Biogeochemical and kinetics characterization of sulfate reducing microbial communities enriched from mine drainages
| dc.contributor.advisor | Van Heerden, E. | |
| dc.contributor.advisor | Castillo, J. C. | |
| dc.contributor.author | Moloantoa, Karabelo Macmillan | |
| dc.date.accessioned | 2015-08-18T12:04:04Z | |
| dc.date.available | 2015-08-18T12:04:04Z | |
| dc.date.issued | 2015-02 | |
| dc.description.abstract | English: Acid mine drainage (AMD) or acid rock drainage (ARD) is a global challenge contaminating a lot of the fresh surface and groundwater. The drainage is characterized by low pH, high metal and sulfate concentrations. It is a consequence of most mining activities as lead source of AMD. The metals and sulfate in the drainage result from oxidation of metal sulfide containing rocks also referred to as host rock. Oxidation of the host rock occurs during mining by water and oxygen or naturally where the oxidation process occur through weathering and both processes can be accelerated by iron oxidising bacteria such as Acidithiobacillus ferrooxidans and Leptospirillum ferrooxidans. The A. ferrooxidans catalyses the oxidation of iron containing sulfide minerals such as pyrite and arsenopyrite releasing iron, arsenic and sulfur which gets oxidized into sulfate generating sulfuric acid that lowers the pH of the water. The acidic environment induces dissolution of other metals. In most coal mines, AMD contains some neutralizing minerals such as calcium oxide (lime), calcium carbonate (calcite) and sodium carbonate which raise the pH of the water but do not precipitate all of the metals and sulfate. The pH of water can be raised to between 5 and 8 hence the drainage is termed Non-acid mine drainage (NMD). Toxic metals in AMD and NMD contaminate streams and rivers where they affect aquatic and terrestrial life. Treatments of these drainages have been developed and characterized into biotic and abiotic systems where chemical and biological methods are used. In this study, attention was given to extending knowledge that can contribute to developing and extending biotic remediation systems where sulfate-reducing bacteria (SRB) are employed to interact with AMD or NMD. SRB are a diverse group of microorganisms used in bioremediation and have been studied widely. SRB play a major role in the reversal process of AMD and NMD formation by reducing the sulfate concentrations. SRB use sulfate as their terminal electron acceptor releasing sulfide in a gas form that dissolves in the solution when the pH is above 4. This process occurs optimally in anaerobic environments and the dissolved sulfide, reacts with dissolved metals in the drainage forming metal sulfide precipitates. The sulfate reduction and metal removal processes from the drainages can be affected by numerous factors and a few discussed were explored and the knowledge base extended in this study. Environmental conditions always have an effect on most activities performed by biological entities. For sulfate reduction, pH, temperature, carbon source type and availability, metal concentrations and redox conditions have direct effects on SRB activities. In this study, three mine drainage study sites with generic names: Site-Ex, Site-Ka and Site-Po were selected. Water samples from the three sites as well as a sludge sample from Site-Po were collected and characterized chemically. Drainage from Site-Ex had characteristics of NMD while Site-Ka and Site-Po water samples had AMD characteristics. High concentrations of sulfate and transition metals were detected in the AMD samples. Microscopic analysis revealed high microbial cell counts in NMD and lower cell counts in AMD samples that could most probably be directly related to the diversity and toxicity of metals and low pH of the drainages. Molecular analysis revealed the presence of various SRB species in the drainages and sludge samples including the well-studied Desulfovibrio sp. Two media compositions: Postgate medium B (PSGM) and Anaerobic sulfate reducing medium (ASRM) were used to enrich anaerobic bacterial communities. Acclimation process with three passaging intervals of 20 days was conducted in anaerobic serum vials. After the third passaging stage, molecular identification of the enriched cells was performed and results revealed successful enrichment of SRB and other anaerobic (some novel) bacteria within the consortia. Scanning electron microscopy (SEM) was used to morphologically characterize the biogenic precipitates from the tertiary cultures. The SEM results showed bacterial biofilm associated with precipitates similar to those identified as framboid pyrite precursors. The enriched consortia from the three study sites were co-cultured in PSGM and ASRM respectively. The best bacterial growth was achieved in PSGM which was then used for subsequent experiments as the medium of choice. The sulfate reducing capabilities of the enriched SRB were tested in PSGM with sulfate concentrations ranging between 2 000 mg/L to 4 000 mg/L. An average of 72% sulfate reduction was achieved in all experiments with a positive response of SRB to higher sulfate concentrations. Effects of pH and temperature on sulfate reduction were evaluated at pH of 3.5 and 6.2 and temperatures of 10ºC and 25ºC. Low pH conditions showed negative effects on sulfate reduction activity and bacterial growth even when temperature was raised to 25ºC. Optimum SRB activity was observed in the experiment where pH was 6.2 at 25ºC. The results confirmed higher sulfate reducing conditions at higher pH (6.2) and temperature (25ºC). The carbon source utilisation by the enriched SRB between glycerol and sodium lactate was evaluated in batch operated bioreactors. The best sulfate reduction activity by SRB was observed when glycerol was used as a sole carbon source yielding greater amounts of dissolved sulfide concentrations. Glycerol was then used further as the main carbon source in PSGM. Metal-microbe interactions were evaluated where higher concentrations of zinc (Zn2+) and iron (Fe2+) were introduced in the bioreactors. Results showed 100%, 85% and 40% sulfate reduction in experiments where no metals, 200 mg/L of iron and 200 mg/L of zinc were added respectively. Effects of high Zn2+ concentrations were similar to those exerted by low pH conditions. However, 90% zinc and 97% iron were removed from the medium through biogenic precipitation. Precipitates were characterized by SEM, Transmission Electron Microscopy (TEM), X-ray Diffraction (XRD) and Energy Dispersive X-ray Spectroscopy (EDX) which confirmed the presence of biologically induced precipitates. Results in this study can be used to model the activity of SRB in evaluated conditions that affect sulfate reduction. The enriched bacterial communities also showed great potential to be used in the up-scaled “reactors” to reduce sulfate concentration while indirectly precipitating dissolved metals in AMD. | en_ZA |
| dc.description.abstract | Afrikaans: Suur myn water (SMW) is ‘n globale probleem wat baie vars oppervlak- en grondwater kontamineer. Die water word gekarakteriseer deur lae pH en hoë metaal- en sulfaat konsentrasies. Dit is die nagevolg van meeste myn aktiwiteite as die hoofsaaklike bron van SMW. Die metale en sulfate in die water is as gevolg van die oksidasie van metaal sulfied wat in sekere minerale voorkom. Die oksidasie van SMW geskied tydens mynwese aktiwiteite as gevolg van die teenwoordigheid van water en suurstof of natuurlik waartydens die oksidasie proses plaasvind deur verwering. Beide prosesse kan versnel word deur yster-oksiderende bakterië soos Acidothiobacillus ferrooxidans en Leptospirillum ferrooxidans. A. ferrooxidans kataliseer die oksidasie van yster bevattende sulfied minerale soos piriet en arsenopiriet, wat lei tot die vrystelling van yster, arseen en swael wat op sy beurt geoksideer word om swaelsuur te produseer wat lei tot die daling van die water se pH. Hierdie lae pH induseer die oplossing van ander metale. In meeste steenkool myne bevat die SMW neutraliserende minerale soos kalsium oksied (kalk), kalsium karbonaat (kalsiet) en natrium karbonaat wat lei tot die styging van die water pH. Hierdie styging in pH verminder egter nie die hoë metaal- en sulfaat konsentrasies nie. Die pH van die water kan verhoog word na tussen 5 en 8, waarna dit verwys word na Nie-suur myn water (NMW). Toksiese metale wat in SMW en NMW voorkom kontamineer strome en riviere met ‘n negatiewe impak op die akwatiese en terestriële lewe. Behandeling van hierdie water is al ontwikkel en kan gekarakteriseer word as biotiese en abiotiese sisteme waar biologiese en chemiese metodes gebruik word. In hierdie studie, was die aandag gefokus op die uitbreiding van kennis wat kan bydrae tot die ontwikkeling en uitbreiding van biotiese remediasie sisteme waartydens sulfaat-reduserende bakterië (SRB) gebruik word in interaksies met SMW en NMW. SRB is 'n diverse groep mikroorganismes wat in bioremediasie gebruik word en is al wyd bestudeer. SRB speel ‘n groot rol in die omkerings proses van SMW en NMW formasie deurdat dit die sulfaat konsentrasie in die water verlaag. SRB gebruik sulfaat as ‘n terminale electron akseptor, wat lei tot sulfied wat as ‘n gas vrygestel word en opgelos word in die water wanneer die pH steig bo 4. Hierdie proses vind optimaal plaas in anaërobiese toestande waartydens die sulfied dan ook reageer met die opgeloste metale in die water wat lei tot die vorming van metaal sulfied presipitate. Die sulfaat reduksie en metaal presipiterings prosesse in die water kan geaffekteer word deur verskeie faktore en 'n paar wat bespreek is, is ondersoek en die kennis uitgebrei tydens hierdie studie. Omgewings toestande het altyd ‘n invloed op die meeste aktiwiteite wat deur biologiese entiteite uitgevoer word. Vir sulfaat reduksie om plaas te vind, kan die pH, temperatuur, koolstof bron tipe en beskikbaarheid, metaal konsentrasies en redox toestande ‘n direkte effek hê op SRB aktiwiteite. Tydens hierdie studie was drie myn water studie areas met generiese name, Site-Ex, Site-Ka en Site-Po geselekteer. Water monsters van die studie areas, asook 'n slyk monster vanaf Site-Po is geneem en chemies gekarakteriseer. Die water vanaf Site-Ex is as NMW en beide die vanaf Site-Ka en Site-Po as SMW gekarakteriseer. Hoë konsentrasies sulfaat en transisie metale was opgespoor in die SMW monsters. Mikroskopiese analiese het gewys dat hoë mocrobiese sel getalle in die NMW voorkom, terwyl laer getalle in die SMW voorkom, wat waarskynlik direk verband kan hou met die diversiteit en toksisiteit van die metale en lae pH van die water. Molekulêre analiese het die teenwoordigheid aangedui van verskeie SRB spesies in die water en slyk monsters wat die wyd bestudeerde Desulfovibrio spesie insluit. Twee groei medium komposisies, Postgate medium B (PSGM) en Anaërobiese sulfaat-reduserende medium (ASRM), is gebruik om die anaërobiese bakteriële populasies te verryk. ‘n Akklimasie proses is gevolg met drie passerings intervalle van 20 dae in anaërobiese serum bottels. Na die derde passering, is molkulêre identifikasie van die verrykte selle uitgevoer. Die resultate het gewys op suksesvolle verryking van die SRB asook ander anaërobiese (sommige uniek) bakterië in die konsortium. Skanderings Elektron Mikroskopie (SEM) is gebruik om die biogeniese presipitate van die tersiêre kulture morfologies te karakteriseer. Die SEM resultate het gewys op ‘n bakteriële biofilm wat geassosieer is met presipitate soortgelyk aan die wat geïdentifiseer is as framboïde piriet voorgangers. Die verrykte konsortiums van die drie studie areas is afsonderlik gekweek in PSGM en ASRM onderskeidelik. Die beste bakteriële groei is bereik in PSGM wat dus in die gevolglike eksperimente gebruik is as die groei medium van keuse. Die sulfaat reduserende vermoë van die verrykte SRB is getoets in PSGM met afsonderlike sulfaat konsentrasies tussen 2000 mg/L en 4000 mg/L. ‘n Gemiddeld van 72% sulfaat reduksie kon gehandhaaf word in alle eksperimente, met ‘n positiewe reaksie van die SRB op hoër sulfaat konsentrasies. Die effek van pH en temperatuur op sulfaat reduksie is geëvalueer by ‘n pH van 3.5 en 6.2 en temperature van 10°C en 25°C. Lae pH toestande het ‘n negatiewe impak gehad op die sulfaat reduksie aktiwiteit sowel as bakteriële groei, selfs toe die temperatuur toegeneem het na 25°C. Optimale SRB aktiwiteit kon waargeneem word by ‘n pH van 6.2 by 25°C. Die resultate het die verhoogde sulfaat reduserende toestande teen hoër pH en temperature bevestig. Die koolstof bron gebruik deur die SRB tussen gliserol en natrium laktaat is geëvalueer in bioreaktors. Die beste sulfaat reduserende aktiwiteit is waargeneem met die gebruik van gliserol as die enigste koolstof bron, met die hoogste konsentrasie van sulfied in oplossing. Gliserol is verder gebruik as die koolstof bron van keuse in die PSGM. Metaal-mikrobe interaksies is geëvalueer deur hoër konsentrasies Zn2+ en Fe2+ by die bioreaktors te voeg. Resultate het gewys dat 100%, 85% en 40% sulfaat reduksie plaasgevind het toe 0 mg/L en 200 mg/L yster en 200 mg/L zink onderskeidelik bygevoeg is. Die effek van hoë Zn2+ konsentrasies was soortgelyk aan die effek wat lae pH toestande gehad het, terwyl 90% zink en 97% yster egter verwyder kon word deur biogeniese presipitasie. Presipitate is kerakteriseer deur SEM, Transmissie Elektron Mikroskopie (TEM), X-straal Diffraksie (XSD) en Energie Dispersiewe X-straal Spektroskopie (EDX) wat die teenwoordigheid van biologies-geïnduseerde presipitate bevestig het. Die resultate van hierdie studie kan gebruik word om die aktiwiteit van die SRB vir sulfaat reduksie te modelleer. Die verrykte bakteriële populasies het die potensiaal getoon om gebruik te word in groter skaal “reaktore” om sulfaat konsentrasies te reduseer, terwyl opgeloste metale indirek gepresipiteer word in SMW. | |
| dc.identifier.uri | http://hdl.handle.net/11660/907 | |
| dc.language.iso | en | en_ZA |
| dc.publisher | University of the Free State | en_ZA |
| dc.rights.holder | University of the Free State | en_ZA |
| dc.subject | Acid mine drainage | en_ZA |
| dc.subject | Sulfate-reducing bacteria | en_ZA |
| dc.subject | Batch bioreactors | en_ZA |
| dc.subject | Enrichments | en_ZA |
| dc.subject | sulfate reduction | en_ZA |
| dc.subject | Metal microbe interactions | en_ZA |
| dc.subject | Mine drainage | en_ZA |
| dc.subject | Dissertation (M.Sc. (Microbial, Biochemical and Food Biotechnology))--University of the Free State, 2015 | en_ZA |
| dc.subject | Groundwater -- Pollution | en_ZA |
| dc.title | Biogeochemical and kinetics characterization of sulfate reducing microbial communities enriched from mine drainages | en_ZA |
| dc.type | Dissertation | en_ZA |