Department of Soil, Plant, Environmental and Animal Production Sciences, School of Biotechnological Sciences, University of Naples Federico II, Portici, ItalyDepartment of Biological Sciences, University of Naples Federico II, Naples, Italy
r t i c l e i n f o
rticle history:eceived 31 January 2011eceived in revised form 31 May 2011ccepted 3 June 2011
eywords:
a b s t r a c t
Olive oil mill wastewaters (OMWs) are characterised by low pH and a high content of mono- and polyaro-matic compounds that exert microbial and phytotoxic activity. The laccase cDNA of the poxA1b gene fromPleurotus ostreatus, carrying a signal peptide sequence for enzyme secretion and driven by the CaMV 35Spromoter, was cloned into a plant expression vector. Nuclear genetic transformation was carried out byco-cultivation of Agrobacterium tumefaciens with tobacco cv Samsun NN leaves and cells of five different
microalgae accessions belonging to the genera Chlamydomonas, Chlorella and Ankistrodesmus. Transgenicplants and microalgae were able to express and secrete the recombinant laccase in the root exudates andthe culture medium, respectively. In comparison to untransformed controls, the ability to reduce phenolcontent in OMW solution was enhanced up to 2.8-fold in transgenic tobacco lines and by up to about 40%in two microalgae accessions. The present work provides new evidence for metabolic improvement ofgreen organisms through the transgenic approach to remediation.
. Introduction
Industrial activities release a wide range of toxic chemicals intohe biosphere. Among such chemicals, phenolic compounds rep-esent a major risk for human health and biodiversity mainly dueo their relative persistence in the environment. In the Mediter-anean area the olive oil industry accounts for about 95% of thentirely world olive oil production. It generates a large amount ofarmful effluent (olive oil mill wastewaters; OMWs), estimated atbout 3 × 107 m3 per year [1]. OMWs are one of the most complexndustrial by-products, due its low pH, high content of minerals andrganic constituents represented by sugars, nitrogen compounds,ectins, fats and phenols [2,3]. The phenolic fraction accounts for–15% by weight and includes low and high molecular phenolicompounds, tannins, anthocyanins and lignans [1,4]. OMWs alsoontain phytotoxic components that inhibit microbial growth [5,6],ffecting soil bacterial communities [7] as well as the germina-ion and vegetative growth of plants [8]. In several Mediterranean
Please cite this article in press as: Chiaiese P, et al. Engineered tobacco and min olive oil mill wastewater. Enzyme Microb Technol (2011), doi:10.1016/j
ountries OMWs are released into the environment, causing severeamage to the biosphere [9,10]. Hence there is enormous interest
Physical, chemical and biological treatments have been appliedto remove the hazardous compounds in such industrial wastewa-ters [11]. Physico-chemical approaches are considered inadequatebecause they are both complex and costly [12]. By contrast, reme-diation by biological agents has been reported to be economicaland to show considerable potential. In this context, research hasbeen carried out to evaluate the performance of phenol degra-dation by bacteria (bioremediation), fungi (mycoremediation),algae (phycoremediation) and higher plants (phytoremediation)[6,11,13,14].
White-rot fungi have been proved to secrete a large arrayof ligninolytic enzymes which are highly efficient in degradingaromatic compounds, such as lignin peroxidases, manganese per-oxidase and laccases [15]. However, mycoremediation needs anabundant supply of nutrients to maintain such organisms [16],while remediation based on photosynthetic organisms is self-sustaining since they use sunlight, water, inorganic ions and carbondioxide. On the other hand, since the growth of plants and microal-gae does not depend on organic compounds as a source of energy,they may lack highly efficient enzymes for phenol degradation[17].
A biotechnological approach to enhance the efficiency of xenobi-
icroalgae secreting the fungal laccase POXA1b reduce phenol content.enzmictec.2011.06.002
otic removal from green organisms is to overexpress genes involvedin metabolism, uptake or transport of specific organic pollutants.However, a useful approach is to secrete the phenol-degradingenzymes in the soil or water. The main advantage of this strategy is
the Cauliflower Mosaic Virus 35S cassette present in the pGreen 0029 vector(http://www.pgreen.ac.uk/a cst fr.htm).
The nucleotide sequence of the expression cassette CaMV35S pro-moter/poxA1b/CaMV35S terminator was determined by BigDye Terminator v3.1
Fig. 1. Molecular analysis of green organisms. (A) PCR analysis of genomic DNAisolated from putative transgenic plants. Lanes 1–11 represent putative transgeniclines; C− represents wild-type plants; C+: plasmid pFFP001; M: molecular marker.Total soluble proteins isolated from tobacco plants were analysed by Western blot
−
L + − − −
, solid medium; L, liquid medium.
o avoid any uptake of the toxic pollutants by the green organism18–20]. Among xenobiotic-degrading enzymes, white-rot laccasesbenezediol: oxygen oxidoreductase, EC 1.10.3.2) catalyse oxi-ation of a broad range of substrates. Moreover, they are ableo oxidise non-phenolic compounds in the presence of higheredox mediators. As demonstrated by Giardina et al. [21] POXA1bs the most thermostable isoenzyme among Pleurotus ostreatusaccases, exerting a high stability under several environmentalonditions.
Laccase from Coriolus versicolor has been expressed into tobaccoor the rhizoremediation of bisphenol A and pentachlorophenol22]. Furthermore, Arabidopis thaliana expressing a cotton laccaseene showed enhanced resistance to 2,4,6-trichlorophenol [23].n both studies the feasibility of such an approach was verifiedy growing transgenic plants in solutions containing only a singlehenol.
The ability of some species of green microalgae to remove or bio-ransform hazardous organic pollutants is well recognized [24]. Inarticular, species belonging to the genera Chlorella, Ankistrodesmusnd Scenedesmus have been suggested as bioremediation agentsor OMW treatment [25–27]. To date, no advancement has beeneported in using microalgae as remediation agents in any organicollutant system through the transgenic approach. Our aim is to
mprove the ability of plants and microalgae to reduce the OMWhenolic content by the expression of heterologous genes. In thistudy we report the expression of the white rot P. ostreatus poxA1baccase gene in tobacco plants and in five accessions of green
icroalgae. The efficiency of plants and microalgae in removingMW phenol content is also reported.
. Materials and methods
.1. Plant genotype, microalgae accessions and growth condition
Nicotiana tabacum cv Samsun NN were micropropagated onto solid growthedium containing Murashige and Skoog mineral salt formulation [28], sucrose
30 gl−1) and Plant Agar (10 g l−1; Duchefa). Cultures were incubated at 24 ◦C under6 h of light at 100 �E m−2 s−1.
The following microalgae accessions, namely Chlamydomonas pitschmanniittl no. 238/79, Chlorella emersonii Shihira and Krauss 061-103, 317-24, and53-95, and Ankistrodesmus braunii (Naegeli) Brunnthaler strain 160-146, werevailable at the Algal collection at the University of Naples Federico II (ACUF,ttp://www.biologiavegetale.unina.it/acuf.html). Microalgae were grown in Boldasal medium (BBM) [29] at 24 ◦C, 100 rpm under 16 h/8 h light/dark photoperiodt 100 �E m−2 s−1.
Please cite this article in press as: Chiaiese P, et al. Engineered tobacco and min olive oil mill wastewater. Enzyme Microb Technol (2011), doi:10.1016/j
.2. In silico analysis of cellular localisation
The computational prediction of subcellular localisation of POX A1b in plantsas carried out by ProtComp 9.0 (http://www.softberry.com).
− − − − − −
2.3. Expression vector construction
The fungal poxA1b cDNA (AJ005018.1) cloned into the vector pGEM 7zF waskindly gifted by Prof. Giovanni Sannia (University of Naples Federico II). The poxA1bcDNA fragment was obtained from digestion with SacI/XbaI followed by gel purifi-cation (Promega). It was then cloned directionally in the SacI/XbaI sites within
icroalgae secreting the fungal laccase POXA1b reduce phenol content.enzmictec.2011.06.002
(B) and Nondenaturing PAGE (C). Lanes 1–8 represent transgenic lines; C representswild-type plants; C+: POXA1b purified protein. Microalgae cell-free supernatantwas analysed by Dot blot. Panel (D) shows identification of A. braunii colonies; squareM1 represents POXA1b purified protein; from square A8 to N8 wild-type cell-freesupernatant.
P. Chiaiese et al. / Enzyme and Microbial Technology xxx (2011) xxx– xxx 3
F fresha For ed
Cert0B
2
aBdwotd
2
ct(pgdim
2
PAtt
ig. 2. Laccase activity. (A) For root exudates this was calculated as mU per gram ofctivity of the highest microalgae cell-free supernatant is reported as mU mg FW−1.ifferent (p < 0.05).
ycle Sequencing Kit (Applied Biosystem) and 3100 Genetic Analyzer ABI PRISM. Thexpression cassette was excised by EcoRV digestion followed by gel purification. Theesultant EcoRV fragment was then inserted into the pGreen 0029 vector carryinghe nptII gene as selectable marker (http://www.pgreen.ac.uk/JIT/pGreenII/T-029.pdf). The Agrobacterium tumefaciens LBA4404 strain was electroporated byioRad Escherichia coli pulser following the manufacturer’s instructions.
.4. Antibiotics sensitivity assay
To determine the minimum inhibitory concentration of kanamycin sulphatend geneticin (G418) wild-type microalgae cells were left to grow on solid or liquidBM medium. Each algal accession (∼106 cells) was spread over the surface of a Petriish containing 20 ml of solid medium or inoculated into 1.5 ml medium in a 24-ell plate (M-Medical), with the addition of kanamycin (0, 25, 50, 100, 200 mg l−1)
r geneticin (0, 10, 20, 40, 50, 60 mg l−1). After 20 days, growth was assessed byhe appearance of visible colonies on solid medium and by measuring the opticalensity of the liquid culture at 550 nm.
.5. Nuclear genetic transformation
Plant genetic transformation was carried out according to Horsch et al. [30] byo-cultivation of tobacco leaf explants with A. tumefaciens. Differentiation of puta-ive transgenic shoots was carried out in the presence of 100 mg l−1 kanamycinDuchefa). Six weeks after co-cultivation, putative transgenic shoots were trans-lanted onto solid medium with the addition of 100 mg l−1 kanamycin. Microalgaeenetic transformation was performed as reported by Kumar et al. [31]. After 2ays of co-cultivation, algae were collected and plated onto BBM medium contain-
ng geneticin at 50 mg l−1 (BBMG). The number of colonies growing on selectiveedium was scored 22 days after co-cultivation.
.6. Molecular analysis
Please cite this article in press as: Chiaiese P, et al. Engineered tobacco and min olive oil mill wastewater. Enzyme Microb Technol (2011), doi:10.1016/j
The screening of putative transgenic rooted shoots was carried out byCR analysis. PCR primer 1 (5′ TGCTGGGTGCATTTATGAC 3′) and primer 2 (5′
GGAGTTTCGATGGGTTCG 3′) were designed according to the coding region ofhe poxA1b gene. Genomic DNA was isolated from young leaves of 3–4-week-oldobacco plants as described by Edwards et al. [32].
weight. Columns with different letters are significantly different (p < 0.05). (B) Theach cluster of microalgae accession, columns with different letters are significantly
Total soluble proteins (TSP) were extracted from tobacco plants according toNagel et al. [33]. Bradford reagent was used to determine protein concentration.For Western blots, 50 �g of plants TSP were analysed using anti-POX A1b poly-clonal antibodies. A chemiluminescence detection system (West Pico, Pierce) wasused to visualise immuno-positive proteins. Non-denaturing PAGE was performedas reported by Palmieri et al. [34]. The POXA1b purified protein was kindly donatedby Prof. Giovanni Sannia (University of Naples Federico II).
In order to identify POX A1b-expressing transgenic microalgae colonies dot blotassays were performed. For each accession line at least 84 colonies were inocu-lated in liquid BBMG in a 96-well plate. After 15 days of incubation, plates werecentrifuged at 4000 rpm for 20 min and then 100 �l of cell-free supernatant werevacuum-infiltrated onto a nitrocellulose membrane (Amersham). The detection ofimmune-reactive spots was detected as described above for plants.
2.7. Enzymatic activity
Laccase activity was evaluated at 25 ◦C using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid; ABTS) as substrate. The assay was carried outin sodium citrate buffer (0.2 M pH 3.0) containing ABTS at 20 mM. Oxidation of thesubstrate was followed by absorbance increase at 420 nm (ε420 = 36,000 M−1 cm−1)as described by Piscitelli et al. [35]. Enzymatic activity was expressed as inter-national units (U), where 1 U corresponds to 1 �mol of substrate oxidised perminute.
To assess the rhizosecretion of heterologous protein in culture medium, threeplants for each line were grown hydroponically in vivo in Hoagland solution [36]in a growth chamber at 24 ◦C, under a 16 h/8 h light/dark photoperiod. After 30days of culture, fresh medium was added, avoiding any damage to root apparatus.Laccase activity was then determined on liquid growth medium collected withoutany concentration step after other 10 days of cultivation. Plants were carefully wipedand excess moisture from the roots was removed with absorbent paper. Plant freshweight (g) was determined on a technical balance.
icroalgae secreting the fungal laccase POXA1b reduce phenol content.enzmictec.2011.06.002
Fifteen immune-reactive positive colonies for each microalgae accession (seeSection 2.6) were inoculated in 20 ml of liquid BBMG for 10 days. Enzymatic assaywas performed on 20 �l of cell-free supernatant using a Wallac Victor Multireader(Perkin Elmer). The growth of microalgae was estimated by measuring the opticaldensity of the culture at 550 nm. The absorbance values were then converted into
Table 2Genetic transformation frequency of three accession of Chlorella emersonii,Ankistrodesmus braunii, and Chlamydomonas pitschmannii.
Microalgae Accession No. of growingcolonies
Transformationfrequency
A. braunii (#146) 386 7.72 × 10−6
C. pitschmannii (#079) 29 1.16 × 10−6
C. emersioni (#103) 468 9.36 × 10−6
ARTICLEMT-8272; No. of Pages 7
P. Chiaiese et al. / Enzyme and Mi
resh weight (mg). Enzymatic activity was calculated using a Beckman spectropho-ometer in kinetic mode and expressed as mU g fresh weight−1 for plants and as
U mg fresh weight−1 for microalgae.
.8. Phenolic reduction in OMWs
Olive oil mill wastewaters (OMWs) were collected from a mill in Campania,outhern Italy. In order to remove the corpuscular fraction, the OMWs were cen-rifuged at 10,000 rpm for 45 min. The liquid phase was filtered with 0.44 �m using
vacuum pump system. The OMWs were made axenic by filter sterilization with.22 �m nitrocellulose filter under a laminar flow hood.
Transgenic tobacco plants and control plants were grown hydroponically in vivo,n a growth chamber under the same conditions as previously reported. Each plant
as transferred to a culture vessel containing 500 ml of diluted OMW whose finalhenol content was set at 4.2 g l−1. After 10 days, phenolic concentration wasvaluated as described by Folin and Ciocalteu [37] on 5 ml aliquots of the liquidulture.
The best microalgae expressing laccase for each accession was cultivated iniluted OMW in a 24-well plate under 16 h/8 h light/dark photoperiod at 24 ◦Cithout shaking. OMW phenol content and catechol as standard were quantified
s described above.
.9. Statistical analysis
In all experiments three replicates were performed for each sample and eacheported value represents the mean of three experiments. Statistical analysis wasarried out using the ANOVA test (Systat v. 12, Systat Software Inc.).
. Results and discussion
.1. Genetic transformation of green organisms
In silico analysis was performed on the signal peptide of the P.streatus poxA1b gene encoding the laccase. From targeting predic-ion analysis it may be inferred that this signal peptide should beecognised by the plant cell machinery with the highest score (dataot shown). Microalgae subcellular localisation prediction was notarried out due to the absence of bioinformatic data. Consequently,he native poxA1b cDNA was inserted into the plant expressionassette.
In green microalgae, a few species have been successfullyanipulated, namely Volvox, Chlorella, Dunaliella, Haemotococcus
nd Chlamydomonas, by using several methods which requiredhe absence of cell walls [38]. A recent advancement in microal-ae genetic manipulation was obtained by Kumar et al. [31] byo-cultivation of Chlamydomonas reinhardtii with A. tumefaciensearing a plant expression vector. In addition, the capability of A.umefaciens as a genetic transforming agent has been confirmed onaemotococcus pluvialis [39,40].
In this study, the microalgae accessions were evaluated to beenetic transformed by A. tumefaciens. The genetic transforma-ion protocols involved the use of the nptII resistance gene [41]s a selectable marker. The cassettes of the poxA1b gene wereloned into the pGreen 0029 plant binary vector containing theos:nptII:nos. The main advantage of using this vector was thebsence of a single point mutation in the nptII gene which is a morefficient enzyme [42].
To select transgenic colonies it is essential to identify the sensi-ivity of algae to the selective agent. Antibiotic kill curve assaysere performed on solid and liquid medium to establish theinimal inhibitory concentration of two aminoglycoside antibi-
tics, geneticin and kanamycin, on microalgae growth. Microalgaeell growth was inhibited by both antibiotics at different con-entrations (Table 1). After 20 days of culture on solid medium,icroalgae were able to tolerate up to kanamycin 25 mg l−1 but
nly accessions #79 and #95 were able to grow until kanamycin
Please cite this article in press as: Chiaiese P, et al. Engineered tobacco and min olive oil mill wastewater. Enzyme Microb Technol (2011), doi:10.1016/j
00 mg l−1. Above this concentration, no colonies were detected.eneticin was more effective than kanamycin at inhibiting microal-ae growth since colonies were detected up to the concentrationf 20 mg l−1, although accession #79 was susceptible at 10 mg l−1.
C. emersioni (#24) 429 8.50 × 10−6
C. emersioni (#95) 1215 24.30 × 10−6
Both antibiotics in liquid selection medium were more effec-tive at inhibiting cell growth than in solid medium, as observedfor H. pluvialis [40]. This might be due to a difference in mem-brane permeability between the cells grown in the two media[40]. On the basis of these results, in subsequent microalgaegenetic transformation experiments, geneticin 50 mg l−1 was cho-sen as a selective agent in both solid and liquid medium. After 10days of incubation on this medium, a large number of putativetransgenic microalgae colonies became visible. The transforma-tion frequency for each algae accession was calculated after 20days of incubation under selective conditions. The highest num-ber of colonies was scored in accession #95, and for accessions#146, #103, #24 the transformation frequencies were not sta-tistically different (p < 0.05). No green colonies were observed onplates containing the selection medium inoculated by control cells(Table 2).
In plants, after six weeks of in vitro cultivation on selectivemedium, 22 shoots differentiated from 190 tobacco leaf explants.Only 11 shoots were able to root on growth medium containingkanamycin at 100 mg l−1. No adventitious shoots were regeneratedon control leaf explants on selective medium.
Identification of the laccase transgene in primary plant transfor-mants was performed by PCR on plant genomic DNA. As shown inFig. 1, panel A, the poxA1b specific products of 400 bp were detectedin only 8 of 11 rooted shoots, as well as in the lane of the posi-tive control expression vector. PCR analysis showed the absence ofA. tumefaciens contamination on growing rooted plants (data notshown).
The PCR positive transformants were further propagated on invitro rooting medium to identify the laccase-producing plants. Afterone month of node propagation, stems and leaves were collectedand total soluble proteins were assayed for immunoblot. A uniqueimmunoreactive band similar in molecular mass of the purified POXA1b from P. ostreatus was observed in transgenic plant samples.The above results suggest that these transgenic tobacco plants pro-duced POX A1b protein in their leaf and stem tissues (Fig. 1, panel B).Furthermore, zymogram analysis indicated that all eight indepen-dent transgenic plants produced the active fungal laccase (Fig. 1,panel C). No bands were observed in either detection analysis fromthe control tobacco plants (WT).
To identify POXA1b expressing microalgae colonies,immunoblot assay was performed on cell-free supernatantfrom the microalgae colonies grown in selective BBMG medium.Immunoreactive spots were observed in some colonies (Fig. 1,panel D). From these results we infer the ability of microalgae cellsto recognise the fungal signal peptide as a suitable secretory tag.Hence heterologous laccase was found in supernatant.
3.2. Laccase activity in transgenic green organisms
On the basis of the ABTS assay, control and transgenic plants
icroalgae secreting the fungal laccase POXA1b reduce phenol content.enzmictec.2011.06.002
secrete laccases into the growth liquid medium (Fig. 2, panel A).It has been shown elsewhere that the roots of several plant fami-lies are able to secrete enzymes [43,44]. The ability of 12 terrestrialplant species to secrete oxidoreductase enzymes including laccase
P. Chiaiese et al. / Enzyme and Microbial Technology xxx (2011) xxx– xxx 5
Fig. 3. Phenol removal by transgenic organisms from olive oil mill wastewaters. (A) Transgenic plant reduction of phenol contents is measured by phenol removed per mgo (B) Rei n, col
wfttPtoseappc
crmddsiapa5i#iTgp
f fresh weight. Columns with different letters are significantly different (p < 0.05).
s reported as phenol removed per mg of fresh weight. For each microalgae accessio
as reported by Gramss et al. [44]. Among these plants, exudatesrom tobacco showed a low detectable activity against ABTS, lowerhan that we detected (5.3 ± 1.4 mU mg FW−1). This is probably dueo a different plant tobacco cultivar and growing conditions. TheOX A1b rhizosecretion enhanced total laccase activity comparedo control plants. Plants were grouped into six classes on the basisf their exudate laccase activity. Three transgenic plants were nottatistically different from the control plants at p < 0.05. The high-st laccase activity was detected in plant #7 exudates, showingn activity 46-fold higher than that detected in the control sam-le (Fig. 2, panel A). These results confirmed the in silico analysisrediction of the poxA1b signal peptide as excrete tag in plantells.
Laccase activity in microalgae cell-free supernatant mediumulture is reported in Fig. 2 (panel B). For each accession weeport the results from selected transgenic colonies. All wild-typeicroalgae were able to oxidise ABTS; the values were statistically
ifferent (p < 0.05). Otto et al. [45] neatly demonstrated the pro-uction of an extracellular laccase-like enzyme in coccoid greenoil algae Tetracystis aeria. Our results strongly support the abil-ty of green microalgae to release laccase into the environmentnd that this trait varied among accessions. Overexpression ofoxA1b in such microalgae accessions increased the oxidoreductasectivity. Accession #24 showed the highest activity, an average of0.4 mU mg FW−1, while accession #103 was the lowest perform-
ng with an average of 10.8 mU mg FW−1. The microalgae accessions 103, #79, and #95 showed a respective 4.5, 4.9 and 4.2-fold
Please cite this article in press as: Chiaiese P, et al. Engineered tobacco and min olive oil mill wastewater. Enzyme Microb Technol (2011), doi:10.1016/j
ncrease in supernatant activity compared with their wild types.he greatest improvement was obtained in the transgenic microal-ae #79-1 with a 6.5-fold increase in supernatant activity (Fig. 2,anel B).
duction in phenol content by the highest enzymatic activity transgenic microalgaeumns with different letters are significantly different (p < 0.05).
3.3. Reduction of phenol content in OMWs
To establish the efficiency of phenolic removal on the part oftransgenic plants, assays were performed in vivo in hydroponic cul-ture with OMWs. As shown in Fig. 3 (panel A) all tobacco lines wereable to reduce the OMW phenolic content in only 10 days of treat-ment. However, the degree of such reduction significantly differedamong lines. Lines #3, #6, and #4 did not perform statistically dif-ferently from WT, showing the lowest ability to reduce the totalphenolic content in such a complex matrix. Of the others, lines #1and #7 showed the highest phenolic removal; their figures werenot statistically different. On average, they showed a reduction of64.9 mg of phenols per gram of fresh weight, about 2.8-fold morethan WT.
Reduction of single aromatic compounds driven by expressionof the oxidoreductase enzyme in plants has been reported: tobaccoplants expressing a fungal laccase [22] or an Mn–peroxidase [16]from C. versicolor were able to reduce pentachlorophenol 2-foldmore than control plants. Hirai et al. [46] reported the expres-sion of a codon usage optimised fungal laccase from Schizophyllumcommune in tobacco plants; the removal of trichlorophenol inliquid medium culture was 1.6-fold higher than the WT. Interest-ingly, in this study poxA1b-expressing tobacco plants challengedwith a harsh environment deriving from the complex matrix ofOMW compared to the one-phenol solution tested by these authors[16,22,46].
Evaluation of phenol removal by microalgae was carried out in
icroalgae secreting the fungal laccase POXA1b reduce phenol content.enzmictec.2011.06.002
sterile OMW. Wild-type microalgae cultured in OMW or in BB solu-tion medium did not show any statistically significant difference ingrowth. After genetic transformation, for each accession the highestexpressing POXA1b laccase colony was chosen. Accession #103 was
ot tested, given its poor performance with the ABTS assay. Again,he complex phenol mixture of OMW did not affect the growth ofny transgenic accession (data not show).
As reported in the literature green microalgae are able tohycoremediate a wide range of chemical pollutants, includinghenolic compounds discharged in soil or water by industries suchs tanning or olive-oil production [26,47]. This metabolic abilityas proved by all five microalgae accession assayed to decrease thehenol content in a complex matrix, namely OMW, in only 10 daysf treatment. Analysis by the Folin Ciocalteu procedure showed thatild-type microalgae were able to reduce total phenol content to
aried extents. Since the biomass produced in the 10 days of cultureere lower in accessions #79 and #24, those two microalgae acces-
ions showed the highest rates of phenol content reduction with aalue of 0.009 and 0.011 mg phenols mg FW−1, respectively (Fig. 3,anel B). By contrast, accession #95 was able to reduce the initialhenol content by about 60% in our condition (data not shown).he ability of A. braunii to grow and remove about 6% of phenolontent in OMW after 5 days of culture is reported elsewhere [26].n comparison, the OMW initial concentration of phenols tested inhe above reported experiments was 0.15 g l−1, 28-fold lower thanhat we assayed.
The overexpression and secretion in culture medium of POX1b did not increase the capability of transgenic accessions #95nd #146 to reduce the phenolic content with respect to theirild types. Transgenic microalgae strains #24 and #79 were par-
icularly effective at reducing the phenol pool. Therefore, theaccase-producing microalgae were able to decrease phenols moreffectively than their controls. Although the ability to biotrans-orm phenolic compounds has been demonstrated in Chlorella sp.,cenedesmus sp. [48] and in A. braunii [25], the catabolic sequencesnvolved in transforming these compounds are still not well under-tood. By contrast, several classes of enzymes are known to benvolved in the biodegradation of single aromatic compoundsr a complex matrix such as OMW by root fungi and bacteria11,49].
A less clear response for phenolic removal on the part of microal-ae accessions compared to tobacco plants might be correlatedo the different genetic background and therefore be species-pecific. According to Lei et al. [50] the removal of fluoranthene andyrene varied among microalgae species belonging to the Chlorella,cenedesmus and Selenastrum genera.
. Conclusion
In this study we observed enhanced catabolic ability of higherlants and green microalgae by manipulating their genomesowards phenolic content in OMW. Genetic engineering by A.umefaciens is a robust procedure for breeding improvement oflants and microalgae. The fungal laccase gene poxA1b was cloned
nto a plant expression vector and A. tumefaciens-mediated trans-ormation was carried out on tobacco leaves and microalgaeells. As a result, five microalgae accessions belonging to thehlamydomonas, Ankistrodesmus and Chlorella genera were success-ully transformed. The transgenic plants and microalgae correctlyxpressed the fungal gene poxA1b. The release of the heterolo-ous laccase significantly increased enzymatic activity in the rootxudates and in cell-free microalgae supernatant. This is the firstime that a fungal laccase was expressed in several microalgaepecies. Moreover, the removal ability vis-à-vis phenols in the plantransgenic lines and in C. emersonii and C. pitschmannii accessions
Please cite this article in press as: Chiaiese P, et al. Engineered tobacco and min olive oil mill wastewater. Enzyme Microb Technol (2011), doi:10.1016/j
as enhanced significantly compared with their wild type. Furthertudies could be performed by using a consortium of algae or a com-ination of plants and microalgae expressing laccase for phenolicemoval in OMW.
[
PRESSl Technology xxx (2011) xxx– xxx
To our knowledge, this is the first study to evaluate the use ofgreen organisms as bioremediation agents in such a complex matrixas olive oil mill wastewaters. Using the heterologous approach,green organisms in which oxidoreductase enzymes are expressedwill be a useful tool for phyto- or phycoremediation of hazardousphenolic pollutants.
Acknowledgments
The authors wish to thank Sara Siervo and Rosario Nocerino fortheir technical assistance.
References
[1] Kapellakis I, Tsagarakis K, Crowther J. Olive oil history, production and by-product management. Reviews in Environmental Science and Biotechnology2008;7:1–26.
[2] Cabrera F, López R, Martinez-Bordiú A, Dupuy de Lome E, Murillo JM.Land treatment of olive oil mill wastewater. International Biodeterioration &Biodegradation 1996;38:215–25.
[3] Aktas ES, Imre S, Ersoy L. Characterization and lime treatment of olive millwastewater. Water Research 2001;35:2336–40.
[4] Sierra J, Martí E, Garau MA, Cruanas R. Effects of the agronomic use ofolive oil mill wastewater: field experiment. Science of the Total Environment2007;378:90–4.
[5] Ben Othman N, Ayed L, Assas N, Kachouri F, Hammami M, Hamdi M. Ecologicalremoval of recalcitrant phenolic compounds of treated olive mill wastewaterby Pediococcus pentosaceus. Bioresource Technology 2008;99:2996–3001.
[6] McNamara CJ, Anastasiou CC, O’Flaherty V, Mitchell R. Bioremediationof olive mill wastewater. International Biodeterioration & Biodegradation2008;61:127–34.
[7] Karpouzas DG, Ntougias S, Iskidou E, Rousidou C, Papadopoulou KK, Zervakis GI,et al. Olive mill wastewater affects the structure of soil bacterial communities.Applied Soil Ecology 2010;45:101–11.
[8] Casa R, D’Annibale A, Pieruccetti F, Stazi SR, Giovannozzi Sermanni G, Lo Cas-cio B. Reduction of the phenolic components in olive-mill wastewater by anenzymatic treatment and its impact on durum wheat (Triticum durum Desf.)germinability. Chemosphere 2003;50:959–66.
[9] Mahmoud M, Janssen M, Haboub N, Nassour A, Lennartz B. The impact of olivemill wastewater application on flow and transport properties in soils. Soil andTillage Research 2010;107:36–41.
10] Chartzoulakis K, Psarras G, Moutsopoulou M, Stefanoudaki E. Application ofolive mill wastewater to a Cretan olive orchard: effects on soil properties, plantperformance and the environment. Agriculture, Ecosystems & Environment2010;138:293–8.
11] Cao B, Nagarajan K, Loh K-C. Biodegradation of aromatic compounds: currentstatus and opportunities for biomolecular approaches. Applied Microbiologyand Biotechnology 2009;85:207–28.
12] Contreras EM, Albertario ME, Bertola NC, Zaritzky NE. Modelling phenolbiodegradation by activated sludges evaluated through respirometric tech-niques. Journal of Hazardous Materials 2008;158:366–74.
13] Verkleij JAC, Golan-Goldhirsh A, Antosiewisz DM, Schwitzguébel J-P, SchröderP. Dualities in plant tolerance to pollutants and their uptake and translocationto the upper plant parts. Environmental and Experimental Botany 2009;67:10–22.
14] Rawat I, Ranjith Kumar R, Mutanda T, Bux F. Dual role of microalgae: phy-coremediation of domestic wastewater and biomass production for sustainablebiofuels production. Applied Energy 2011;88:3411–24.
15] Singh Arora D, Kumar Sharma R. Ligninolytic fungal laccases and theirbiotechnological applications. Applied Biochemistry and Biotechnology2010;160:1760–88.
16] Iimura, Iimura Y, Ikeda, Ikeda S, Sonoki, Sonoki T, et al. Expression of a genefor Mn-peroxidase from Coriolus versicolor in transgenic tobacco generatespotential tools for phytoremediation. Applied Microbiology and Biotechnology2002;59:246–51.
17] Abhilash PC, Jamil S, Singh N. Transgenic plants for enhanced biodegrada-tion and phytoremediation of organic xenobiotics. Biotechnology Advances2009;27:474–88.
18] Glick BR. Phytoremediation: synergistic use of plants and bacteria to clean upthe environment. Biotechnology Advances 2003;21:239–44.
19] Gerhardt KE, Huang X-D, Glick BR, Greenberg BM. Phytoremediation and rhi-zoremediation of organic soil contaminants: potential and challenges. PlantScience 2009;176:20–30.
20] Kawahigashi H. Transgenic plants for phytoremediation of herbicides. CurrentOpinion in Biotechnology 2009;20:225–30.
21] Giardina P, Palmieri G, Scaloni A, Fontanella B, Faraco V, Cennamo G, et al. Pro-
icroalgae secreting the fungal laccase POXA1b reduce phenol content.enzmictec.2011.06.002
tein and gene structure of a blue laccase from Pleurotus ostreatus. BiochemicalJournal 1999;341(Pt 3):655–63.
22] Sonoki T, Kajita S, Ikeda S, Uesugi M, Tatsumi K, Katayama Y, et al. Transgenictobacco expressing fungal laccase promotes the detoxification of environmen-tal pollutants. Applied Microbiology and Biotechnology 2005;67:138–42.
23] Wang G-D, Li Q-J, Luo B, Chen X-Y. Ex planta phytoremediation of trichlorophe-nol and phenolic allelochemicals via an engineered secretory laccase. NatureBiotechnology 2004;22:893–7.
25] Pinto G, Pollio A, Previtera L, Temussi F. Biodegradation of phenols by microal-gae. Biotechnology Letters 2002;24:2047–51.
26] Pinto G, Pollio A, Previtera L, Stanzione M, Temussi F. Removal of low molecularweight phenols from olive oil mill wastewater using microalgae. BiotechnologyLetters 2003;25:1657–9.
27] Tarlan E, Dilek FB, Yetis U. Effectiveness of algae in the treatment of awood-based pulp and paper industry wastewater. Bioresource Technology2002;84:1–5.
28] Murashige T, Skoog F. A revised medium for rapid growth and bio assays withtobacco tissue cultures. Physiologia Plantarum 1962;15:473–97.
29] Bischoff HN, Bold HC. Some Soil Algae from Enchanted Rock and Related AlgalSpecies. Austin, TX: Phycol. Stud., IV. Univ. Texas Publs; 1963.
30] Horsch RB, Fry JE, Hoffmann NL, Eichholtz D, Rogers SG, Fraley RT. A simple andgeneral method for transferring genes into plants. Science 1985;227:1229–31.
31] Kumar SV, Misquitta RW, Reddy VS, Rao BJ, Rajam MV. Genetic transforma-tion of the green alga Chlamydomonas reinhardtii by Agrobacterium tumefaciens.Plant Science 2004;166:731–8.
32] Edwards K, Johnstone C, Thompson C. A simple and rapid method for thepreparation of plant genomic DNA for PCR analysis. Nucleic Acids Research1991;19:1349.
33] Nagel R, Manners J, Birch R. Evaluation of an ELISA assay for rapid detection andquantification of neomycin phosphotransferase II in transgenic plants. PlantMolecular Biology Reporter 1992;10:263–72.
34] Palmieri G, Giardina P, Bianco C, Fontanella B, Sannia G. Copper induction oflaccase isoenzymes in the ligninolytic fungus Pleurotus ostreatus. Applied andEnvironment Microbiology 2000;66:920–4.
Please cite this article in press as: Chiaiese P, et al. Engineered tobacco and min olive oil mill wastewater. Enzyme Microb Technol (2011), doi:10.1016/j
35] Piscitelli A, Giardina P, Mazzoni C, Sannia G. Recombinant expression of Pleu-rotus ostreatus laccases in Kluyveromyces lactis and Saccharomyces cerevisiae.Applied Microbiology and Biotechnology 2005;69:428–39.
36] Hoagland DR, Arnon DI. The water-culture method for growing plants withoutsoil. Berkley: Univ. of Calif. Agric. Exp. Station; 1950.
[
[
PRESSl Technology xxx (2011) xxx– xxx 7
37] Folin O, Ciocalteu V. On tyrsoine and tryptophane determinations in proteins.Journal of Biological Chemistry 1927;73:627–50.
38] Potvin G, Zhang Z. Strategies for high-level recombinant protein expres-sion in transgenic microalgae: a review. Biotechnology Advances 2010;28:910–8.
39] Kathiresan S, Chandrashekar A, Ravishankar GA, Sarada R. Agrobacterium-mediated transformation in the green alga Haematococcus pluvialis (Chloro-phyceae, volvocales). Journal of Phycology 2009;45:642–9.
40] Kathiresan S, Sarada R. Towards genetic improvement of commercially impor-tant microalga Haematococcus pluvialis for biotech applications. Journal ofApplied Phycology 2009;21:553–8.
41] Miki B, McHugh S. Selectable marker genes in transgenic plants: applications,alternatives and biosafety. Journal of Biotechnology 2004;107:193–232.
42] Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM. pGreen: a versatileand flexible binary Ti vector for Agrobacterium-mediated plant transformation.Plant Molecular Biology 2000;42:819–32.
43] Muratova A, Pozdnyakova N, Golubev S, Wittenmayer L, Makarov O, MerbachW, et al. Oxidoreductase activity of sorghum root exudates in a phenanthrene-contaminated environment. Chemosphere 2009;74:1031–6.
44] Gramss G, Voigt KD, Kirsche B. Oxidoreductase enzymes liberated by plant rootsand their effects on soil humic material. Chemosphere 1999;38:1481–94.
45] Otto B, Schlosser D, Reisser W. First description of a laccase-like enzyme in soilalgae. Archives of Microbiology 2010.
46] Hirai H, Kashima Y, Hayashi K, Sugiura T, Yamagishi K, Kawagishi H, et al.Efficient expression of laccase gene from white-rot fungus Schizophyllum com-mune in a transgenic tobacco plant. FEMS Microbiology Letters 2008;286:130–5.
47] Andreozzi R, Canterino M, Di Somma I, Lo Giudice R, Marotta R, Pinto G, et al.Effect of combined physico-chemical processes on the phytotoxicity of olivemill wastewaters. Water Research 2008;42:1684–92.
48] Lika K, Papadakis IA. Modeling the biodegradation of phenolic compounds by
icroalgae secreting the fungal laccase POXA1b reduce phenol content.enzmictec.2011.06.002
microalgae. Journal of Sea Research 2009;62:135–46.49] Haritash AK, Kaushik CP. Biodegradation aspects of polycyclic aromatic hydro-
carbons (PAHs): a review. Journal of Hazardous Materials 2009;169:1–15.50] Lei AP, Hu ZL, Wong YS, Tam NF. Removal of fluoranthene and pyrene by dif-
![[Production of insulin secreting cells from stem cells]](https://static.documents.page/doc/80x56/6361633b1b0df99ce10218b5/production-of-insulin-secreting-cells-from-stem-cells.jpg)
