Biological process and Bioremediation

Metabolisme Mikroorganisme Sistem energi mikroorganisme Reaksi redoks dalam proses metabolisme Klasifikasi mikroorganisme Konsorsium mikroorganisme Enzim

Sistem energi mikroorganisme ATP : bentuk penyimpanan energi yang dapat digunakan secara biologis dalam metabolisme mikroorganisme dan disimpan di dalam sel Terbentuknya ATP melalui proses katabolisme Hidrolisa ATP menghasilkan energi untuk tumbuh dan reproduksi dalam proses anabolisme Mineralisasi : konversi dari suatu unsur dari bahan organik menjadi suatu keadaan anorganik sebagai akibat proses dekomposisi oleh mikroba

Metabolisme Mikroorganisme Sistem energi mikroorganisme Reaksi redoks dalam proses metabolisme Klasifikasi mikroorganisme Konsorsium mikroorganisme Enzim

Akseptor elektron dan pendonor elektron Dalam kaitan dengan degradasi kontaminan, secara konseptual, dapat ditulis sebagai berikut : Kontaminan + Xoks Produk + Xred

Contoh soal

Reaksi redoks dalam proses metabolisme Akseptor elektron dan pendonor elektron Fosforilasi dan kemiosmotis Fosforilasi pada tingkat substrat dilakukan oleh mo yang tumbuh dan hidup dalam kondisi anaerob Mekanisme transpor proton dan elektron melewati dinding sel yang bersifat sebagai membran Urutan penggunaan elektron penerima dalam reaksi redoks Urutan akseptor elektron : O2 > NO3 > Mn > Fe > SO4 > CO2 d. Reaksi generik degradasi senyawa organik Metode prakiraan berdasarkan pendekatan stoikiometrik berbasis reaksi redoks dilakukan untuk perkiraan perbaikan tanah dan pertimbangan biaya operasi. Untuk itu reaksi generik antara kontaminan sebagai pendonor elektron dan akseptor elektron dalam bentuk reaksi redoks dapat digunakan untuk memperkirakan jumlah akseptor elektron yang diinginkan. Lihat halaman 197-200 buku Pencemaran Tanah dan Air Tanah oleh Suprihanto Notodarmojo.

Reaksi penting yang dimediasi mikroorganisme Reaksi transformasi biotis - reaksi hidrolisa - Reaksi cleavage (pemecahan atau pemutusan rantai) - Reaksi oksidasi reduksi - Reaksi dehidrogenasi - Reaksi dehidrohalogenasi - Reaksi substitusi Jalur proses dalam transformasi biotis Langkah pertama dalam proses degradasi oleh metabolisme mikroorganisme adalah bagaimana molekul target atau substrat masuk ke dalam sel membran melalui mekanisme difusi Selanjutnya reaksi di sitoplasma untuk menguraikan senyawa organik dan keberadaan elektron donor dan elektron akseptor

Degradasi Hidrokarbon - PAH (polyciclic aromatic hydrocarbon) - Metabolisme didahului oleh white rot fungi dan alga hijau

Degradasi Pestisida DDT menjadi perhatian karena sifat persistensi dan sifat racunnya Ada dua jalur yaitu aerob dan anaerob

Faktor faktor yang mempengaruhi proses biologis dalam tanah Mikroorganisme sebagai pelaku Substrat dan nutrien Penerima elektron (akseptor elektron) Faktor lingkungan : kelembaban tanah, temperatur, pH tanah dan kandungan garam

Interaksi mikroorganisme dengan logam Spesies logam dan bioavailability Spesies logam dalam tanah dipengaruhi oleh kondisi redoks, pH dan kekuatan ion Mikroorganisme mempunyai kemampuan mempengaruhi potensial redoks (mengubah spesies logam dalam larutan tanah), misal kehaidran bakteri Desulfovibrio yg dalam kondisi tereduksi mereduksi sulfat menjadi sulfida. Logam terbentuk endapan yang non toxic.Dalam kondisi teroksidasi hampir semua logam terlarut dalam bentuk ion bebas. pH akan turun karena aktivitas thiobacillus thioooxidans yang mengoksidasi belerang menjadi asam sulfat. pH rendah kelarutan logam meningkat. Bioavailability merupakan spesies logam yang terlarut, yang tidak terikat atau tersorpsi oleh partikel padat tanah. Toksisitas logam terhadap mo tgt bioavailability logam. Semakin tinggi kelarutan , maka semakin tinggi bioavailability dan toksisitas semakin tinggi

Interaksi logam dengan mikroorganisme 3 proses utama yang menyebabkan interaksi antara mikroorganisme dengan logam” oksidasi-reduksi Contoh Pseudomonas spp. dapat mengoksidasi arsenite menjadi arsenate 2. Kompleksasi. 2 mekanisme komplekasi logam oleh bakteri : Logam ikut aktif dalam pengikatan (non specific binding) pada permukaan dinsing sel, lapisan lendir atau pada bagian luar sel lainnya (extracelullar matrix) Logam mungkin masuk ke bagian dalam sel dan terikat 3. Metilasi Penambahan metil atau gugus alkil pada logam membentuk metil logam. Metilasi logam membentuk senyawa yang mudah menguap, selain itu logam termetilasi akan meningkatkan kemampuan menembus membran biologis sel

BIOREMEDIATION

Bioremediation Defined Any process that uses microorganisms, fungi, green plants or their enzymes to break down harmful chemicals and pollutants in order to return the environment to its original natural condition. “Bioremediation is not only about genetics and enzymology but also about physiology and ultimately ecology.”—de Lorenzo V: Systems biology approaches to bioremediation. Curr Opin Biotechnol 2008, 19:579-589.

Alleviating Pollution Ex situ or in situ intervention Natural attenuation Example: phytoremediation (hyperaccumulators) store heavy metals in vacuoles Sebertia acuminata 20% dry weight is nickel. Plants on side of freeways are taking up lead from gas exhaust Bio-stimulation Add nutrients (nitrate/sulfate) that cause blooms of naturally occurring microbial bioremediators. Example: bacteria that metabolize polycyclic aromatic hydrocarbons or polychlorinated biphenyls Bio-augmentation Genetically Modified Bioremediators Alter organisms to manufacture proteins for desired metabolism Yellow poplar tree given enzyme mercuric reductase thrives in mercury soil, cadmium, TCE Bacteria gene breaks down TNT is linked to jellyfish gene that glows. Bacteria spread on soil glows green near explosives Chakrabarty first patented oil eater bacterium. Combined 4 plasmids in one bacterial cell gave it the ability to degrade four components of crude oil.

Why do we even need it? We can’t seem to stop polluting Inorganics Uranium, technicium, sulfur, slfuric acid Explosives RDX, TNT Polyaromatic hydrocarbons creosote Chlorinated hydrocarbons Trichlorethylene, PCBs, pentachlorophenol Petroleum hydrocarbons Gas, gas additives (MTBE), deisel From mid-1980’s up to 90’s numerous attempts were made to design GMO for environmental release for pollutants and heavy metals (USGS). Failures to program: bacteria doesn’t behave in a predictable fashion from the lab.

Case Study 1 Most failures at bioremediation are due to failure of introduced organisms to thrive in the natural environment or a failure to access the contaminant. This could be due to: Lack of nutrients Predation or parasitism Competition (GEM’s tend to compete poorly with indigenous populations). Immobility of introduced bacteria Contaminant concentrations below threshold for organism survival Organisms may feed on alternative substrates (E. coli and Pseudomonas diverge genetically from initial inoculum in field trials). A few examples of failed bioremediation attempts: Inoculation of soil with aliphatic hydrocarbon degrading bacteria did not enhance degradation of fuel oil Venosa AD, Wrenn BA (1996) Selective enumeration of aromatic and aliphatic hydrocarbon degrading bacteria by a most‑probable number procedure. Can. J. Microbiol.42: 252‑258 A Pseudomonas sp. shown in lab cultures to degrade 1,4-dichlorophenol failed to degrade the compound when added to surface soils Sayler GS, Ripp S (2000) Field applications of genetically engineered microorganisms for bioremediation processes. Curr Opin Biotechnol 11:286-289

Case study 2: degradation of crude oil by halophilic Archaea

Defining bioremediation by natural attenuation what is the environment? what is the pollutant? are bacterial, Archaeal, and/or plant species present that degrade the pollutant of interest? what conditions (nutrient, temperature, pH, salt etc) are necessary for that activity?

Defining bioremediation by natural attenuation: hydrocarbon degradation in the Arabian Gulf hypersaline coast what is the environment? what is the pollutant? the hypersaline coast of the Arabian coast crude oil (hydrocarbons) West, Ian. 2008. Qatar - sabkhas, evaporites and some other desert features: an introduction. http://www.soton.ac.uk/~imw/Qatar-Sabkhas.htm

Defining bioremediation by natural attenuation: hydrocarbon degradation in the Arabian Gulf hypersaline coast what is the environment? what is the pollutant? the hypersaline coast of the Arabian coast crude oil (hydrocarbons) are bacterial, Archaeal, and/or plant species present that degrade the pollutant of interest? 2 Haloferax strains 1 Halobacterium strain 1 Halococcus strain environmental samples grow on minimal mineral plates with crude oil vapor as sole carbon/energy source (Al-Mailem et al., 2010 Extremophiles)

Defining bioremediation by natural attenuation: hydrocarbon degradation in the Arabian Gulf hypersaline coast are bacterial, Archaeal, and/or plant species present that degrade the pollutant of interest? C18 hydrocarbon autoclaved control Haloferax isolate Halobacterium isolate Halococcus isolate gas-liquid chromatography to measure hydrocarbon degradation (Al-Mailem et al., 2010 Extremophiles)

Defining bioremediation by natural attenuation: hydrocarbon degradation in the Arabian Gulf hypersaline coast what conditions (nutrient, temperature, pH, salt etc) are necessary for that activity? increased salt increased hydrocarbon degradation Haloferax isolate Halococcus isolate Halobacterium isolate Haloferax isolate (Al-Mailem et al., 2010 Extremophiles)

How could this hydrocarbon degradation activity by Haloarchaea be improved? General strategies for improving microbial bioremediation: stimulation or augmentation Some existing bio-engineering tools University of Minnesota Biocatalysis/Biodegradation Database (UMBBD): enzymes, pathways, reactions, compounds from hundreds of bacterial species of interest (Gao et al., 2010) MetaRouter: tracks possible breakdowns from a starting point using all possible reactions (Pazos et al., 2005) in silico modeling of altered strains: ex. Optstrain (Pharkya et al. 2004), DESHARKY (Rodrigo et al. 2008)

How could this hydrocarbon degradation activity by Haloarchaea be improved? How could systems level knowledge help design stimulation or augmentation strategies? need to know network topology of the pathway being added or altered as well as the influence of the environment on that pathway understand coupling of pathways to better integrate the engineered or altered pathway into the rest of the host system understand demands created by the new flux on resources needed for growth/survival: energy, carbon, redox balance, transcriptional and translation capacity is it possible to compensate by altering regulation by TFs etc, or by adding or deleting other pathways? understand effect of environment on pathway flux understand role of noise in pathway regulation if cooperation between multiple microbial species is used, then systems analysis can evaluate impact of biodegradative flux on the multispecies consortia