Karakterisering af en anaerob termofil ... carboxymethyl cellulose (CMC) with the Somogyi-Nelson assay

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  • Karakterisering af en anaerob

    termofil cellulosenedbrydende

    bakteriekultur fra en papirfabrik

    Maria Giltoft Jørgensen

    Specialerapport April 2008

    Mikrobiologi

    Biologisk Institut

    Aarhus Universitet

    Vejleder:

    Kjeld Ingvorsen

    Titel på engelsk:

    Characterisation of an anaerobic thermophilic cellulose-degrading bacterial culture obtained from a

    paper factory

    Forside:

    Krystalstrukturen af cellobiohydrolase CelS fra Clostridium thermocellum (Guimaraes et al., 2002),

    transmission-elektronmikroskopibilleder af AN60-blandingskultur og Dalum Papirfabrik A/S, 1948

    (http://www.dalumpapir.dk/).

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    Abstract

    Because of human activity cellulose containing materials, such as straw, paper and wood chips has

    become a waste product. This material is accessible in great amounts, it is cheap and so far does not

    have a lot of applications. The future utilization of cellulosic waste is a matter of great interest, as is the

    production of fuels from cellulosic biomass. There are still a lot of obstacles for the production of fuels

    from cellulosic biomass both technical and microbial.

    The purpose of this project was to isolate and characterise cellulose-degrading bacteria from a paper

    factory (Dalum Paper A/S in Maglemølle, Næstved, Denmark) in order to get a better understanding of

    how to utilize cellulosic waste. Four aerobic bacterial strains were isolated from 40°C incubations, but

    they were not further characterised because of their low potential to hydrolyze cellulose substrate. A

    stable and efficient cellulose-degrading mixed culture was obtained by anaerobic incubations at 60°C

    (AN60). Several non-successful attempts were made to isolate a cellulose-degrading bacterium from

    AN60. The different kinds of bacteria in the mixed culture were then sequenced using PCR and cloning

    techniques. According to the 16S rRNA genes of the clones AN60 contained two different species. One

    (the CP-strains) related to a thermophilic, anaerobic and proteolytic bacterium which does not grow on

    cellulose, Coprothermobacter proteolyticus (Kersters et al., 1994). The other (the Cel60-strains) related

    to a thermophilic anaerobic cellulose-degrading clostridia, Clostridium sp. EBR-02E-0045 isolated by

    Shiratori et al. (2006). It was assumed that the CP-strains were not able to degrade cellulose and the CP-

    strains were named the contaminants of AN60. AN60 was characterized with particular reference to its

    cellulose degrading capabilities. AN60 showed both exo- and endocellulase activity but no β-

    glucosidase activity. It degraded both crystalline (Avicel, Whatman No. 1) and amorphic (α-cellulose)

    cellulose substrates in 1-2 days at 60°C and grew equally well on all cellulose substrates tested.

    Cellulase synthesis and activities were not inhibited by growth on 0.4 % [w/v] cellobiose. Furthermore

    the culture fermented glucose but showed no cellulase activity by growth on glucose. Exo- and

    endocellulase activities were assayied by measuring the liberation of reducing sugars from Avicel and

    carboxymethyl cellulose (CMC) with the Somogyi-Nelson assay and the dinitrosalicylic acid (DNS)

    assay, respectively. For the purpose of determining the pH- and temperature optima a colorimetric assay

    was used (Cellazyme C assay). The endocellulases had optima at pH ∼ 4.8-8.8 and ∼62°C. The

    measured maximum exo- and endocellulase activities were 5.7 and 76 mU glucoseequivalents per ml

    culture, respectively. The doubling times during growth on cellulose and cellobiose were ∼6 and 4.1

    hours, respectively. The cellulases from AN60 were extracellular and did not adhere to the cells during

    all stages of growth on cellulose, but the cellulases were to some point adsorbed to the cellulose during

    breakdown. The cellulases from AN60 were very stable, since they still showed activity after 5 months

    of incubation at 60°C, and they can still be used for inoculation of new cultures after several months at

    4°C.

  • 3

    Forord

    Formålet med dette projekt var at isolere og karakterisere bakteriestammer med cellulytisk aktivitet

    fra papirfabrikken Dalum Papir A/S Afdeling Maglemølle i Næstved. Idéen opstod i forbindelse

    med et tidligere projekt i samarbejde med fabrikken (Tang & Jørgensen, 2005), hvor det blev

    opdaget, at der var cellulytisk aktivitet i procesvandet. Procesvandet fra afsværtningsanlægget i

    Maglemølle var fyldt med papirfibre, som er et godt substrat for mange cellulosenedbrydende

    bakterier (Freier et al., 1988), og der er vandindtag fra Susåen, hvorfra anlægget konstant inokuleres

    med nye bakterier. På denne baggrund blev det besluttet at anvende procesvandet til isolering af

    cellulosenedbrydende bakterier.

    Karakterisering af aerobe isolater blev ikke foretaget da forsøg viste, at isolerede aerobe bakterier

    havde et lavt potentiale til hydrolyse af cellulose. I stedet blev en stabil, effektiv og

    cellulosenedbrydende blandingskultur karakteriseret. Denne blandingskultur opstod ved en anaerob

    berigelse af procesvandet ved 60°C, og benævnes AN60. Adskillige forsøg på at isolere en

    cellulosenedbrydende bakteriestamme fra kulturen slog fejl, og PCR og kloningsteknikker blev

    anvendt for at identificere bakterierne i kulturen. Efter sekventering af AN60 fremgik det af

    sekvensresultaterne, at der var tale om en kultur antageligt bestående af to bakteriearter. De to

    forskellige typer af bakterier i kulturen blev benævnt Cel60-kloner og CP-kloner. Den ene art var

    tæt beslægtet med Coprothermobacter proteolyticus, som er en anaerob proteolytisk bakterie

    (Ollivier et al., 1985). Denne art betragtes ikke som værende i stand til at nedbryde cellulose, da

    dens nærmeste beslægtede ikke er beskrevet som cellulosenedbrydende (Kersters et al., 1994), og

    benævnes i rapporten som kontaminanten eller CP-stammerne (Tabel 7). Den anden bakterieart i

    blandingskulturen AN60 var tæt beslægtet med anaerobe termofile cellulytiske clostridier. Den

    benævnes i rapporten som den cellulytiske bakterie i AN60 kulturen eller Cel60-stammerne. AN60

    blev derefter karakteriseret specielt med henblik på dens cellulosenedbrydende egenskaber.

    Tak til

    Jeg vil gerne give en stor tak til alle de søde mennesker, der har hjulpet mig i udarbejdelsen af dette

    specialeprojekt. En speciel stor tak til min vejleder Kjeld Ingvorsen, som fik mig involveret i dette

    ”umulige” projekt: at isolere en anaerob termofil cellulytisk bakterie. Derudover tak til Tove og

    Britta for mange hjælpende hænder i laboratoriet samt til Ditte og Kristina for meget kritisk

    gennemlæsning af rapporten og til Marie for ARB-arbejde. Sidst men ikke mindst tak til alle på

    afdelingen for god stemning i bygning 540 og interessante samtaler over frokosten, og til Rasmus

    og Anton derhjemme for den sidste tids tålmodighed med en til tider fraværende kæreste og mor.

  • 4

    1. INTRODUKTION ............................................................................................................. 8

    1.1 Cellulose ....................................................................................................................................................................... 9

    1.2 Bakteriel cellulosenedbrydning ............................................................................................................................... 11

    1.3 Cellulaser ................................................................................................................................................................... 12

    1.4 Analyse af cellulaseaktivitet ..................................................................................................................................... 14 1.4.1 Metoder til måling af cellulaseaktivitet ............................................................................................................... 15 1.4.2 Cellulosehydrolysemekanisme ............................................................................................................................ 16 1.4.3 Celle- og cellulaseadhæsion til cellulose ............................................................................................................ 17 1.4.4 Substrater anvendt i cellulaseaktivitetsassays ..................................................................................................... 17

    1.5 Cellulaser og deres anvendelsesmuligheder ........................................................................................................... 18 1.5.1 Cellulaser anvendt til fremstilling af biobrændstof ............................................................................................. 19 1.5.2 Processer ............................................................................................................................................................. 20 1.5.3 Consolidated Bioprocessing (CBP) ..................................................................................................................... 21 1.5.4 Hvor langt er vi med teknologierne? .................................................................................................................