Bioprospecting for novel biosurfactants and biosurfactant producing bacteria in wastewater

Thesis (PhD)--Stellenbosch University, 2017.ENGLISH ABSTRACT: Biosurfactants are surface active amphiphilic compounds, synthesised by numerous bacteria, fungi and yeast. They are known to exhibit broad spectrum antimicrobial activity and are currently applied as antimicrobial agents, antiadhesives, foaming agents, emulsifiers etc. in the cosmetic, food, pharmaceutical and biotechnology industries. The primary aim of the study was thus to bioprospect for novel biosurfactants and biosurfactant-producing bacteria in a wastewater treatment plant (WWTP). Wastewater was selected as it is a suitable environment for the growth of diverse microorganisms and the presence of numerous organic and inorganic contaminants were postulated to enable the flourishing of biosurfactant-producing microorganisms. Chapter 1 then outlined literature pertaining to biofurfactants, their characterisation and mode of action, amongst many other topics. Chapter 2 of this study focused on the distribution and diversity of biosurfactant-producing bacteria isolated from wastewater. Wastewater samples were collected from various points of the Stellenbosch WWTP and culturable isolates were screened for possible biosurfactant production using the oil spreading and drop collapse methods. Surface tension and emulsification activities were then used for the partial characterisation of the produced biosurfactant compounds. Thirty-two of the 667 bacterial isolates were regarded as biosurfactant producers and were classified into the Aeromonadaceae, Bacillaceae, Enterobacteriaceae, Gordoniaceae and the Pseudomonadaceae families using 16S rRNA analysis. Bacillus and Pseudomonas were among the most dominant genera, which constituted 21.8% (7/32) and 12.5% (4/32) of all isolates, respectively. High surface tension reduction of the growth medium (71.1 mN/m) was also observed for the Bacillus ST34 (34.4 mN/m) and the Pseudomonas ST5 (32.3 mN/m) isolates. In addition, the Bacillus ST34 and Pseudomonas ST5 isolates tested positive for the sfp and rhlB genes involved in the biosynthesis of surfactin and rhamnolipid biosurfactants. While numerous studies have reported on the isolation of biosurfactant-producing bacteria from contaminated soil and terrestrial environments, the current study indicated that municipal wastewater could be exploited for the isolation of diverse biosurfactant-producing bacterial strains. In chapter 3, 32 biosurfactant-producing isolates were then genotypically differentiated utilising repetitive element PCRs (rep PCRs) [targeting the repetitive extragenic palindromic (REP) and the BOX element sequences]. This molecular differentiation was performed as the genetic diversity amongst bacterial species is known to produce different concentrations and proportions of various homologues of biomolecules such as biosurfactants and antibiotics. With the use of the conventional PCR assays, some of the isolates were identified as Bacillus subtilis (n = 4), Aeromonas hydrophila (n = 3) and Bacillus amyloliquefaciens (n = 2), amongst others. These bacterial species were genotypically differentiated into four, three and two sub-species (strains), respectively, utilising rep PCRs. The BOX AIR and REP primers utilised for rep PCR in the current study thus provided a powerful tool to discriminate between biosurfactant-producing bacterial isolates identified as the same species. Chapter 4 focused on the characterisation and antimicrobial activity of the biosurfactant extracts produced by the isolates B. amyloliquefaciens ST34 and Pseudomonas aeruginosa ST5. Crude biosurfactants from ST34 and ST5 culture broth were extracted using solvent extraction based methods. Thereafter, the high resolution ultra-performance liquid chromatography (UPLC) coupled to electrospray ionisation mass spectrometry (ESI-MS) method, developed in the current study, was utilised to characterise the produced compounds. Results indicated that B. amyloliquefaciens ST34 primarily produced the C13, C14, C15 and C16 surfactin analogues when grown on mineral salt medium (MSM) supplemented with glycerol. For P. aeruginosa ST5, high resolution ESI-MS linked to UPLC confirmed the presence of dirhamnolipid congeners, specifically Rha-Rha-C10-C10 as well as monorhamnolipid congeners, specifically Rha-C10-C10. The crude surfactin and rhamnolipid extracts were also assessed for their antimicrobial activities and displayed significant antimicrobial activity against a broad spectrum of opportunistic and pathogenic microorganisms, including antibiotic resistant Staphylococcus aureus and Escherichia coli strains. The quantitative and qualitative effects of various substrates utilised for the surfactin and rhamnolipid production by B. amyloliquefaciens ST34 and P. aeruginosa ST5 strains, respectively, were assessed in chapter 5. For B. amyloliquefaciens ST34, maximum biosurfactant production was observed in the MSM supplemented with fructose (28 mg/L). In addition, four surfactin analogues were produced by B. amyloliquefaciens ST34 using the different substrates, however, the Srf2-4 (C13-15 surfactins) were the most dominant in all the B. amyloliquefaciens ST34 extracts. For P. aeruginosa ST5, maximum biosurfactant production was observed in the MSM supplemented with glucose (307 mg/mL). In addition, six rhamnolipid congeners were produced by P. aeruginosa ST5 using the different substrates, however, similar to results obtained in Chapter four, the dRL2 (Rha-Rha-C10-C10) and mRL2 (Rha-C10-C10) were the most abundant compounds produced in all P. aeruginosa ST5 extracts.AFRIKAANSE OPSOMMING: Biosurfaktante is oppervlak aktiewe amfifiliese verbindings, gesintetiseer deur talle bakterieë, swamme en giste. Hierdie verbindings is bekend vir hul breë spektrum antimikrobiese aktiwiteit en word tans gebruik as antimikrobiese middels, emulsifiseerders, surfaktant agente, ens. in die kosmetiese, kos, farmaseutiese en biotegnologie-industrieë. Die primêre doel van hierdie studie was dus om te bioprospekteer vir nuwe biosurfaktante en biosurfaktant-produserende bakterieë teenwoordig in 'n riool-suiweringsaanleg. Afvalwater is gekies omdat dit as 'n geskikte omgewing dien vir die groei van diverse mikro-organismes en daar word gepostuleer dat die teenwoordigheid van talle organiese en anorganiese stowwe die biosurfaktant-produseerende mikro-organismes laat floreer. Hoofstuk een was uiteengesit met literatuur wat betrekking hou tot, onder andere, biosurfaktante, hul karakterisering en metode van werking. Hoofstuk twee fokus op die diversiteit en verspreiding van biosurfaktant-produserende bakterieë wat vanuit afvalwater geïsoleer is. Om hierdie doelwit te bereik, is afvalwatermonsters by verskeie punte van die Stellenbosch rioolsuiweringaanleg geneem en groeibare isolate is getoets vir moontlike biosurfaktant produksie met behulp van die olie- verspreiding- en die druppel-ineenstortings- metodes. Oppervlakspanning en emulsifiseringsaktiwiteit is daarna gebruik vir die gedeeltelike karakterisering van die geproduseerde biosurfaktante. Twee-en-dertig van die 667 bakteriese isolate is geïdentifiseer as biosurfaktant produseerders en is geklassifiseer in die Aeromonadaceae, Bacillaceae, Enterobacteriaceae, Gordoniaceae en Pseudomonadaceae families, met behulp van 16S rRNS analises. Bacillus en Pseudomonas is geïdentifiseer as die mees dominante genera, met 21.8% (7/32) en 12.5% (4/32) van die isolate wat onderskeidelik tot hierdie genera behoort. `n Groot vermindering in die oppervlakspanning van die groeimedium (71.1 mN/m) is waargeneem vir die Bacillus ST34 (34.4 mN/m) en die Pseudomonas ST5 (32.3 mN/m) isolate. Die Bacillus ST34 en Pseudomonas ST5 isolate het verder ook positief getoets vir die ‘sfp’ en ‘rhlB’ gene wat betrokke is by die biosintese van surfaktien en rhamnolipied biosurfaktante. Terwyl talle studies verslag doen oor die isolasie van biosurfaktant produserende bakterieë uit besmette grond en landelike omgewings, dui die huidige studie aan dat munisipale afvalwater gebruik kan word vir die isolasie van diverse biosurfaktant produseerende bakteriese stamme In Hoofstuk drie is hierdie 32 biosurfaktant-produseerende isolate verder geïdentifiseer (tot op spesie vlak) met behulp van genus en spesie spesifieke polimerase kettingreaksies (PKR). Bakteriese isolate wat as dieselfde spesie geïdentifiseer is, is genotipies onderskei deur gebruik te maak van herhalende element PKRs [gerig op die herhalende ekstrageniese palindromiese (HEP) en die “BOX” element DNS volgordes]. Hierdie molekulêre differensiasies is uitgevoer omdat die genetiese diversiteit onder bakteriese spesies kan lei tot die produksie van verskillende konsentrasies en proporsies van verskeie homoloë van biomolekules soos biosurfaktante en antibiotika. Met die gebruik van konvensionele PKR toetse, is sommige van die isolate geïdentifiseer as ondere andere Bacillus subtilis (n = 4), Aeromonas hydrophila (n = 3) en Bacillus amyloliquefaciens (n = 2). Hierdie bakteriese spesies is genotipies onderverdeel, met behulp van herhalende element PKRs, in vier, drie en twee sub-spesies (stamme), onderskeidelik. Die ‘BOX AIR’ en ‘REP’ inleiers wat gebruik is vir die herhalende element PKRs in die huidige studie, is dus 'n kragtige toepassing wat gebruik kan word om te onderskei tussen biosurfaktant-produseerende bakteriese isolate, wat as dieselfde spesie geïdentifiseer is. Hoofstuk vier het gehandel oor die karakterisering en antimikrobiese aktiwiteit van die biosurfaktant ekstrakte wat deur die Bacillus amyloliquefaciens ST34 en Pseudomonas aeruginosa ST5 isolate geproduseer is. Ru-biosurfaktante wat deur die ST34 en ST5 isolate geproduseer is, is vanuit die vloeibare medium geisoleer met behulp van oplosmiddel-ekstraksie metodes. Daarna is hoë resolusie ultra-verrigting vloeistofchromatografie gekoppel aan elektrosproei-ionisasie massaspektrometrie (ESI-MS) (waarvoor ‘n metode in die huidige studie ontwikkel is) gebruik om die geproduseerde verbindings te karakteriseer. Die resultate het aangedui dat B. amyloliquefaciens ST34 hoofsaaklik die K13, K14, K15 en K16 surfaktien analoë produseer wanneer dit op`n minerale sout medium, waarby gliserol gevoeg is, gegroei word. Vir P. aeruginosa ST5 is die hoë resolusie ultra-verrigting vloeistofchromatografie gekoppel aan ESI-MS gebruik om die teenwoordigheid van dirhamnolipied verwante produkte, spesifiek Rha-Rha-K10-K10, asook monorhamnolipied verwante produkte, spesifiek Rha-K10-K10, te bevestig. Die ru-surfaktien en rhamnolipied ekstrakies is ook geëvalueer vir hul antimikrobiese aktiwiteite en het beduidende antimikrobiese aktiwiteit teen 'n wye verskeidenheid opportunistiese en patogeniese mikro-organismes, insluitende antibiotika weerstandige Staphylococcus aureus en Escherichia coli stamme, getoon. Die kwantitatiewe en kwalitatiewe effek van verskeie substrate wat gebruik is vir die produksie van surfaktien en rhamnolipiede deur B. amyloliquefaciens ST34 en P. aeruginosa ST5 stamme, onderskeidelik, is in Hoofstuk vyf geëvalueer. Vir B. amyloliquefaciens ST34, is maksimale biosurfaktant produksie waargeneem in die minerale sout medium wat met fruktose aangevul is (28 mg/L). Daarbenewens is B. amyloliquefaciens ST34 daartoe instaat om vier surfaktien analoë te produseer deur gebruik te maak van verskillende substrate. Die Srf2-4 (K13-15 surfaktiene) is egter steeds die mees dominante verbindings in al die ekstrakte van die B. amyloliquefaciens ST34 stam gewees. Vir P. aeruginosa ST5 is maksimale biosurfaktant produksie waargeneem in die minerale sout medium wat met glukose aangevul is (307 mg/L). Daarbenewens is ses rhamnolipied verwante produkte deur P. aeruginosa ST5 geproduseer deur gebruik te maak van verskillende substrate. Die dRL2 (RHA-RHA-K10-K10) en mRL2 (RHA-K10-K10) was egter steeds die mees algemene verbindings wat in al die ekstrakte van die P. aeruginosa ST5 stam geproduseer is