on human detached nails (data notshown). To date, no report shows that S. fimicarius infects or doesharm to human, animal or plant. Therefore, we had the mostinterest in S. fimicarius YUCM 310038. Strain S. fimicarius YUCM310038 has been deposited in SCMGB–YUMRC, with an accessionno. YUCM 310038, and in China Center for Type Culture Col-lection (CCTCC) as well (accession no. M2011248). Its myceliumand spore morphology and 16S rDNA (GenBank accession numberJQ696990) phylogenesis were shown in Figs. 3 and 4, respec-tively.

Similar to our results, the phorbol esters in some J. curcas geno-types in Mexico [21] and Thailand [38] were also undetectable.These varieties were classified as “nontoxic”. However, as demon-strated in this work, even though phorbol esters were undetectable,the kernel cake was severely toxic to carp fingerlings and stronglyinhibited plant seed germination and the growth of some microor-ganisms. Seed germination and seedling establishment are criticalstages in plant life cycle. Germination test is commonly used forthe assessment of phytotoxicity [39,40]. The kernel cake severelyinhibited the seed germination of pumpkin, Chinese cabbage andgarden radish, indicating its phytotoxicity. The toxic agents mightbe the derivatives of phorbol esters and/or other toxins, in addition

to the undetectable phorbol esters. For instance, phorbol com-pounds without any side chain also have tumor promoting activity[41]; Jatropherol-I, a phorbol-type compound, is the main toxinof J. curcas to silkworm [42]. Consequently, using the content of

242 X.-H. Wang et al. / Journal of Hazardous Materials 260 (2013) 238– 246

AU

0.00

0.10

0.20

0.30

0.40

5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00 50.00 55.00 60.00

A

AU

0.00

0.50

1.00

1.50

2.00

2.50

3.00

10.00 20.00 30.00 40.00 50.00 60.00

B

Retention time (min)

curcas

pcphtefatdfresdawsnts

Fig. 2. HPLC chromatograms of the methanol extract of the fresh Jatropha

horbol esters as the sole criterion for evaluating the toxicity of J.urcas kernel cake is not correct. Toxicity evaluation by animal andlant tests is essential. The toxicity evaluation on animal and plantas the advantage to reveal the general toxicity and does not needo quantify the individual components of the toxins. This kind ofvaluation is particularly essential for future development of theermented kernel cake as animal feed or bio-safe fertilizer. In fact,s mentioned previously, due to the large variety of the toxins andhe existence of unknown toxins it is impossible to evaluate theetoxification effect by determining all the individual toxins of theermented kernel cake in the strain screening program. In previouseports, by the determination of phorbol esters the authors onlyvaluated the degradation effects of one [20] or a few microbialtrains [26,43]. We only obtained three strains capable of effectivelyetoxifying the kernel cake from the 578 strains. This indicates that

large strain resource is the prerequisite for the strain screening,hich makes a strain screening process a huge project. The efficient

tep by step target oriented strain screening strategy especially theovel detoxification evaluation methods by plant seed germina-ion and carp fingerling lethality tests facilitated the large-scalecreening process.

kernel cake (A) and standard phorbol 12-myristate-13-acetate (PMA) (B).

3.3. A mathematic model to estimate the general toxicity of thekernel cake

The relationship between the concentration of the methanolextract of the unfermented kernel cake in the water (c) and thecorresponded survival time of the carp fingerlings (t) is shown inFig. 5. When the concentration of the methanol extract below athreshold value (cTmin

) in the water, the toxins did not affect theviability of the carp fingerlings, so the survival time reaches themaximum (Tmax), i.e., the same as in tap water. That is

If t(c) = Tmax (1)

c ≤ cTmin(2)

When the concentration of the methanol extract above a thresh-old value (cTmax ) in the water, the toxins reached saturation in thewater or reached the concentration to the maximal toxicity, mani-festing the maximal lethality and resulting in the minimal survival

X.-H. Wang et al. / Journal of Hazardous Materials 260 (2013) 238– 246 243

F 8. Cos ation

tr

I

c

I

FGB

ig. 3. Morphological characteristics of strain Streptomyces fimicarius YUCM 31003pore under scanning electronic microscopy (C, magnification 12,000×; D, magnific

ime (Tmin); a higher concentration of the methanol extract did noteduce the survival time anymore. That is

f t(c) = Tmin (3)

≥ cTmax (4)

f Tmin < t(c) < Tmax (5)

ig. 4. Neighbor-joining phylogenetic tree constructed with the 16S rRNA gene sequence oenBank database. GenBank accession numbers are given in the parentheses for referencar, substitutions per nucleotide position.

lonial morphology on potato dextrose agar (A, surface; B, reverse), mycelium and 60,000×).

t reduced exponentially against c

t(c) = aekc (6)

t kc

a= e (7)

kc = ln(

t

a

)= ln t − ln a (8)

f strain YUCM 310038 and those of other related Streptomyces strains retrieved frome sequences. Numbers at nodes were bootstrap values based on 1000 resamplings.

244 X.-H. Wang et al. / Journal of Hazardous Materials 260 (2013) 238– 246

Fig. 5. The relationship between the carp fingerling survival time and the concen-tration of the methanol extract of the Jatropha curcas kernel cake in the water. Thesurvival time was the average of the fish (3 × 3, triplicates). Within the tested con-ci

c

wn

TT

otvv

b

p

wctt

3

lcceseiicow

1.E+05

1.E+06

1.E+07

1.E+08

1.E+09

1.E+10

0 24 48 72 96 120 144 168 192 216 240 264

Fermentatioin time (h)

ekaclenrekg/ slleclaiborci

mfor ebmuN

0

10

20

30

40

50

60

70

80

90

Carp

fing

erlin

g su

rviv

al ti

me

(h)

Numbe r of mic rob ia l c e ll s

Surviva l time (h)

Fig. 6. The relationship between the fermentation time of Jatropha curcas kernel

Collectively, the kernel cake was highly toxic to both animal

entrations, at each concentration point the differences of the survival time of threendependent tests were not significant at P < 0.01 level.

(t) = ln t − ln a

k(9)

here a = 71.863, k = −0.0085 (R2 = 0.991), calculated by an expo-ential regression.

For fish from the same batch or with the same viability, Tmax,min, cTmin

and cTmax were constants. In the case shown in Fig. 5,max = 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

r calculated by formula (2), (4) or (9). In other words, the generaloxin concentration in the kernel cake was determined by the sur-ival time of the carp fingerlings. The determination of the t wasery simple, quick and repeatable.

The percentage (p) of the toxins degraded by an SSF process cane calculated by

= cf − cunf

cf× 100% (10)

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

.4. Time course of the SSF and detoxification

As shown in Fig. 6, during the SSF process, there was no obviousag phase; the maximal microbial cell density appeared at 168 h; theell density declined sharply afterwards. The survival time of thearp fingerlings incubated in the water that contained the methanolxtract of the unfermented kernel cake (Tmin) was 12 h, while theurvival time of those in the water that contained the methanolxtract of the kernel cake fermented for 24 h extended to 24 h. Thisndicated that the microbial detoxification started as soon as thenitiation of the fermentation. The survival time in the water that

ontained the extract of the kernel cake fermented for 192 h, i.e.,ne day after the peak density of the bacteria in the kernel cake,as the same as that in the tap water (Tmax = 80 h). This indicated

cake by strain Streptomyces fimicarius YUCM 310038 and the average survival timeof carp fingerlings living in the 200 ml water that contained 1000 mg of the methanolextract of the fermented kernel cake. Values were mean of three replicates.

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

∵ 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 contain-

ing 1000 mg/l of the methanol extract of the kernel cake fermentedby S. fimicarius YUCM 310038 for 9 d was equivalent to that in thewater containing ≤30 mg/l of the methanol extract of the unfer-mented 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 increasecf 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 kernelwas not toxic to the carp fingerlings. Whether the fermented kernelcake is safe as animal feed may need further chronic toxicity teston domestic animals and birds, such as pigs and chickens.

3.5. Improved tobacco plant growth by the application of thefermented kernel cake

The kernel cake contained rich N, P, K and other essential ele-ments for plant growth (Table 1). However, as shown in Table 2,the application of the unfermented kernel cake to the soil didnot significantly improve tobacco seedling growth but inhibitedthe root growth by 48% as compared with the control. This indi-cates that the phytotoxicity of the unfermented kernel cake alsoincluded root growth inhibition, in addition to germination inhi-bition mentioned previously. Interestingly, the application of thekernel cake fermented by S. fimicarius YUCM 310038 significantlyimproved tobacco seedling growth by 80% based on biomass in 14days (Table 2), indicating the potential of the fermented kernel cakeas safe organic fertilizer.

and plant even though the phorbol esters were undetectable.Consequently, any uncontrolled disposal or spread of J. curcasseed cake either in aquatic or terrestrial environment may cause

X.-H. Wang et al. / Journal of Hazardous Materials 260 (2013) 238– 246 245

Table 2Comparison of tobacco seedling growth in pots applied with unfermented Jatropha curcas kernel cake (unfermented) and with that after 9 day fermentation by Streptomycesfimicarius 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 samecolumn 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)

b b c b b

11

edfignkmmc

ifTmlrboPsPap3cer3etJ

4

nesetcvsdtfifs

A

ou(P

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Control 14.4 ± 2.8 2.4 ± 0.3Unfermented 15.2 ± 2.6b 2.5 ± 0.4b

Fermented 25.8 ± 2.9a 4.3 ± 0.5a

cological concern. Presently, most Jatropha industries directlyispose the kernel cake to the soils as fertilizer without any detoxi-cation treatment because of not knowing that doing so will pose areat risk to the environment and human health. The present workot only greatly improved our understanding of the toxicity of theernel cake but also provided strategies to detoxify it. The toxicityodel and the strain screening strategy may be significant for othericrobial detoxification/decomposition systems for substrate with

omplex and/or unknown toxic ingredients.The ultimate goal of the detoxification of J. curcas kernel cake

s effectively converting it from toxic waste to bio-safe animaleed or organic fertilizer to reduce the cost of Jatropha industry.o fulfill this goal via an efficient and environmentally friendlyicrobial fermentation, the microbial strain should have the fol-

owing characters: (1) the effective detoxification capacity, (2) aapid growth rate in the kernel cake, (3) not producing any toxinsy itself, in particular, (4) not being an animal (especially human)r plant pathogen. Phorbol esters could be degraded by SSF throughseudomonas aeruginosa [20]. Nevertheless, no animal or plant testhows that the fermented kernel cake was nontoxic. In addition,. aeruginosa is a notorious human and animal (both vertebratend invertebrate) opportunistic pathogen [44], as well as plantathogen [45], causing diseases fatal to the hosts. S. fimicarius YUCM10038 is the only known and available strain that has all these 4haracters. It is able to directly and efficiently detoxify the moist-ned kernel cake under benign condition without any additionaleagent. The application of the kernel cake fermentation by YUCM10038 has the potential to transform the kernel cake with a vari-ty of toxins to bio-safe animal feed or organic fertilizer, not onlyo remove the environmental concern but to reduce the cost of theatropha industry as well.

. Conclusions

The J. curcas kernel cake strongly inhibited plant seed germi-ation and root growth and was highly toxic to carp fingerlingsven though the phorbol esters were undetectable. Therefore, ithould be detoxified before release to the environment. The math-matic model enabled us to estimate the concentration of the totaloxins in the kernel cake by determining the survival time of thearp fingerlings without the determination of the (unknown) indi-idual toxins. The effective strain screening strategy facilitated thecreening process. Strain S. fimicarius YUCM 310038 was able toegrade the total toxicity by more than 97% in a single fermenta-ion process, conferring the fermented kernel cake nontoxic to carpngerlings and plants, and promoting tobacco plant growth. There-

ore, the present work represents a novel method for effectivelyimultaneous detoxification of multiple toxins in plant biomass.

cknowledgements

This work was financed by the Ministry of Science and Technol-

gy for the China National Science and Technology Pillar Programnder the project 2011BAD30B00, and by European Union FundsFEDER/COMPETE–Operational Competitiveness Programme) andortugal national funds (FCT – Portuguese Foundation for Science

[

8.6 ± 1.1 5.8 ± 0.7 5.8 ± 0.81.3 ± 1.6b 6.8 ± 0.7b 3.9 ± 0.6c

5.6 ± 1.9a 8.2 ± 0.9a 10.2 ± 1.2a

and Technology) under the project FCOMP-01-0124-0124-FEDER-022692.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.jhazmat.2013.05.012.

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