Effects of Penicillium chrysogenum var. halophenolicum on kraft lignin : color stabilization and cytotoxicity evaluation

ORIGINAL ARTICLE E ec s o Penicillium ch ysogenum a . halophenolicum on k a lignin: colo s abiliza ion and cy o oxici y e alua ion Ma lene Reme ´dios 1 •Filomena A. Ca alho 2 •F ancisco J. Engui a 2 • Ca los Ca doso 3 •I o C. Ma ins 2 •Nuno C. San os 2 •Ana Lu ´cia Lei a ˜o 1 Recei ed: 11 No embe 2015 / Accep ed: 21 Ma ch 2016 ÓThe Au ho (s) 2016. This a icle is published wi h open access a Sp inge link.com Abs ac Wood indus ies and ag icul u al c ops gene - a e an inexhaus ible supply o by-p oduc s like lignin, which cons i u es an en i onmen al p oblem. Inc easing e o s ha e been done o ind new applica ions o lignin. One o hem is as a ood addi i e, bu i s chemical na u e makes i sensi i e o b owning which cons i u es a majo d awback o his ype o lignin applica ion. In he p esen s udy we a e documen ing how colo s abiliza ion o a comme cial k a lignin was achie ed a e he ea men wi h Penicillium ch ysogenum a . halophenolicum. In addi ion he ungal capaci y o emo e lignin is s udied oge he wi h he e ec o i s ea men on cy o oxici y o lignin. P. ch ysogenum a . halophenolicum was able o ans o m lignin, ensu ing i s colo s abili y o mo e han 24 mon hs. Dynamic ligh sca e ing and a omic o ce mic oscopy showed ha he ungus con ibu ed o homogenize pa icle size and hyd odynamic p ope ies in lignin suspensions wi hou inc ease he oxici y o e HeLa cells and human p ima y ib oblas s. These indings sug- ges new uses o k a lignin a e P. ch ysogenum a . halophenolicum ea men p o iding an e ec i e app oach o imp o e colo s abili y. Keywo ds Penicillium ch ysogenum a . halophenolicum Colo Lignin emo al Toxici y  T ans o ma ion In oduc ion Lignin is he second mos abundan na u al p oduc o ege al o igin, con e ing impe meabili y and s uc u al suppo o plan s (Bau hoo e al. 2008;Pe ´ ez and Mo - aleda-Mun ˜oz 2011). I s high-molecula weigh s uc u e is gene ally achie ed by dehyd ogena i e polyme iza ion o h ee p ima y hyd oxycinnamyl alcohols (monolignols): coni e yl alcohol (guaiacyl p opanol, G), couma yl alcohol (p-hyd oxyphenyl p opanol, H), and sinapyl alcohol (sy- ingyl p opanol, S), being classi ied as a h ee-dimensional amo phous polyme . As a majo componen o lignocel- lulosic ma e ial, lignin is ound in was es ha a e p oduced in la ge amoun s by many indus ies including o es y, ag icul u e and ood. Global wood consump ion is a ound 3.5 910 9 m 3 /yea , being b oadly applied o pulp and pape p oduc s, p oduc ion o uel and building ma e ials (Ma inez e al. 2005). This huge p oduc ion gene a es a la ge amoun o by-p oduc s wi h nega i e impac s o he aqua ic, and consequen ly o he e es ial ecosys em. The ad e se e ec is due no only o he o ganic load and oxici y o e luen s, bu also o he es he ically unaccep - able colo o wa e bodies la gely due o lignin and i s de i a i es. The b ownish da k colo a ion o wa e bodies could be he esul o enzyma ic ac i i ies o e a oma ic compounds p esen in lignocellulose-de i ed ma e ials. Among hem, polyphenol oxidases ca alyze he oxida ion &Ana Lu ´cia Lei a ˜o [email p o ec ed] 1 Depa amen o de Cie ˆncias e Tecnologia da Biomassa, Faculdade de Cie ˆncias e Tecnologia, Uni e sidade NOVA de Lisboa, Quin a da To e, Campus de Capa ica, 2829-516 Capa ica, Po ugal 2 Ins i u o de Medicina Molecula , Faculdade de Medicina, Uni e sidade de Lisboa, A . P o . Egas Moniz, 1649-028 Lisbon, Po ugal 3 Ins i u o Nacional do Ma e A mos e a, Unidade de Valo izac¸a ˜o dos P odu os da Pesca da Aquicul u a, L-IPIMAR, IP-INRB, A . B ası ´lia, 1449-006 Lisbon, Po ugal 123 3 Bio ech (2016) 6:102 DOI 10.1007/s13205-016-0414-x o o ho-dihyd oxyl phenols in he p esence o molecula oxygen o he co esponding o ho-quinones, which spon- aneously polyme ize o p oduce da k o b own pigmen s (Walke 1977). One o cu en challenges aced by a mo e en i on- men al- iendly science and indus y is o employ na u al esou ces and was e p oduc s maximizing economic pe - o mance. Indeed, new uses o lignin as uel, bioma e ials, biocides, bios abilise s, c op cul i a ions and animal eed is now a eali y (Lo a and Glasse 2002). Mo eo e , i was sugges ed ha pu i ied lignins may also b ing new heal h bene i s o animals (Bau hoo e al. 2008). Howe e , he colo s abili y o lignin p oduc s cons i u es a majo d awback o i s gene al use as addi i e. In his con ex , he colo p ese a ion o ood p oduc s du ing p ocessing and s o age is one o he main challenges o ood indus y; since hei o ganolep ic p ope ies a e an impo an equi- si e ha con ibu es decisi ely o ood accep ance, based on he ini ial pe cep ion o ood condi ion and deg ee o p ocessing, among o he cha ac e is ics (Lopez-Nicolas and Ga cia-Ca mona 2007). Due o i s a oma ic na u e and s uc u al complexi y, lignin is esis an o he a ack o he majo i y o mic oo - ganisms (Bau hoo e al. 2008;Pe ´ ez and Mo aleda-Mun ˜oz 2011). Indeed, in na u e, whi e- o ungi a e he only e i- cien lignin deg ade s, since hey ha e he abili y o com- ple ely mine alize his polyme , and consequen ly hey a e qui e impo an in he global u no e o ca bon om woody plan s (Wong 2009). The bes s udied a e Phane ochae e ch ysospo ium,Pleo o us os ea us,Bje kande a adus a, Pycnospo us cinnaba inus,Ce ipo iopsis sub e mispo a, Phlebia sp., o T ame es e sicolo (Pe ´ ez and Mo aleda- Mun ˜oz 2011). Meanwhile, he e a e o he ungi ha ha e he abili y o pa ially deg ade lignin. Fo ins ance, a Penicillium ch ysogenum s ain was able o using k a , o ganosol , and syn he ic dehyd ogena i e polyme ised lignins wi h low deg ada ion a es (Rod iguez e al. 1994). Despi e o ha , only a ew epo s ha e discussed lignin deg ada ion by Penicillium s ains (Polman e al. 1994; Rod iguez e al. 1994; Hao e al. 2006; Yada and Yada 2006;Dwi edi e al. 2011); howe e , as a as we know k a lignin emo al and colo s abiliza ion by impe ec ungi has no ye been desc ibed. We ha e p e iously cha ac e ized a halo ole an P. ch ysogenum s ain, P. ch ysogenum a . halopheno- licum, isola ed om a sal mine (Lei a ˜oe al.2012), and able o me abolize phenolic compounds unde osmo ic s ess (Lei a ˜oe al.2007; Guedes e al. 2011). In he p esen wo k, we aimed o s udy he e ec o P. ch ysogenum a . halophenolicum on k a lignin ans- o ma ion and colo s abili y, in es iga ing i his s ain has he po en ial o become a b owning con ol agen , o impa lignin wi h s able colo ha may p omo e i s use as an addi i e o bio echnological applica ions. Ma e ials and me hods S ain and cul u e condi ions P. ch ysogenum a . halophenolicum was used h oughou his s udy; his s ain was isola ed om a sal mine in Alga e, Po ugal, and p e iously cha ac e ized (Lei a ˜o e al. 2012). P. ch ysogenum a . halophenolicum was main ained a 4°C on nu ien aga pla es (Di co, BD diagnos ic sys ems, Hun Valley, MB, USA) wi h 2 % (w/ ) NaCl. P e-cul u es o cells we e ou inely ae obically cul i a ed (160 pm in a Ce oma Ò BS-T Incuba o , Sa o ius s edim bio ech, Goe ingen, Ge many) a 25 ±1°C in 100 mL o complex medium (MC: glucose, 30.0 g/L; NaNO 3 , 3.0 g/L; MgSO 4- 7H 2 O, 0.5 g/L; NH 4 Fe(SO 4 ) 2 12H 2 O, 10.0 mg/L; K 2 HPO 4 , 1.0 g/L; yeas ex ac , 5.0 g/L; NaCl, 20.0 g/L; pH 5.6). To in es iga e he abili y o ans o m k a lignin, he s ain was cul i a ed in 500-mL lasks con aining 100 mL o MC du ing 68 h. Cells we e collec ed by cen i uga ion and washed in 0.85 % (w/ ) o NaCl. A 15 % o he p e- inoculum was inocula ed in modi ied Jansheka medium (Jansheka e al. 1982) con aining NaNO 3 (1.00 g) in subs i u ion o NO 3 NH 4 (0.496 g) and wi hou i amin solu ion and ni ilo iace a e as a componen o ace ele- men solu ion and amended wi h 1700 mg/L k a lignin (alkali, low sul ona e con en was ob ained om Sigma- Ald ich, S . Louis, MO, USA). P. ch ysogenum a . halophenolicum was ae obically incuba ed (160 pm in he Ce oma Ò BS-T Incuba o ) in he da k du ing 96 h, a 25 °C. Th ee eplica es we e used. Abio ic assays we e pe o med in pa allel wi h uninocula ed lasks (duplica es) as nega i e con ols. A e egula imes o cul u e, cells we e ha es ed and 12 mL o supe na an we e kep ozen a -40 °C un il lignin de e mina ion assays, while app oxima ely 12 mL we e main ained a oom empe a u e o lignin colo s a- bili y expe imen s. Mic obial d y biomass was es ima ed g a ime ically by he me hod desc ibed by Gun he e al. (1995). Lignin quan i ica ion and colo s abili y e alua ion The comme cial k a lignin is an ideal subs a e o assessing colo s abili y and biological ans o ma ion o lignin. Fi s ly, he use o comme cial alkali lignin a oids he conce ns abou chemical composi ion, he isola ion me hod, o pu i y, since i is a comme cial p oduc . Sec- ondly, comme cial alkali lignin was employed in o he s udies o assess chemical e ec on he s uc u e o lignins (Kadam and D ew 1986; Supa no e al. 2005; Yuan e al. 2010; DeAngelis e al. 2011; Geo ge e al. 2011; Huang e al. 2013). 102 Page 2 o 10 3 Bio ech (2016) 6:102 123 K a lignin colo s abili y expe imen s we e conduc ed in a lab oom i ed wi h a chambe empe a u e se a 22 °C wi hou any special s o age condi ion. A e a ime o 7, 50 days and 7 mon hs, samples we e il e ed be o e colo measu emen , as desc ibed in he sec ion o colo measu emen . All assays we e pe o med in n=3 epe i- ions. The mean alues and s anda d de ia ions we e e alua ed using analysis o a iance (ANOVA). Lignin concen a ions we e quan i ied by spec opho o- me ic abso p ion a 205 nm in a Spekol Ò 1500 UV VIS Spek alpho ome e (Analy ic Jena AG, Ge many). A omic o ce mic oscopy A NanoWiza d II a omic o ce mic oscope (JPK Ins u- men s, Be lin, Ge many), moun ed on he op o an Axio- e 200 in e ed mic oscope (Ca l Zeiss, Jena, Ge many) was used o imaging he samples. The AFM head is equipped wi h a 15-lm z- ange linea ized piezoelec ic scanne and an in a ed lase . Samples we e dilu ed o 1:200 in Milli-Q wa e and deposi ed on eshly clea ed musco i e mica o 20 min. A e subsequen washes, he sample was allowed o ai d y a oom condi ions. Imaging o he samples componen s we e pe o med in ai apping mode ( o he 1s day ungal ea ed samples) and in con- ac mode ( o he 4 h day ungal ea ed samples). Oxi- dized sha pened silicon ips (ACL ips om Applied Nanos uc u es, CA) wi h a ip adius o 6 nm, esonan equency o abou 190 kHz and sp ing cons an o 45 N/m we e used o he measu emen s. Imaging pa ame e s we e adjus ed o minimize he o ce applied on he scanning o he opog aphy o he samples. Scanning speed was op i- mized o 0.4 Hz (on he i s day o ea men sample) and 0.8 Hz (on he ou h days o ea men sample), and acquisi ion poin s we e 512 9512 and 360 9360, espec i ely. Imaging da a we e analyzed wi h he JPK image p ocessing .3 (JPK Ins umen s). The wid h and heigh o he samples we e calcula ed om he c oss sec- ion plo s. All complex dimensions measu emen s we e pe o med using he Gwyddion so wa e (Czech Me ology Ins i u e, B no, Czech Republic), e sion 2.19. Dynamic ligh sca e ing (DLS) measu emen s Dynamic ligh sca e ing expe imen s we e ca ied ou a 25 °C on a Mal e n Ze asize Nano ZS (Mal e n, UK), wi h a backsca e ing de ec ion a 173°, equipped wi h a He–Ne lase (k=632.8 nm), using glass cu e es wi h ound ape u e. K a lignin samples we e dilu ed 1/10 in MiliQ wa e The samples we e le equilib a ing o 15 min a 25 °C be o e each measu emen s se (10 measu emen s; each one being he a e age o 10 uns, wi h 10 s pe un). No malized in ensi y au oco ela ion unc ions we e analyzed using he CONTIN me hod (P o enche 1982a,b), yielding a dis ibu ion o di usion coe icien s (D). The measu ed D(m 2 /s) was used o he calcula ion o he hyd odynamic diame e , D H (nm), h ough he S okes– Eins ein ela ionship (Be ne and Peco a 1990): DH¼jT 3pgD whe e jis he Bol zmann cons an (J/K), T he absolu e empe a u e (K), and g he medium iscosi y (Pa s). The da a we e s a is ically analyzed wi hin each se o 10 measu emen s by obse ing he a e age and s anda d de ia ion, and disca ding ou lie s. The a e age wi hou ou lie s became close o he median in all he size poin s. Colo measu emen Samples colo was de e mined using a Macbe h eye 3000 colo ime e . The colo in he CIELAB sys em is cha ac- e ized by h ee pa ame e s, L*,a* and b*. The ligh ness alue (L*), aking alues om 0 % (black) o 100 % (whi e), a* om g een (-a) o ed (?a) and b* om blue (-b) o yellow (?b). The L*,a* and b* colo coo dina es o each g oup o samples we e measu e a e s abili y expe imen . These alues we e hen used o calcula e he colo change DE* as a unc ion o he s abili y expe imen du a ion acco ding o he ollowing equa ions: DL¼L L i Da¼a a i Db¼b b i DE¼ðDL2þDa2þDb2Þ1=2 whe e DL*, Da* and Db* a e he changes be ween he ini ial (i) and he inal ( ) alues and DE* co esponds o a colo change. The colo e olu ion assays we e pe o med using he measu emen s a ime 7 days as s anda ds, which co espond o he i s measu emen (i) ha was made a e he ungal ea men , mycelia emo al and s o age a 22 °C. Cy o oxici y e alua ion Quan i ica ion o he oxic e ec s o k a lignin and ungal ea ed samples a he cellula le el was pe o med by he use o Alama Blue Ò es o quan i y HeLa and human p ima y skin ib oblas s (Co iell Ins u u e o Medical Resea ch, e . GM05565) iabili y (In i ogen) using a modi ied e sion o an al eady desc ibed p o ocol (Al-Nasi y e al. 2007). B ie ly, HeLa and ib oblas cells we e pla ed in 96 well pla es a 10 5 cells pe well in RPMI medium con aining 10 % FBS, and incuba ed a 37 °C and 3 Bio ech (2016) 6:102 Page 3 o 10 102 123 5%CO 2 du ing a leas 8 h o allow cell a achmen o he pla e su ace. A e his incuba ion ime, RPMI was sub- s i u ed by Op i-MEM medium o a oid se um in e e ence wi h he assay. Cells we e hen exposed o di e en dilu- ions o he P. ch ysogenum a . halophenolicum cul u e supe na an s, p e iously bu e ed wi h 10 9PBS and s e ilized by il a ion hough 0.22 lm memb anes. A e 48 h, HeLa and ib oblas cells we e washed wice wi h PBS and incuba ed du ing 1 h a 37 °C wi h Alama Blue Ò ollowing he ecommenda ions om he manu ac u e . Cell iabili y was quan i ied by de e mina ion o he luo- escence o Alama Blue Ò a 590 nm a e exci a ion a 530 nm in a luo escence pla e eade (TECAM In ini e M200). EC 50 we e de e mined by non-linea eg ession using an equa ion o a sigmoid cu e (G aphPad P ism 5.02). Resul s and discussion Lignin emo al To de e mine he abili y o P. ch ysogenum a . halophe- nolicum o emo e k a lignin, he mic oo ganism was cul u ed in he p esence o 1700 mg/L o comme cial k a lignin. Since no abio ic loss o lignin was de ec ed in con ol samples, he dec ease o lignin concen a ion in he p esence o ungus mus be due o i s biological ac ion. The g ow h o P. ch ysogenum a . halophenolicum and lignin emo al is depic ed in Fig. 1. A he ini ial concen a ion o 1700 mg/L o k a lignin no lag phase was obse ed. A lignin emo al o 68.0 % was achie ed a e 96 h o ungal ea men , showing a clea co ela ion wi h ungal g ow h. Indeed, da a indica e ha he e we e wo peaks in biomass, co esponding o exponen ial g ow h phase. The i s one should be due o he p esence o glucose in he cul u e media composi ion besides lignin, since he ungal inocu- lum was p epa ed in a complex medium wi h glucose, and i s enzyma ic sys em was al eady ac i e and eady o use his subs a e. This ac was co obo a ed by he absence o lag phase on ungal g ow h. A e 24 h o cul u e, a sig- ni ican inc ease on ungal biomass and a concomi an dec ease in he lignin concen a ion we e obse ed, indi- ca ing ha P. ch ysogenum a . halophenolicum g ow h was due o lignin ans o ma ion. In gene al, mos o ligninoly ic ungi epo ed equi e a high le el o con- sump ion o easily me abolized cosubs a e o be e ec i e in he ligninolysis. The P. ch ysogenum a . halopheno- licum was able o emo e mo e han 1000 mg/L o k a lignin in he i s 24 h o ba ch cul u e, which is qui e in e es ing; whe eas he he e opolyme concen a ion did no signi ican ly dec eased du ing he las h ee days o ungal ea men , p obably due o he C–C bonds o alka- line lignin ha a e highly esis an o hyd oly ic b eaking (Yuan e al. 2010). K a lignin is en iched wi h guaiacyl g oups, whe e he C5 posi ion in he a oma ic ing ep e- sen s he mos abundan C–C linkage in lignin molecules (El Mansou i e al. 2011). Fungi a e he only mic oo - ganisms ex ensi ely s udied o he deg ada ion o lignin. A s ain o Penicillium ch ysogenum is able o deg ade some syn he ic and na u al lignins, and mine alize 7.9 % o [ 14 C b ] DHP in 29 days (Rod iguez e al. 1996). Recen ly se e al s udies we e published using com- me cial k a lignin. Among hem, Huang e al. epo ed ha 500 and 700 mg/L o comme cial k a lignin is deg aded by Bacillus pumilus and Bacillus a ophacus, espec i ely, a e 18 days o incuba ion (Huang e al. 2013). A compa a i e s udy o na u al and comme cial k a lignin deg ada ion by Ci obac e sp. was desc ibed, showing i s highe capaci y o use k a lignin han com- me cial k a lignin (252 and 186 mg/L, espec i ely) (Chand a and Bha aga a 2013). Ou esul s indica e ha P. ch ysogenum a . halophenolicum e icien ly emo ed k a lignin. E ec o ungal ea men on he e olu ion o lignin colo To e alua e he e ec o P. ch ysogenum a . halopheno- licum on lignin colo , scala pa ame e s (L*,a*, and b*) we e de e mined, a e 7 days o s o age, in samples wi h di e en imes o ungal ea men . The ini ial L* alue o un ea ed sample was 56.45. Fungal ea men did no induce any signi ican change in L* alues (Table 1). A e 96 h o ungal ea men he L* alue was 56.88, indica ing ha he componen s abso bing isible ligh emain cons an . Fig. 1 K a lignin emo al by P. ch ysogenum a . halophenolicum du ing 96 h unde ae obic condi ions. Squa es cell g ow h; ci cles, k a lignin concen a ion. Da a shown ep esen s a e age o ipli- ca es ±s anda d de ia ions 102 Page 4 o 10 3 Bio ech (2016) 6:102 123 In he samples ea ed wi h he ungus du ing 24 h he a* alues sligh ly inc eased, possibly due o he p esence o deg ada ion and/o oxida ion p oduc s, ollowed by a dec ease a la e cul u e imes. Ne e heless, he di e en imes o cul u e led o lowe alues o a*, wi h he ini ial g een colo s ill emaining a e 96 h o ea men (Table 1). These esul s a e compa ible wi h chemical changes o lignin. The b* alues o samples inc ease a e ungal ea - men . The inc ease in yellowness could be caused by he low-molecula -weigh phenolic compounds ob ained by he ac ion o ungal enzymes. AFM s udies In na u e, lignin is an amo phous polyme ic ma e ial o en associa ed o cellulose ibe s as main cons i uen s o plan cell walls. Since he exac s uc u e o lignin i sel is no known (Chaka and Ragauskas 2004; Geo ge e al. 2011), a omic o ce mic oscopy has been applied o cha ac e ize he opog aphy and oughness o lignocellulosic ma e ials in hei na u al o ms (Chundawa e al. 2011). The AFM scanning images o lignin samples wi h one (a, b) and 4 days (c, d) o ea men wi h P. ch ysogenum a . halophenolicum a e shown on Fig. 2. F om he igu e, i can be e iden ly no iced ha ungal ea men on he lignin samples leads o mo phological changes wi hin he polyme . F om ai apping mode AFM e o (a) and heigh (b) images o he sample ea ed h ough only 24 h, i could be seen ha i is e y he e o- geneous in size. Opposing his, he lignin sample wi h 96 h o ea men is much mo e homogenous and only one size popula ion could be isualized. To quan i a i ely analyze AFM heigh images o hese popula ions, we measu ed he diame e and he heigh o hei su ace p o iles (Fig. 2b, d, igh images). C oss sec ion analysis o each ea u e on he images was done, yielding dis inc p o iles. Examples o hese c oss sec ions a e also shown (Fig. 2b, d, le ima- ges). The analysis indica es ha he sample wi h 96 h o ea men had a unique popula ion, wi h an a e age diam- e e o 321.2 ±53.7 nm and heigh o 28.8 ±8.6 nm (n=11). We could dis inguish h ee di e en popula ions on he lignin sample wi h only 24 h o ungal ea men , wi h di e en dimensions: 42.5 ±6.0 nm (diame e ) and 5.1 ±1.0 nm (heigh ) on popula ion 1 (n=9); 124.2 ±17.3 nm (diame e ) and 16.8 ±2.7 nm (heigh ) on popula ion 2 (n=16); and, 221.9 ±36.9 nm (diame- e ) and 34.8 ±10.9 nm (heigh ) on popula ion 3 (n=23). The e we e no signi ican di e ences in he isualized oughness o bo h analyzed samples (Fig. 2b, d, igh images). F om he calcula ed dimensions o he h ee popula ions o he 24 h ea men sample, we could hypo hesize ha popula ions 2 and 3 we e o med by oligome s o he lignin s uc u es o popula ion 1. A e 96 h o ea men , he subs an ial inc ease in size o he lignin pa icles achie ed on he en i e popula ion e eals he exis ence o polyme - iza ion o majo agg ega ion phenomena. DLS analysis DLS expe imen s o un ea ed lignin and ungal ea ed samples a e shown in Table 2. Un ea ed lignin showed a g ea a ia ion in pa icle sizes and poo ly esol ed peaks, wi h h ee majo ones obse ed, one co esponding, oughly, o 6–20 nm sized species, he o he o 20–1000 nm sized species and a inal one co esponding o species well o e 3 lm. Taking only he i s wo (which a e be e esol ed), he maxima o he peaks co espond o a e age hyd odynamic diame e s (D H ) o 16.6 nm (sca - e ing in ensi y 34.4 %) and 629.7 nm (sca e ing in ensi y 65.6 %). Upon 24 and 48 h o ea men wi h he ungus, he sample sizes become mo e uni o m and o e all smalle . A e 24 h o ea men h ee majo peaks we e obse ed, bu be e esol ed han in he con ol, since he i s one co esponds mos ly o, app oxima ely, 10–20 nm sized species, he second one o 150–400 nm sized species and he hi d one o species wi h mo e han 2 lm. Taking again only he i s wo (which a e much be e esol ed), he maxima o he peaks co esponded o a e age hyd ody- namic diame e s (D H ) o 15.5 nm (sca e ing in ensi y 44.9 %) and 301.3 nm (sca e ing in ensi y 42.3 %). The Table 1 E olu ion o L* and a* and b* coo dina es o k a lignin samples a e he absence and p esence o P. ch ysogenum a . halophe- nolicum. A e ea men , ungal micelia we e emo ed and he samples we e s o ed a 22 °C du ing 7 days be o e colo es ima ion (n=3) Fungal ea men (h) L* a* b* Con ol 56.45 ±0.04 a -0.81 ±0.03 a 1.87 ±0.03 a 24 56.53 ±0.22 a -0.74 ±0.02 a 2.10 ±0.15 ab 48 56.82 ±0.02 b -0.91 ±0.06 ab 2.38 ±0.01 c 72 56.64 ±0.10 a -0.98 ±0.02 b 2.18 ±0.02 b 96 56.88 ±0.05 b -0.87 ±0.04 a 2.47 ±0.03 c Di e en le e s ( e ically) indica e signi ican di e ences (p 0.05) 3 Bio ech (2016) 6:102 Page 5 o 10 102 123 ea men wi h P. ch ysogenum a . halophenolicum appea ed o elimina e he la ge pa icles (cen e ed a ound 629.7 nm) esul ing in much smalle s uc u es, wi h abou hal o he hyd odynamic diame e o he con ol samples (hyd odynamic diame e cen e ed a ound 301.3 nm). These esul s suppo he hypo hesis ha he ungus had he abili y o use di e en ly sized lignin molecules, as desc ibed ecen ly by Wang e al. in he lignosul ona e biodeg ada ion p ocess by Sphingobac e ium sp. HY-H (Wang e al. 2013). The hi d peak did no ha e a good esolu ion, howe e , i is clea ha he o e all sca e ing pa icles size dec eased, and ha also in pa allel, he amoun o pa icles wi h sizes abo e 1 lm is diminished, esul ing on an o e all ex ensi e educ ion and uni- o miza ion in pa icles size. The same p o ile was obse ed a e 96 h o ea men , bu in such an ex en ha he sample can be said o be essen ially homogeneous, wi h one peak a 245.7 nm (sca e ing in ensi y 92.4 %), wi h obse ed easonable ag eemen wi h he AFM da a (321.2 ±53.7 nm). A small un esol ed peak o pa icles wi h mo e han 1 lm was also isible, bu wi h a limi ed ac ion o he in ensi y. Mean- while, in he un ea ed samples h ee peaks clea ly emains a e 96 h. These esul s we e consis en wi h he Fig. 2 AFM scanning images o k a lignin wi h 1 and 4 days o ea men wi h P. ch ysogenum a . halophenolicum. Ai apping mode AFM e o (a) and heigh (b) images o k a lignin wi h 1 day o ungal ea men (ho izon al scale 1lm91lm; heigh scale up o 27.6 nm). Ai con ac mode AFM e o (c) and heigh (d) images o k a lignin wi h 4 days o ungal ea men (ho izon al scale: 700 nm 9700 nm; heigh scale up o 66.5 nm). C oss sec ion analysis o he AFM heigh images could also be pe o med. Th ee examples o c oss sec ions o he di e en isualized popula ions (b igh image) and one example o a homogeneous popula ion (d, igh image) o he k a lignin p o iles a e shown. Wi h his ype o analysis, lignin size, heigh , shape and oughness can be de e mined Table 2 A e age pa icles size o ungal ea men samples (hyd odynamic diame e ) es ima ed by dynamic ligh sca e ing (DLS) Incuba ion ime (h) Samples Peak 1 Peak 2 Peak 3 24 Fungal ea men 15.5 nm (44.9 %) 301.3 nm (42.3 %) 3590.0 nm (12.8 %) Con ol 16.6 nm (34.4 %) 629.7 nm (65.6 %) No majo peak 48 Fungal ea men 15.2 nm (5.0 %) 191.5 nm (80.6 %) 3551.0 nm (12.8 %) Con ol 17.0 nm (29.5 %) 718.4 nm (64.0 %) 4202.0 nm (6.5 %) 96 Fungal ea men No peak 245.7 nm (92.4 %) 4397.0 nm (7.6 %) Con ol 13.0 nm (35.3 %) 250.5 nm (43.0 %) 2053.0 nm (21.7 %) Size ep esen s he peak a e age based on size dis ibu ion by olume; he pe cen ages ep esen he peak a ea, da k shadowed a ea highligh s homogeneously sized samples (e.g., samples wi h o e 80 % o he pa icles included wi hin a single size dis ibu ion peak) 102 Page 6 o 10 3 Bio ech (2016) 6:102 123 hypo hesis ha he p esence o P. ch ysogenum a . halophenolicum could p omo e he polyme iza ion o lig- nin in o well-de ined and homogenously sized samples. O e all, and in spi e o he small di e ences be ween obse ed AFM and DLS pa icles size due o he di e en p ecision le el o he wo echniques (DLS size es ima ion may be dis o ed by la ge pa icles, while he la ening o he sample o a discoidal shape on he AFM subs a e and/o con olu ion wi h he AFM ip dimensions also a ec s size de e mina ions), he esul s ob ained using AFM opog aphic images we e in acco dance wi h hose achie ed ia DLS, showing ha no only he low-molec- ula -weigh o ganic compounds, bu also he polycyclic a oma ic compounds can be deg aded by P. ch ysogenum a . halophenolicum wi h he concomi an uni o miza ion o he lignin samples in o a single sized species a e 96 h o ea men wi h he ungus. Lignin colo s abili y As desc ibed abo e, DLS and AFM show ha , a e 96 h o ungal ea men , he lignin samples become highly homogenized in e ms o size, es ima ed o be below 390 nm, he app oxima e lowe limi o ligh de ec ion by he human eye (S a 2005). This ac p e en s, among o he phenomena ha may in e e e wi h colo s abili y, he occu ence o ligh sca e ing phenomena. This can occu in ela i ely dense solu ions, especially, i hey o m di - e en ly sized pa icles ha migh e en ually modi y he colo pe cep ion. Howe e , in his pa icula case, ligh sca e ing by hese uni o mly sized lignin pa icles ( 390 nm) would no be a p oblem since i would be al eady in he ul a iole ligh spec um, in isible o he human eye, which helps s eng hen he po en ial o lignin ea ed samples as colo s abilize . To u he e alua e his po en ial, he ligh ness alues o samples ea ed wi h he P. ch ysogenum a . halophenolicum a e 50 days and 7 mon hs unde s abili y assay condi ions, we e compa a- ble o sligh ly highe han he ini ial L* alues (Table 3). Mo eo e , colo s abili y inc eased a e 50 days o s o age, since he o e all colo change (DE*) alues o he samples ea ed wi h he ungus we e smalle han hose o he con ol samples (Table 3). Fo ea ed ungal samples, he colo change o 24 h was 0.55. The colo change alue was 0.17 o 96 h in he ea ed ungal samples. These esul s showed a ace isual change in samples ea ed mo e han 48 h wi h P. ch ysogenum a . halophenolicum, acco ding o he scale p oposed by Di ckx e al. (Di ckx e al. 1992). Fu he - mo e, he o e all colo change dec eased as ungal con- ac ime inc eased, sugges ing heigh ened colo s abili y. A he end o 7 mon hs s o age, a sligh inc ease o DE* in he ea ed samples was obse ed, when compa ed o he alues ob ained a 50 days o s o age. Ne e heless, in all samples ea ed wi h ungus a e 7 mon hs o s o age, he o e all colo change was lowe han ha o he con- ol wi h 50 days. Fu he mo e, in he cul u e ea ed du ing 96 h no signi ican colo change was obse ed be ween 7 and 26 mon hs o s o age, which is in excellen ag eemen wi h he DLS and AFM size es ima ion showing s able uni o m molecules (a e ungal ea - men ). O e all, his s ongly suppo s he hypo hesis ha he ea men wi h P. ch ysogenum a . halophenolicum s abilizes he lignin molecules p ope ies, especially in wha ega ds o colo and o i s possible use as a colo modi ies/s abilize . Cy o oxici y o k a lignin be o e and a e ungal ea men The e alua ion o he po en ial oxic e ec s o lignin me aboli es was conduc ed by he de e mina ion o he ela i e p opo ion o iable cells, by he Alama Blue Ò assay. This es is based on he con inuous con e sion by iable cells o esazu in, a non- luo escen cell pe meable pigmen , o eso u in, a luo escen compound ha can be de e mined by luo ime ic assay, yielding a quan i a i e measu emen o cell su i al. We used HeLa cells and human p ima y skin ib oblas s iabili y as an indica o o he po en ial oxic e ec s o lignin and i s me aboli es. The EC 50 alues o k a lignin showed ha i had cy o oxic e ec s, bu only a e y high concen a ions (Fig. 3). In incuba ions wi h ib oblas s, he EC 50 o cy o oxici y was highe han HeLa cells (EC 50 o 5177 ±1726 and 1223 ±328 mg/L, espec i ely in ib oblas s and HeLa cells). A he concen a ions o 750, 1000, 2000 mg/L, we obse ed a signi ican dose-dependen inc emen in he HeLa cells iabili y. A good co ela ion be ween he cy o oxic e ec s on HeLa cells and on ib oblas s cells a e 48 h was obse ed ( =0.9775 and =0.9509, espec i ely). As i is p e iously epo ed he di e ences be ween a cance cell line and p ima y ib oblas s can be a ibu ed o di e ences in cell sensi i i y o he compound Table 3 E olu ion o colo di e ence (DE*) a e 50 days, 7 and 26 mon hs o s o age Fungal ea men (h) DE* 50 days/ 7 days 7 mon hs/ 7 days 26 mon hs/ 7 days Con ol 0.59 ±0.52 1.20 ±0.62 n.d. 24 0.55 ±0.19 0.90 ±0.67 1.06 ±0.19 48 0.26 ±0.01 0.60 ±0.04 n.d. 96 0.17 ±0.04 0.61 ±0.05 0.68 ±0.05 n.d. no de e mined 3 Bio ech (2016) 6:102 Page 7 o 10 102 123 ha is assayed and would be mainly ela ed wi h he cell di ision a e (Uga ondo e al. 2008). To assess i lignin me aboli es gene a ed om P. ch ysogenum a . halophenolicum biological ac i i y a e non oxic o HeLa and ib oblas cells, new expe imen s we e done. The o iginal samples we e dilu ed in cell cul- u e medium be o e o cy o oxici y es ing o ob ain a concen a ion ange simila o hose whe e s anda d lignin concen a ion begins o be oxic o he es ed cells. Resul s shown in Fig. 4clea ly indica e ha bo h HeLa and ib oblas cells iabili y we e no signi ican ly modi ied by he exposu e o P. ch ysogenum a . halophenolicum, in compa ison wi h he s anda d lignin a he same concen- a ions, indica ing ha he lignin me aboli es gene a ed by P. ch ysogenum a . halophenolicum we e no cy o oxic. These esul s we e qui e impo an since i is epo ed ha many dyes a e belie ed o be oxic, some o hem as a esul o mic obial me abolism (Cou o 2009). Addi ionally, Penicillium species a e known o p oduce a high abun- dance o seconda y me aboli es including myco oxins such as oc a oxin A, ci inin, e ucosidin, pa ulin, among o he s, which ha e di e en deg ees o oxici y on a ge o gans o a e ca cinogenic (Cha ´ ez e al. 2011). The e o e, i is impo an o assess he cy o oxic e ec s o lignin samples a e ea ed wi h P. ch ysogenum a . halophe- nolicum o disca d any possible cy o oxic p ope ies. Despi e he po en ial o biological haza d is low o ungi con e ed eed as so a , assess cell iabili y is o majo impo ance o ensu e consume heal h and sa e y. The p esen da a indica ed ha P. ch ysogenum a . halophenolicum, in his es condi ions, did no inc ease lignin oxici y, showing ha k a lignin can be used o e an e ec i e concen a ion ange ha is sa e o no mal and cance cell lines s udied. The en i onmen al p o ec ion agencies a e becoming mo e es ic i e ega ding wa e discha ge om indus ial e luen s. Se e al indus ies, besides he classical p ima y and seconda y ea men s, use physical and chemical e - ia y ea men s o emo e he colo cause by lignin and lignin de i a i es, which a e expensi e and no always lead o high pe o mance. Biological echnology is a easible and p omising al e na i e. Fungi ha e a ac ed a g ea deal Fig. 3 K a lignin dose esponse cu es using he Alama Blue assay. Cu es a e examples ob ained by a e age o i e expe imen al eplica es and illus a e he esponse o he human p ima y skin ib oblas s (a) and HeLa cells (b) Mock Lignin s anda d 24 h 48 h 96h 0 20 40 60 80 100 Cell su i al (%) BA Mock Lignin s anda d 24h 48h 96 h 0 20 40 60 80 100 Cell su i al (%) Fig. 4 HeLa and ib oblas cells iabili y quan i ied by Alama Blue assay a e incuba ion wi h ungal ea ed and un ea ed k a lignin p epa a ions. aHeLa cells, bhuman p ima y skin ib oblas s. Mock, un ea ed cells; K a lignin s anda d, cells incuba ed wi h soluble lignin a a concen a ion o 50 mg/L; 24, 48 and 96 h ep esen he ime o ungal ea men . Cell su i al was calcula ed as he a io o luo escence be ween sample o s anda d and mock 102 Page 8 o 10 3 Bio ech (2016) 6:102 123 o in e es as po en ial biomass and ecalci an compounds deg ade s such as lignin due o hei abili y o p oduce a b oad di e si y o ex acellula ligninoly ic enzymes some o hem wi h lack speci ici y o a pa icula subs a e. I whi e- o ungi comple ely mine alize lignin, o he ungi and bac e ia lead o he p oduc ion o b own pigmen s which limi s he p ac ical use o such ag o esidues. Meanwhile, al hough lignin was gene ally conside ed o be nu i ionally ine , new scien i ic da a on heal h-p o ec i e mechanisms o ce eals endo se he opposi e idea (Fa de 2010). Meis e ad anced a new possible use o modi ied lignin o pe and human ood as oughage, a ibe sou ce, o a cance p o ec ion agen (Meis e 2002). Recen ly, To o a e al. desc ibed he use o k a lignin as he aw ma e ial o mic ocapsula ion p ocesses assembled by ul asound in o lignin mic ocapsules o s o age and elease Couma in- 6 (To o a e al. 2014). Se e al esea che s poin ed ou ha a lignin wi h a mo e cons an s uc u e and size will enhance i s po en ial u iliza ion in high- alue p oduc s (No g en and Edlund 2014; Qu e al. 2015). In summa y, his wo k showed ha P. ch ysogenum a . halophenolicum was able o ans o m k a lignin, dis- playing a highe capaci y o g ow on lignin subs a es. AFM and DLS assays indica ed ha k a lignin bio ans- o ma ion p oceeds ei he ia low-molecula phenolic compounds o oligome s. P. ch ysogenum a . halophe- nolicum ea men esul ed in a s abiliza ion o comme cial soluble k a lignin colo . I is also ema kable ha lignin colo s abili y was eco ded o mo e han 24 mon hs a 22 °C unde ligh condi ions o s o age. These esul s we e compa ible wi h he hypo hesis ha P. ch ysogenum a . halophenolicum could be a po en ial ool o s abilizing lignin agains colo change by ans o ming he he e oge- neous lignin composi ion in a mo e homogenous s uc u e. The inc eased lignin s abili y was achie ed wi hou inc easing oxici y o e HeLa and ib oblas cells. These indings could p omo e he applica ion o P. ch ysogenum a . halophenolicum o he de elopmen o lignin- o i ied o mula ha would imp o e ood ibe con en . Compliance wi h e hical s anda ds Con lic o in e es The au ho s decla e ha hey ha e no con lic o in e es in he publica ion. Open Access This a icle is dis ibu ed unde he e ms o he C ea i e Commons A ibu ion 4.0 In e na ional License (h p:// c ea i ecommons.o g/licenses/by/4.0/), which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided you gi e app op ia e c edi o he o iginal au ho (s) and he sou ce, p o ide a link o he C ea i e Commons license, and indica e i changes we e made. Re e ences Al-Nasi y S, Geusens N, Hanssens M, Luy en C, Pijnenbo g R (2007) The use o Alama Blue assay o quan i a i e analysis o iabili y, mig a ion and in asion o cho ioca cinoma cells. Hum Rep od 22:1304–1309 Bau hoo B, Ruiz-Fe ia CA, Zhao X (2008) Pu i ied lignin: nu i ional and heal h impac s on a m animals—a e iew. Anim Feed Sci Technol 144:175–184 Be ne BJ, Peco a R (eds) (1990) Dynamic ligh sca e ing—wi h applica ion o chemis y, biology and physic. 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