F A C U L T Y O F S C I E N C E

U N I V E R S I T Y O F C O P E N H A G E N

PhD thesis

Lea Ellegaard-Jensen

Fungal degradation of pesticides

- construction of microbial consortia for bioremediation

Academic advisor: Søren Rosendahl

Submitted: November 2012 The PhD School of Science, Faculty of Science, University of Copenhagen, Denmark.

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Name of department: Department of Biology Author: Lea Ellegaard-Jensen Title: Fungal degradation of pesticides - construction of

microbial consortia for bioremediation Academic advisor: Søren Rosendahl Submitted: November 2012

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Preface

This thesis presents the results of my three years as a PhD student at the section of Terrestrial

Ecology at the University of Copenhagen. The PhD was part of the project on Microbial

Remediation of Contaminated Soil and Water Resources (MIRESOWA) funded by the Danish

Council for Strategic Research. The MIRESOWA project provided excellent opportunities for

collaboration between partners, and the research presented in this thesis was especially a result of

close collaboration between the section of Terrestrial Ecology at University of Copenhagen and the

department of Geochemistry at the Geological Survey of Denmark and Greenland (GEUS). The

work was therefore carried out both at the University of Copenhagen and at GEUS.

Acknowledgments

First of all, I wish to thank my two supervisors Søren Rosendahl and Jens Aamand for invaluable

guidance, support and also thoughtfulness during rough periods. I am truly grateful to you both for

the great experience and pleasure it has been working on this project: A project with bioremediation

as the core focus, which is one of my favorite scientific subjects.

I also want to thank all the kind people at the section of Terrestrial Ecology, many of whom I

already knew well from my years as a master student – starting my PhD work at the section really

made me feel at home though in a new role. Special thanks go to the very kind technician Lis and to

my fellow PhD students in the Journal Club for including me even though I spend much of my time

at GEUS.

To all the people at the department of Geochemistry at GEUS I am grateful for making me feel

welcome and for a great working environment. I appreciate the way that everyone there takes the

time to listen and discuss problems of both scientific and non-scientific nature. My great thanks go

to the skillful and caring technicians at GEUS Spire and Pia. I also wish to thank my fellow PhD

students (at the time): Meric, Erkin, Tina, Nora, Christian, Sarka and of course above all Berith.

Starting my PhD I knew that it was predetermined that 1/3 of the work should be in collaboration

with another PhD student on creating consortia of fungi and bacteria, which could create quite a

challenge. Amazingly this turned out to be not only one of the most interesting scientific parts, but

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also yielded lots of very enjoyable hours in the laboratory (and also outside it…) thanks to the way

two minds think alike.

I would also like to thank all the MIRESOWA partners for fruitful discussions and collaborations in

particular at our seminars and scientific meetings. Special thanks go to Anders Johansen from the

department of Environmental Science at Aarhus University and to Birthe B. Kragelund from the

Structural Biology and NMR Laboratory at the University of Copenhagen. Birthe thank you for

letting me visit and work in your laboratory.

Last but not least, I am very thankful to my family for all their support, and especially Michael and

Noa for their every-day loving support and understanding!

Lea Ellegaard-Jensen

Copenhagen, November 2012

Table of Contents

Preface ..................................................................................................................................... III

Acknowledgments ............................................................................................................................ III

List of manuscripts ..................................................................................................................... 3

English summary ........................................................................................................................ 5

Dansk resumé ............................................................................................................................ 8

Introduction and aims .............................................................................................................. 11

Background .............................................................................................................................. 13

Pesticide pollution – why should we care .......................................................................................... 13 Diuron, dichlobenil and BAM .................................................................................................................................. 13

Biodegradation, bioremediation and bioaugmentation ..................................................................... 15 Challenges for bioaugmentation ............................................................................................................................. 16

Biodegradation by fungi ................................................................................................................... 17 Filamentous fungi .................................................................................................................................................... 17 Mortierella .............................................................................................................................................................. 19 Fungal degradation of pesticides ............................................................................................................................ 20

Consortia for bioremediation ........................................................................................................... 24 Fungal-bacterial interactions .................................................................................................................................. 24 Fungal highway ....................................................................................................................................................... 25 Degradation by consortia ........................................................................................................................................ 26

Conclusions and perspectives ................................................................................................... 29

Reference list ........................................................................................................................... 31

Manuscript I ............................................................................................................................. 41

Manuscript II ............................................................................................................................ 61

Manuscript III ........................................................................................................................... 79

Appendix ................................................................................................................................ 104

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List of manuscripts

List of enclosed article manuscripts:

Manuscript I

Ellegaard-Jensen, L., Aamand, J., Kragelund, B.B., Johnsen, A.H., and Rosendahl, S.

Degradation of the phenylurea herbicide diuron by soil fungi: different degradation potentials

of five Mortierella strains.

In revision for Biodegradation

Manuscript II

Knudsen, B.E., Ellegaard-Jensen, L., Albers, C.N., Rosendahl, S., and Aamand, J.

Transport of degrader bacteria via fungal hyphae increases degradation of 2,6-

dichlorobenzamide.

Submitted to Environmental Pollution

Manuscript III

Ellegaard-Jensen, L., Knudsen, B.E., Johansen, A., Albers, C.N., Aamand, J., and Rosendahl, S.

Fungal-bacterial consortium overcomes dry patches and increases diuron degradation.

In prep. for submission to Science of the Total Environment

Article manuscript not included in this thesis:

Knudsen, B.E., Ellegaard-Jensen, L., Albers, C.N., Harder, C.B., Ekelund, F., Aamand, J., and

Rosendahl, S. Characterization of the biota in sandfilter material from 11 Danish water works.

In prep. for submission to Water Research

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Conference presentations:

Ellegaard-Jensen, L., Aamand, J., & Rosendahl, S. Fungal degradation of phenylurea herbicide

diuron. Presented at Symposium of the Danish Microbiological Society, Copenhagen 7.11.2011.

Poster Presentation

Knudsen, B.E., Ellegaard-Jensen, L., Albers, C., Rosendahl, S., & Aamand, J. Transport of BAM

degrading bacteria facilitated by fungal hyphae increases BAM mineralization in sand. Presented at

Symposium of the Danish Microbiological Society, Copenhagen 7.11.2011. Poster Presentation

Ellegaard-Jensen, L., Aamand, J., Kragelund, B.B., Johnsen, A.H., & Rosendahl, S. Degradation of

the herbicide diuron by Mortierella. Presented at The International Symposium on Microbial

Ecology – ISME14, Copenhagen 19.- 24.08.2012. Poster Presentation

Knudsen, B.E., Ellegaard-Jensen, L., Albers, C., Rosendahl, S., & Aamand, J. Transport of BAM

degrading bacteria facilitated by fungal hyphae increases BAM mineralization in sand. Presented at

The International Symposium on Microbial Ecology – ISME14, Copenhagen 19.- 24.08.2012. Poster

Presentation

Ellegaard-Jensen, L., Knudsen, B.E., Albers, C.N., Aamand, J., & Rosendahl, S. Three-member

consortium overcomes dry patches and increases diuron degradation. Presented at Copenhagen

Microbiology Center – CMC Symposium, Copenhagen 5.10.2012. Poster and Flash Presentation

Ellegaard-Jensen, L., Knudsen, B.E., Johansen, A., Albers, C.N., Aamand, J., & Rosendahl, S.

Three-member consortium overcomes dry patches and increases diuron degradation. Presented at

Symposium of the Danish Microbiological Society, Copenhagen 5.11.2012. Poster Presentation

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English summary

Pesticides are used worldwide on agricultural land as well as in urban areas. This use has often led

to contamination of the environment with serious effects on our natural resources. Frequent

pesticide use and spills have led to deterioration of soil quality and pesticide leaching has resulted in

groundwater contamination. New technologies are therefore needed for cleaning up contaminated

soil and water resources. This PhD was part of the project entitled Microbial Remediation of

Contaminated Soil and Water Resources (MIRESOWA) where the overall aim is to develop

new technologies for bioremediation of pesticide contaminated soil and water. The objectives of

this PhD were to investigate fungal degradation of pesticides and following to construct microbial

consortia for bioremediation.

In Manuscript I the fungal degradation of the phenylurea herbicide diuron was studied.

Isolates of soil fungi of the genus Mortierella were tested for their ability to degrade diuron. The

fungi were incubated in liquid culture with diuron on an orbital shaker at 8

oC. The results showed

that three of the five strains tested could degrade diuron, and molecular analysis revealed that these

three strains constituted a closely related phylogenetic group, while the two non-degraders were

located more distantly on the phylogenetic tree. In addition, it was examined whether the fungi

utilized diuron as a carbon or nitrogen source. Here the most efficient diuron degrading strain

Mortierella sp. LEJ701 was applied in liquid cultures with different levels of carbon and nitrogen.

Degradation of diuron was fastest in carbon and nitrogen rich media while degradation was very

restricted at low nutrient levels, making it unlikely that Mortierella utilize diuron as carbon or

nitrogen source. The degradation kinetics of these experiments showed that diuron degradation was

followed by formation of the metabolites 1-(3,4-dichlorophenyl)-3-methylurea, 1-(3,4-

dichlorophenyl)urea and an hitherto unknown metabolite. This metabolite was subsequently

biosynthesised, purified and analyzed by nuclear magnetic resonance (NMR) and mass

spectroscopy (MS). On the basis of these analyses the metabolite was suggested to be 1-(3,4-

dichlorophenyl)-3-methylideneurea. This study especially brings new insights into the phylogenetic

link between fungal diuron degraders, but also to the fungal degradation pathway of diuron.

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The next two manuscripts dealt with constructing fungal-bacterial consortia and examining

whether their degradation would be superior to that of the single strains in unsaturated systems. In

Manuscript II a consortium was created for degradation of the pesticide metabolite 2,6-

dichlorobenzamide (BAM). A consortium with Mortierella sp. LEJ702 and the BAM-degrading

Aminobacter sp. MSH1 as well as the single strains were introduced into small sand columns. The

sand was spiked with [ring-U-14

C]-BAM and mineralization could thus be determined from the

amount of evolved 14

CO2. In addition, the effect of moisture content was examined by adding water

corresponding to 0, 1.7, 5 or 10% of water holding capacity (WHC) to the sand. A faster

mineralization of BAM was obtained by the consortium compared to Aminobacter sp. MSH1 alone,

especially at the lower moisture contents. These results were supported by chemical analyses of 14

C

residues extracted from the sand. Additionally, it was investigated whether bacterial transport was

enhanced in the presence of Mortierella. This was done by extracting DNA from the top layer of the

sand followed by quantitative real-time polymerase chain reaction (qPCR) analysis. Results

demonstrated that the number of Aminobacter transported to the top sand was greatly enhanced in

the presence of Mortierella, suggesting that the fungal hyphae act as transport vectors for the

bacteria in the sand. Finally, the distribution of 14

C-BAM in the sand was studied in novel setup. It

was found that the presence of Mortierella slightly enhanced BAM distribution. From this work it is

evident that the fungal-bacterial consortium is capable of enhancing BAM-degradation in

unsaturated systems, and may therefore be a promising application for soil bioremediation.

In Manuscript III two- and three-member consortia were constructed with bacterial and

fungal diuron degraders. The purpose of this was to create a consortium which was superior for

diuron degradation in unsaturated system and to investigate the interactions between the

microorganisms in this consortium. The synergy leading to a more efficient degradation could either

be a result of co-operative catabolism or physical interactions between the organisms improving

growth and/or distribution of fungi and bacteria. The bacterial strains applied were Sphingomonas

sp. SRS2, Variovorax sp. SRS16 and Arthrobacter globiformis D47 and the fungal strains were

Mortierella sp. LEJ702 and Mortierella sp. LEJ703. In the experimental setup a layer of sterile

glass beads was added between the organisms and the sand column above; simulating air-filled gaps

in soil. [Ring-U-14

C]-diuron was mixed into the sand to a concentration of 100 μg diuron kg-1

.

Degradation was measured as the amount of 14

C-diuron mineralized and as 14

C residues in the sand

at experimental termination. Mineralization results established the three-member consortium

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LEJ702/SRS16/D47 as the most efficient transforming 32% of the diuron to 14

CO2, while the single

strains or other consortia mineralized no more than 10%. Furthermore, analyses of 14

C residues in

the sand showed that production of diuron metabolites by this consortium was minimal. The

interactions between the organisms in the consortia were examined by phospholipid fatty acid

analysis (PLFA) and 16S rDNA PCR using strain-specific primers. The molecular results suggested

that the presence of Mortierella sp. LEJ702 enhanced distribution of SRS16 and D47 in the sand.

Only fungal phospholipid fatty acids could be quantified. From those, however, it was apparent that

the fungal growth was severely inhibited in the presence of A. globiformis D47. On the other hand,

this effect was somewhat alleviated in the three-member consortium LEJ702/SRS16/D47. This

study is the first to show that a three-member consortium of both fungal and bacterial degraders can

indeed increase pesticide degradation.

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Dansk resumé

Pesticider anvendes over hele verden på landbrugsjorde såvel som i byområder. Denne anvendelse

har ofte medført forurening af miljøet med alvorlige følgevirkninger for vores naturressourcer.

Hyppig pesticid brug og utilsigtede udslip har ført til forringelse af jordkvalitet og udvaskning af

pesticider har resulteret i grundvandsforurening. Der er derfor brug for nye teknologier til rensning

af forurenet jord og vand. Denne PhD er udført som en del af forskningsprojektet Microbial

Remediation of Contaminated Soil and Water Resources (MIRESOWA), som har det

overordnede mål at udvikle nye teknologier til bioremediering af pesticidforurenet jord og vand.

Formålet med denne PhD var at undersøge svampes nedbrydning af pesticider og efterfølgende at

konstruere mikrobielle konsortier til bioremediering.

I Manuskript I blev svampes nedbrydning af phenylurea herbicidet diuron undersøgt.

Isolater af jordsvampe af Mortierella slægten blev testet for deres evne til at nedbryde diuron.

Svampene blev inkuberet i flydende kulturer med diuron på rystebord ved 8oC. Resultaterne viste at

tre ud af de fem testede stammer kunne nedbryde diuron, og molekulære analyser viste at disse tre

stammer fylogenetisk set udgjorde en nærtbeslægtet gruppe, mens de to ikke-nedbrydere var

placeret længere væk på det fylogenetiske træ. Derudover blev det undersøgt om svampene brugte

diuron som en kulstof eller kvælstof kilde. Her blev den mest effektive diuron-nedbryder

Mortierella sp. LEJ701 inkuberet i flydende medier med forskellige kulstof og kvælstof niveauer.

Diuron nedbrydning var hurtigst i medier med høje kulstof og kvælstof niveauer, mens

nedbrydningen var meget begrænset ved de lave næringsstofniveauer. Dette viser at Mortierella

sandsynligvis ikke bruger diuron som kulstof eller kvælstof kilde. Nedbrydningskinetikken set i

disse eksperimenter viste at diuron blev nedbrudt til metaboliterne 1-(3,4-dichlorophenyl)-3-

methylurea, 1-(3,4-dichlorophenyl)urea samt til et hidtil ukendt nedbrydningsprodukt. Dette

nedbrydningsprodukt blev efterfølgende biosyntesiseret, oprenset og analyseret ved kernemagnetisk

resonans (NMR) og massespektroskopi (MS). Ud fra disse analyser blev metaboliten med stor

sandsynlighed identificeret til at være 1-(3,4-dichlorophenyl)-3-methylideneurea. Dette studie har

bidraget med ny indsigt i fylogenetiske relationer mellem diuron-nedbrydende svampe, samt ny

viden om svampes nedbrydningsvej for diuron.

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De to efterfølgende manuskripter beskæftigede sig med skabelsen af svampe-bakterie

konsortier og undersøge om disses nedbrydning ville være mere effektiv end renkulturernes

nedbrydning under umættede forhold. I Manuskript II blev et konsortium skabt med det formål at

nedbryde pesticidmetaboliten 2,6-dichlorobenzamide (BAM). Et konsortium bestående af

Mortierella sp. LEJ702 og den BAM-nedbrydende bakterie Aminobacter sp. MSH1, samt de

enkelte renkulturer, blev tilført små sandsøjler. Da sandet var blevet tilsat [ring-U-14

C]-BAM kunne

mineraliseringen således bestemmes ud fra mængden af dannet 14

CO2. Derudover blev effekten af

vandindholdet undersøgt ved at tilføre vandmængder svarende til 0, 1.7, 5 eller 10 % af den

vandholdende evne (WHC) til sandet. Konsortiet opnåede en hurtigere mineralisering af BAM

sammenlignet med Aminobacter sp. MSH1 alene, især ved de lavere vandindhold. Disse resultater

blev støttet af de kemiske analyser af 14

C forbindelser i sandet. Det blev yderligere undersøgt om

transporten af bakterier blev forøget når Mortierella var til stede. Dette blev gjort ved ekstrahering

af DNA fra det øverste sandlag efterfulgt af kvantitativ real-time polymerase chain reaction (qPCR)

analyse. Resultaterne viste at antallet af Aminobacter celler i det øverste sandlag var væsentligt

forøget ved tilstedeværelsen af Mortierella. Dette tyder på at svampehyferne fungerer som fysisk

transportvej for bakterierne i sandet. Herudover blev fordelingen af 14

C-BAM i sandet undersøgt i

en ny opstilling. Undersøgelsen viste at tilstedeværelsen af Mortierella forårsagede en svag

forøgelse af BAM spredningen. Dette studie viser tydeligt at det undesøgte svampe-bakterie

konsortium er i stand til at forøge nedbrydningen af BAM under umættede forhold, hvilket gør brug

af dette lovende i forbindelse med bioremediering af jord.

I Manuskript III blev konsortier med to eller tre organismer sammensat af bakterie og

svampe diuron-nedbrydere. Formålet med dette var at danne et konsortium, der var mere effektivt

med hensyn til diuron nedbrydning under umættede forhold og undersøge interaktionerne mellem

mikroorganismerne i dette konsortium. Synergien som fører til en mere effektiv nedbrydning kan

enten skyldes successiv nedbrydning eller fysiske interaktioner mellem organismerne, der forøger

vækst og/eller udbredelse af svampe og bakterier. De anvendte bakteriestammer var Sphingomonas

sp. SRS2, Variovorax sp. SRS16 og Arthrobacter globiformis D47 og svampe-stammerne var

Mortierella sp. LEJ702 og Mortierella sp. LEJ703. I det eksperimentelle set-up blev et lag af sterile

glasperler tilføjet mellem organismerne og den overliggende sandsøjle, for at simulere luftlommer i

jord. [Ring-U-14

C]-diuron blev blandet i sandet givende en koncentration på 100 µg kg-1

.

Nedbrydningen blev målt som mængden af mineraliseret 14

C-diuron og som koncentrationen af 14

C-

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forbindelser i sandet ved eksperimentets afslutning. Mineraliseringsresultaterne påviste tre-

organisme konsortiet LEJ702/SRS16/D47 som det mest effektive, da det omdannede 32% af det

tilsatte diuron til 14

CO2, mens renkulturerne og de andre konsortier alle mineraliserede mindre end

10%. Ydermere viste analyserne af 14

C-forbindelser i sandet at dette konsortium kun havde

produceret minimale mængder af diuron-metaboliter. Interaktionerne mellem organismerne i

konsortierne blev undersøgt ved fosfolipid fedtsyre analyse (PLFA) og 16S rDNA PCR med

specifikke primere. De molekylære resultater indikerede at spredningen af bakterierne SRS16 og

D47 i sandet blev øget ved tilstedeværelsen af Mortierella sp. LEJ702. Kun svampenes fosfolipid

fedtsyrer kunne kvantificeres. Ud fra disse var det imidlertid tydeligt at svampenes vækst blev

kraftigt hæmmet ved tilstedeværelsen af A. globiformis D47. Denne effekt var dog mindsket i tre-

organisme konsortiet LEJ702/SRS16/D47. Dette studie er det første, der viser at et konsortium med

tre organismer af både svampe og bakterie nedbrydere kan forøge pesticidnedbrydning.

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Introduction and aims

This PhD thesis consists of two submitted manuscripts and one manuscript in preparation.

Furthermore, it includes a background discussion of the most relevant literature regarding fungal

degradation of pesticides and what beneficial interactions may be achieved by employing fungal-

bacterial consortia for bioremediation. In these discussions the new findings of this PhD will be put

into context. Finally, there is a conclusion on the work, and perspectives are brought forth on future

research needs in the present area.

This PhD was part of the project entitled Microbial Remediation of Contaminated Soil and

Water Resources (MIRESOWA). In this setting it was predetermined that at least 1/3 of the work

should focus on constructing fungal-bacterial consortia for bioremediation of pesticides. The other

part of the work was focused on fungal degradation of pesticides. As it turned out these two parts of

the work became very well integrated into each other, and as such they both contributed to a deeper

understanding of the other. Another aspect of the work on consortia was that this was carried out in

close collaboration with a PhD student employed at the Geological Survey of Denmark and

Greenland (GEUS). The experimental work was therefore done both at the University of

Copenhagen and at GEUS.

The first aim of this PhD was to study the fungal degradation of pesticides with special focus

on the biology of the pesticide degrading strains found. For the biology behind fungal degradation

of pesticides the following scenarios could be envisioned: (i) utilization of the pesticide as a carbon

or nitrogen source either by uptake or extracellular breakdown of the compound, (ii) that it is a co-

metabolic process occurring by enzymes excreted by the fungus, where the pesticide does not serve

as a carbon and energy source for the fungus, or (iii) that it is a detoxification mechanism for the

fungus when exposed to toxic compounds. Manuscript I uses the phenylurea herbicide diuron as a

model compound to investigate these issues as well as the phylogenetic relationship between

pesticide degrading strains.

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The second aim was to construct fungal-bacterial consortia for bioremediation of pesticides.

The hypothesis being that these would be more efficient than single strains for biodegradation in

water unsaturated heterogeneous environments. The interactions investigated in Manuscript II and

III were beneficial/antagonistic effects on growth and transport of bacteria via fungal hyphae. In

addition, the effects of these interactions on mineralization and metabolite development were

examined. In Manuscript II the pesticide metabolite 2,6-dichlorobenzamide (BAM) served as

model compound to study the effects of a non-degrading fungal strain on the transport and

performance of a BAM-degrading bacterial strain. Additionally, the influence of moisture content

on BAM mineralization by this consortium was examined. In Manuscript III the herbicide diuron

was used and two- and three-member consortia where both fungal and bacterial strains could

degrade diuron were constructed and studied. Giving in this regard a unique synergistic degradation

situation to investigate.

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Background

Pesticide pollution – why should we care

Pesticides are chemical compounds used on agricultural land, but also in private gardens, along

railways and on other public areas. They are targeted to be toxic to certain groups of organisms; e.g.

insecticides target insects to protect crops from being eaten or infected, fungicides target fungi to

protect the crop from fungal attack, and herbicides target specific plants to protect the crops from

weed growth. Pesticide use worldwide is extensive, with the herbicides accounting for the largest

part of overall pesticide use (Grube et al., 2011).

Aside from being toxic to target organisms, pesticides often have side effects on other

organisms. Be it in the same ecosystem, but on non-target organisms, or in other adjacent

ecosystems such as streams and rivers, affecting the organisms there (Ayansina, 2008). Finally

some pesticides may reach the level of human consumption through the contamination of

groundwater from leaching of pesticides. The issue of pesticides in the groundwater is therefore

very serious. In Denmark groundwater is traditionally used for drinking water without any prior

treatment e.g. for pesticide contamination. Consequently, a number of water wells is being closed

annually, because of contamination by pesticides and/or their metabolites in concentrations above

the EU limit value (Brüsch, 2010). The EU maximum permissible value of a single pesticide in

ground- and drinking-water is 0.1 µg l-1

, whereas the sum of several pesticides in the water must not

exceed 0.5 µg l-1

(European Council, 1980). These values are not based upon direct assessments of

health or environmental hazards, but on the precautionary principle since the effects of many

metabolites and effects of mixtures of pesticides are unknown (Brüsch, 2010).

Diuron, dichlobenil and BAM

It is not unusual that pesticides are formulated and thus added as mixtures of two active compounds.

For instance, the two herbicides diuron and dichlobenil may be applied alone or in mixtures with

each other or alternative compounds (Tomlin, 1997). Diuron belongs to the phenylurea herbicides

while dichlobenil is a benzonitrile herbicide, and the properties of these two compounds are rather

different (see Appendix). Although both compounds have been banned from use as pesticides in

Denmark, they still contribute to the contamination of the environment in Denmark as well as in

other countries where they are still in use.

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Diuron is on the EU Water Framework Directive´s list of priority substances (European

Parliament, 2008). One of the reasons being that it has serious effects on the environment and also

on human health as it is suspected of being carcinogenic (Cox, 2003; Giacomazzi and Cochet,

2004). In addition, diuron and its metabolites have been found in surface and groundwater in

concentrations above the EU limit value (Torstensson, 2001; Lapworth and Gooddy, 2006; Struger

et al., 2011).

For dichlobenil the largest problem is that it is readily degraded to the metabolite 2,6-

dichlorobenzamide (BAM), which is highly mobile and therefore leaches to the groundwater

(Clausen et al., 2004). BAM has frequently been detected in surface as well as groundwater in

concentrations above 0.1 µg l-1

(Porazzi et al., 2005; Eriksson et al., 2007; Brüsch, 2010), which has

led to the closure of several hundred water extraction wells in Denmark (Brüsch, 2010).

Though the compounds described above are moderately (diuron) to very (BAM) persistent in

the environment, microorganisms have been isolated that can degrade them. Simonsen et al. (2006)

and Sørensen et al. (2007) have isolated BAM mineralizing Aminobacter strains ASI1 and MSH1.

Holtze et al. (2007) proposed a degradation pathway for dichlobenil and BAM leading to

mineralization through the formation of new metabolites (see Appendix). Degradation of diuron

also occur primarily as a microbial process, and several organisms have been isolated that can

degrade diuron e.g. the bacterial strains Sphingomonas sp. SRS2 (Sørensen et al., 2001), Variovorax

sp. SRS16 (Sørensen et al., 2008), Arthrobacter globiformis D47 (Turnbull et al., 2001) and also a

number of fungal strains (see Table 1).

15

Biodegradation, bioremediation and bioaugmentation

Contamination of the environment with chemicals is a major issue in our time. Chemicals used

domestically, industrially or in agriculture may lead to environmental problems when deteriorating

the quality of soil and groundwater. In these cases bioremediation can be a possible solution for

remediating pollution in the environment. Bioremediation is simply the use of biological organisms

for cleaning up chemical pollution by reducing the concentrations and/or the toxicity of chemical

compounds and restoring natural conditions (Ahemad et al., 2008). The aim is to clean the

environment to protect both biodiversity (e.g. soil habitats and microbial composition) and

resources for human use and consumption (e.g. soil fertility and groundwater quality). As chemicals

are released into the environment in large amounts, both intentionally as is the case with spraying of

pesticides and accidentally through spills, leading to decrease in environmental quality the needs for

bioremediation is growing.

Numerous studies have focused on the use of either plants (see reviews by Alkorta and

Garbisu, 2001; Gerhardt et al., 2009) or microbes (see reviews by Sørensen et al., 2003; Singh,

2006; Ahemad et al., 2008; Das and Adholeya, 2012) for bioremediation of pollutants.

Nevertheless, bioremediation studies have not always yielded successful results showing that

thought bioremediation is a very promising biotechnology it also has its limitations (Juwarkar et al.,

2010). First, some chemicals are not readily susceptible to biological degradation due to their

chemical properties or strong sorption to the environmental matrix. Secondly, microbial degradation

of some compounds may yield metabolites which are more toxic than the starting compound.

Thirdly, many scientific resources have to go into discovering the best microorganism(s) for the job

(Boopathy, 2000). However, with a multidisciplinary approach, involving expertise from

microbiology, chemistry and geology to name a few, it should be possible to achieve satisfactory

bioremediation.

It goes without saying that bioremediation is more environmental friendly than conventional

remediation techniques. Bioremediation is considered a ‗green technology‘ as it only depends on

biological organisms and processes, and it does not require any chemical addition or heating

treatment (Juwarkar et al., 2010). It also presents several advantages over conventional remediation

techniques such as land filling and incineration, e.g. it can be done on site and is therefore less

16

disruptive, it is often less expensive, and it eliminates the pollutant permanently restoring the

vitality of the environment (Boopathy, 2000). Reviews by Juwarkar et al. (2010) and Leung et al.

(2007) describe applied bioremediation technologies e.g. bioaugmentation, biofilters, biostimulation

and establish the many factors that can affect the outcome of bioremediation e.g. temperature,

oxygen level, moisture and nutrients.

Challenges for bioaugmentation

Biodegradation can proceed as a process where the naturally occurring microorganisms degrade

pollutants, however the processes may be enhanced by special conditions in terms of adding

nutrients or electron acceptors (biostimulation) or by addition of specific degrader organisms

(bioaugmentation) (El Fantroussi and Agathos, 2005). The present work focuses on investigating

strains for bioaugmentation, as it is considered a microbial technology with a large potential for

bioremediation, but with great challenges in many regards (Thompson et al., 2005). These

challenges are in the physical and chemical sense the limited bioaccessibility of the compound due

to sorption or physical heterogeneity in the environment. In addition to this, biological challenges

for the introduced organisms are present, such as predation and competition from the naturally

occurring microorganisms. So the success of bioaugmentation not only depends on the ability of the

organism to degrade the pollutant, but very much also on the survival, growth and distribution of

the organism(s). Fungi alone or in combination with bacteria may be better suited to overcome these

challenges, and this subject will be the topic of the following sections.

17

Biodegradation by fungi

Fungal diversity is globally estimated to 1.5 million species (Hawksworth, 1991, 2001) and consist

of an incredibly diverse group of organisms. Organisms studied by mycologists include members of

the fungal Kingdom but also others like Protozoa e.g. slime moulds (Carlile et al., 2001). The

Kingdom of Fungi is divided into three major classes: the zygomycetes, the ascomycetes and the

basidiomycetes. For a thorough review on the fungal diversity see Carlile et al. (2001).

Biodegradation by fungi is also known as mycodegradation. Likewise, bioremediation in

which fungi are employed is sometimes called mycoremediation (Singh, 2006). Fungi are

ubiquitous in the environment, and the literature on fungal ecology is vast. Despite this, the reports

of fungi in bioremediation are under-represented and as such represent the untapped potentials in

fungal bioremediation (Harms et al., 2011). Moreover, the biology and ecology of mycoremediation

have rarely been examined (Singh, 2006).

The overall aim of this chapter is to examine the manner and function of fungal pesticide

degradation. First, biological characteristics of the fungi which are relevant for successful

bioremediation will be highlighted. Then, characteristics of the fungal genus Mortierella, which

was used in all investigations of this thesis, are presented. Lastly, the most relevant studies on

fungal degradation of pesticides will be reviewed.

Filamentous fungi

Studies into the bioremediation potential of microorganisms have for the largest part focused on

bacterial degraders. However, filamentous fungi possess characteristics which are advantageous in

heterogeneous environments. Examples will be given below of fungal traits that in some instances

make them more successful than bacteria.

Although fungi are non-motile they can respond quickly to changing environmental

conditions to survive or escape them (Read, 2007). Abiotic factors that primarily affect the mycelial

growth include temperature, water potential, pH, oxygen accessibility, and nutrient status (Boddy

and Jones, 2007). However, filamentous fungi can escape unfavorable conditions which in

heterogeneous environments give them an advantage. For instance, in soil fungi do not require a

continuous water phase as they can bridge air filled gaps (Wösten et al., 1999; Ritz and Young,

2004), whereas bacteria in general are immobilized and their motility impeded at matric potentials

18

below -2 to -5 kPa (Wong and Griffin, 1976; Dechesne et al., 2010; Wang and Or, 2010). This

might be of importance in coarse soils for instance, where matric potentials may be relatively low,

approximately -5 kPa, causing discontinuities to occur in the water-filled pathways in the soil

(Wong and Griffin, 1976). Fungal hyphae are also able to penetrate solids and reach microhabitats

like water-filled micropores in soil (Gadd, 2007; Thorn and Lynch, 2007). Fungi may in this way

gain a much better access to the nutrients and contaminants in environments where the compounds

are heterogeneously distributed and inaccessible to bacteria in other ways e.g. trapped in micropores

(Harms et al., 2011). In addition, fungal hyphae transport nutrients between the different regions of

the mycelia, which enables fungal growth into otherwise inhospitable zones (Lindahl and Olsson,

2004).

Also, toxic conditions can be overcome by production of long sparsely-branched or

branchless explorative hyphae (Fomina et al., 2003). A growth response which enables expansive

search for less toxic sites with minimal resources spent by the fungus. We also observed this type of

growth when Mortierella sp. LEJ701 was exposed to diuron (Fig. 1D), suggesting that either diuron

or its metabolites were toxic to the fungus (Manuscript I). This type of growth was not observed

for the fungus in control treatments without diuron.

Saprotrophic fungi produce a wide range of extracellular enzymes which are essential for

degradation of plant materials (Kjøller and Struwe, 2002), and these enzymes may also enable

fungal degradation of organic pollutants. White-rot fungi have been considered as top fungal

candidates for bioremediation purposes for many years due to their potent enzymatic arsenal

(Pointing, 2001; Reddy and Mathew, 2001). However there are a number of drawbacks for this

group of organisms, for instance most white rot fungi require strict growth conditions (e.g. high

temperatures) and therefore have low competitive capabilities against indigenous organisms in the

environment (Baldrian, 2008; Gao et al., 2010). Furthermore, it has been shown that a number of

white-rot fungi have negative effects on soil bacteria e.g. inhibiting growth of indigenous bacteria

(Lang et al., 1997; Andersson et al., 2003). For bioremediation of certain compounds fungi from the

zygo- or ascomycetes might therefore be a superior choice. However, the effect on native bacteria

will probably be strain dependent.

19

Mortierella

The zygomycete genus Mortierella consists of numerous species, which are amongst the most

common soil fungi (Domsch et al., 1980). They have frequently been isolated from soil and have a

global distribution (Domsch et al., 1980; Ho and Chen, 2008; Nagy et al., 2011). Morphologically

Mortierella produce white colonies on agar plates (Fig. 1C) and white or pale yellow spherical

cotton-like pellets in liquid media. The mycelium is relatively fast growing and consists of thin and

frequently branching hyphae on which sporangia with spores are formed (Domsch et al., 1980),

these can be visualized microscopically (Fig. 1A and B).

Figure 1. Mortierella; (A) illustration of Mortierella hyphae with sporangia, (B) microscopic

image of DAPI stained Mortierella sp. LEJ702 viewed under UV light depicting hyphae with

sporangia, (C) Mortierella sp. LEJ704 on Potato Dextrose Agar, and (D) Mortierella sp.

LEJ701 in liquid medium supplemented with diuron. Pictures taken by (B): B. E. Knudsen &

L. Ellegaard-Jensen, 2011, (C): L. Ellegaard-Jensen, 2012, and (D): L. Ellegaard-Jensen, 2011.

A B

C D

20

Mortierella are saprotrophic fungi degrading organic materials and utilizing sugars as well as

pectin, cellulose and chitin (Kjøller and Struwe, 1990). This distinguishes Mortierella from other

genera within the Mucorales, which are unable to degrade polysaccharides (Kjøller and Struwe,

2002). In addition, strains of Mortierella have been shown capable of degrading xenobiotic

compounds e.g. polycyclic aromatic hydrocarbons (PAHs) (Salicis et al., 1999) and pesticides

(Tixier et al., 2000; Rønhede et al., 2005; Vroumsia et al., 2005; Badawi et al., 2009; Kataoka et al.,

2011). Because of the abovementioned characteristics with regard to degradation potentials this

PhD focuses on studying the degradation of pesticides by Mortierella to gain a higher

understanding of the biological processes of fungal degradation of these compounds.

Fungal degradation of pesticides

As mentioned, biodegradation by fungi is not widely studied. However, a number of reports on

fungal degradation of pesticides are found in the literature. As it is the fungal degradation potential

of the phenylurea herbicide diuron that is studied in Manuscript I (and III) the literature on this

will be subjected to a short review. This is done with the aim of relating the findings in Manuscript

I to those of previous studies on fungal pesticide degradation, and further seeking to elucidate the

role that the fungal biology plays in diuron degradation.

The literature on fungal degradation of diuron is somewhat inconsistent with regard to what

parameters and which fungal species were studied (see Table 1). Khadrani et al. (1999) and

Vroumsia et al. (1996) both screened a large number of fungal species for their ability to degrade

phenylurea herbicides including diuron. Rhizoctonia solani (Vroumsia et al., 1996) and Bjerkandera

adusta (Khadrani et al., 1999), both species belonging to the basidiomycetes, were identified as the

most efficient degraders of diuron. However, care should be taken when choosing strains for

bioaugumentation and a plant pathogen as Rhizoctonia solani might therefore not be a sound

choice. Instead, others have reported species belonging to the zygomycetes as very efficient

degraders of phenylurea herbicides (e.g. Manuscript I; Tixier et al., 2000; Rønhede et al., 2005;

Hangler et al., 2007).

21

Table 1. Overview of reported studies on fungal degradation of diuron and related studies. Listed

are the compound(s) and parameters studied as well as the fungal species employed. If > 5 species

are tested the number of species is given along with the most efficient diuron degrading species

reported (*)

Compound Fungi Studied Ref.

9 phenylurea herbicides

incl. Diuron

Rhizoctonia solani (b)

Transformation

(Weinberger and Bollag,

1972)

Diuron, Linuron,

Monolinuron, Monuron

& Buturon

Cunninghamella echinulata

Thaxter (z)

Transformation,

metabolites

(Tillmanns et al., 1978)

Diuron, Chlortoluron &

Isoproturon

Rhizoctonia solani (b) *

90 species

Degradation (screening),

biomass

(Vroumsia et al., 1996)

Diuron, Chlortoluron &

Isoproturon

Bjerkandera adusta (b) *

100 species

Degradation

(screening)

(Khadrani et al., 1999)

10 phenylurea herbicides

incl. Diuron

Botrytis cinerea (a) *

8 species

Transformation (Berger, 1998)

Diuron Phanerochaete chrysosporium (b) Degradation, ligninolytic

enzymes

(Fratila-Apachitei et al.,

1999)

Diuron Cunninghamella elegans (z) *

Mortirella isabellina (z)

Beauveria bassiana (a)

Degradation, metabolites,

ecotoxicity

(Tixier et al., 2000)

Diuron Beauveria bassina (a)

Cunninghamella elegans (z)

Aspergillus niger (a)

Mortierella isabellina (z)

Metabolite degradation,

ecotoxicity

(Tixier et al., 2001)

Diuron, Metalaxyl,

Atrazine &

Terbuthylazine

Coriolus versicolor (b) *

9 species

Degradation, ligninolytic

potential

(Bending et al., 2002)

Isoproturon Mortierella sp. (z) *

Mucor sp.(z)

Alternaria sp. (a)

Phoma cf. Eupyrena (a)

Basidiomycete strain Gr177( b)

Degradation, metabolites (Rønhede et al., 2005)

Isoproturon Cunninghamella elegans (z) *

15 species

Degradation, metabolites (Hangler et al., 2007)

Diuron, Chlortoluron,

Isoproturon & Linuron

Mortierella sp. (z) Degradation, metabolites (Badawi et al., 2009)

Diuron 5 Mortierella sp. strains (z) Degradation, metabolites,

phylogenetic relationship,

nutrient effects, biomass

(Manuscript I)

(z): zygomycete, (a): ascomycete, (b): basidiomycete

22

Other authors have focused on the degradation kinetics of phenylurea herbicides (Tillmanns et

al., 1978; Badawi et al., 2009). A fungal degradation pathway has been established where diuron is

transformed by demethylation to 1-(3,4-dichlorophenyl)-3-methylurea (DCPMU) and then further

to 1-(3,4-dichlorophenyl)-urea (DCPU) (Tixier et al., 2000; Badawi et al., 2009). In addition, a

previously unidentified metabolite was reported in Badawi et al. (2009) and Manuscript I. By use

of NMR and mass spectrometry, the compound was suggested to be 1-(3,4-dichlorophenyl)-3-

methylideneurea (Manuscript I). Transformation beyond DCPU appears to occur mainly by

bacterial degradation. Where certain bacterial strains can transform diuron to 3,4-dichloroaniline

(3,4-DCA) (Turnbull et al., 2001) or mineralize it (Sørensen et al., 2008). Tixier et al. (2000; 2001;

2002) found that DCPMU, DCPU and 3,4-DCA were more toxic than diuron when tested in

Microtox assays, with the most toxic being 3,4-DCA. However, it has been shown that certain fungi

can transform 3,4-DCA to the less toxic 3,4-dichloroacetanilide (Tixier et al., 2002; Martins et al.,

2009; Martins et al., 2010; Silar et al., 2011; Manuscript III). Whether diuron or its metabolites are

toxic to fungi remains unclear, thought findings indicate that it could be the case (Manuscript I;

Vroumsia et al., 1996).

So it is clear from the above and from Table 1 that most studies on fungal degradation of

diuron have focused either on finding efficient degraders or on the kinetics of diuron degradation,

whereas fewer studies have investigated the biology to illuminate the function of fungal diuron

degradation. Pesticide degrading fungi have sometimes been shown able to utilize the compound as

a source of both carbon and nitrogen (Kulshrestha and Kumari, 2011). However, studies on

phenylurea herbicides show that fungi do not utilize these as a nutrient source (Berger, 1998;

Rønhede et al., 2005), which was further confirmed to also be the case for diuron (Manuscript I). It

therefore appears that it is a co-metabolic process: A process probably occurring in close proximity

of the fungal hyphae, where enzymes are excreted, since no further degradation occurred after

removal of the fungal mycelium (Manuscript I). Studies so far have not been able to determine

which enzymes are responsible for the process. Also, Bending et al. (2002) could not find any

relationship between ligninolytic activity and degradation of diuron.

Berger (1998) stated that phenylurea herbicides sorbed to the mycelia of certain fungal

species, though the results were somewhat incoherent as contrasting results were obtained when

using non-labeled and 14

C-labeled compounds. In Manuscript I it is shown that the total amount of

23

diuron can be accounted for as parent compound and metabolites at all times during the

degradation, so it is therefore evident that diuron does not sorb to the mycelia of Mortierella.

Previous studies suggest, on the basis of screening a large number of species, that the

degradation potential can vary greatly within a genus. From the results of Vroumsia et al. (2005) it

is seen that the degradation of 2,4-D varies between 0-52% for five Aspergillus species and 11-46%

for four Mortierella species. A similar picture is found for phenylurea herbicides degradation

(Vroumsia et al., 1996). In Manuscript I molecular tools were employed to investigate the

phylogenetic background with regard to diuron degradation within the genus of Mortierella. The

results revealed a link between the phylogenetic position of the fungi and their diuron degradation

potential under the experimental conditions, as the diuron degrading strains constituted a closely

related group. Similar results have also been found for bacterial phenylurea degraders (Hussain et

al., 2009).

All in all, from the above it is evident that fungal degradation of certain pesticides is mainly a

co-metabolic process. A process which is, under certain conditions, linked to specific strains of

Mortierella, probably due to an enzymatic system developed in those polygenetic groups. Whether

this enzyme is continuously produced or onset at the exposure to toxic compounds remains

uncertain.

24

Consortia for bioremediation

Communities of fungi and bacteria have vital functions in the environment, as they separately and

together degrade and mineralize organic compounds (Frey-Klett et al., 2011). It is therefore also

very plausible that consortia consisting of both fungi and bacteria can in some case be more suitable

in a bioremediation context than single strains. However, there are many things to consider when

seeking to construct efficient consortia for bioremediation. Especially the interactions between the

two groups are important to examine to understand the mechanisms within the consortia, avoid

mismatching of organisms, and creation of efficient consortia. In the following section the most

relevant issues are touched upon. Then the specific interaction known as ‗the fungal highway‘ will

be discussed. Finally, studies on consortia of fungal-bacterial degraders are reviewed.

Fungal-bacterial interactions

Fungal and bacterial organisms often share the same microhabitat (Warmink et al., 2009) and as

such interact with each other. In general terms such interactions may be categorized as antagonistic,

neutral or beneficial for the microbes. Antagonism may be the production of antibacterial or

antifungal compounds or simply the competition for nutrients. Competition for nutrients can lead to

great reduction of fungal biomass in the presence of competing bacteria and vice versa (Mille-

Lindblom and Tranvik, 2003), whereas other bacteria may even obtain nutrients by mycophagy i.e.

feeding on living fungi (de Boer et al., 2005; Höppener-Ogawa et al., 2008). A neutral interaction is

one where there is no positive or negative effect of the presence of the other organism, e.g. where

the organisms have different niches. However, what is desirable in a consortium is a beneficial

interaction for it to be efficient for degradation of pesticides. Beneficial interactions can either be

commensal, with one of the organisms benefiting and the other remaining unaffected, or mutual,

with both organisms benefiting from the interaction (Bennett and Feibelman, 2001). It has been

shown that soil fungi can create niches where bacteria can thrive (de Boer et al., 2005). Boersma et

al. (2010) found that the survival of the Variovorax paradoxus-like strain HB44 in sterilized soil

was significantly improved by the presence of the fungus Lyophyllum sp. strain Karsten, and that

the bacterial strain grew readily on compounds, in particular glycerol, released by the fungus. In

addition, Warmink et al. (2009) showed that bacteria found in the mycosphere (fungiphiles) could

utilize specific fungal exudates that bacteria found in the bulk soil could not. This gives the

25

interacting bacteria an advantage in the presence of the fungus where they gain a higher fitness.

However, information about the compounds released by the fungi is still limited (Nazir et al., 2010),

and this area needs further research.

Fungal highway

A more mechanistic type of interaction is the transport of bacteria along the fungal hyphae, termed

the fungal highway (Kohlmeier et al., 2005). Pollutant-degrading bacteria have limited accessibility

to the polluting compound, as well as water and nutrients, in heterogeneous and water-unsaturated

environments e.g. soil. Limited bioavailability is established as perhaps the most critical factor in

bioremediation (Bosma et al., 1996). The fungal highway may, however, improve this in several

ways. First, transport of bacteria can occur in the thin water film around the fungal hyphae (Wick et

al., 2010), consequently leading to a more homogenous distribution of these bacteria in the soil.

Warmink and van Elsas (2009) showed that soil bacteria were transported via the hyphae of

the basidiomycete Lyophyllum sp. strain Karsten. Likewise, Trifonova et al. (2009) found that

certain bacteria were translocated by the hyphae of the ascomycete Conochaeta ligniaria. Besides

transporting bacteria, fungal hyphae have also been shown to enhance transport of chemical

compounds such as sodium salicylate (Furuno et al., 2010), phenanthrene (Furuno et al., 2012), and

2,6-dichlorobenzamide (Manuscript II) in water-unsaturated systems.

Most importantly, it has also been shown that the transport of pollutant-degrading bacteria via

fungal hyphae enhanced the degradation of phenanthrene (Wick et al., 2007), 2,6-

dichlorobenzamide (Manuscript II) and diuron (Manuscript III). Recent findings confirm that

conditions of limited bacterial dispersal also limit degradation and that fungal networks may

drastically improve these adverse conditions for bioremediation in heterogeneous environments

(Banitz et al., 2011a; Banitz et al., 2011b). The ability to increase bacterial dispersal depends,

however, on the fungal strain and the surface hydrophobicity of its hyphae. Studies have shown

bacterial transport to occur almost exclusively along hydrophilic fungal mycelia (Kohlmeier et al.,

2005; Wick et al., 2007). Bacterial transport via the two Mortierella strains LEJ702 and LEJ703

was investigated in Manuscript III, and a clear difference was found for the two strains as bacterial

transport could only be observed in the presence of LEJ702. This could possibly be due to

differences in the surface hydrophobicity of these to Mortierella strains, though this was not

measured.

26

Degradation by consortia

In general, choosing ‗the right‘ organism for bioremediation, be it fungus or bacterium, is of great

importance. Members of both kingdoms have their advantages in a given environment. A possible

means to benefit from this fact could be to create consortia of both bacterial and fungal degraders

(See Fig. 2). Several authors have suggested this as a promising strategy to achieve more efficient

degradation (Bennett et al., 2007; Mikesková et al., 2012). However, very few studies are found in

the literature where isolates of fungi and bacteria are applied as consortia or co-cultures. Boonchan

et al. (2000) examined degradation of PAHs by Penicillium janthinellum in co-culture with S.

maltophilia both isolated from soil. Results showed that the co-culture degraded 5-ringed PAHs

more efficiently than either single strain alone, and from the compounds developed during

degradation the authors concluded that this was due to cooperative catabolism. On the other hand,

Arun and Eyini (2011) found that co-cultures entailing basidiomycetes plus B. pumilus could not

enhance the degradation of 2-4 ringed PAHs.

Examples are seen of fungi or bacteria degrading pesticide-metabolites produced by the other

organism, suggesting that cooperative catabolism could occur in fungal-bacterial consortia.

Rønhede et al. (2007) showed that fungal hydroxylation of isoproturon in soil generally enhanced

the bacterial mineralization of this compound. Furthermore, Tixier et al. (2002) found that fungi

could transform diuron metabolite 3,4-DCA produced by Arthrobacter sp. N2 to the less toxic 3,4-

dichloroacetanilide (3,4-DCAA). We also found 3,4-DCAA to be produced by Mortierella

especially when it was grown in consortia with Arthrobacter globiformis strain D47 (Manuscript

III), and though this transformation might not lead to mineralization the process reduces the

concentration of the most toxic diuron metabolite.

To my knowledge there are no previous reports on fungal-bacterial consortia constructed for

degradation of pesticides. However, it has been shown that bacterial co-cultures greatly enhance

degradation of phenylurea herbicides (Sørensen et al., 2002; Sørensen et al., 2008). In Manuscript

III it was examined whether consortia of fungal and bacterial diuron degraders could achieve more

efficient degradation than the single strains and the interactions between the organisms were

investigated. Diuron mineralization results showed that the three-member consortium entailing

Variovorax sp. SRS16, Arthrobacter globiformis strain D47 and Mortierella sp. LEJ702 was

27

superior to both the single strains and the other consortia tested in sand columns. In this experiment

the same advantage could not be found with the other fungal strain tested. This is somewhat in

agreement with Machín-Ramírez et al. (2010) who found that certain defined co-cultures of fungi

and bacteria could enhance the degradation of a 5-ringed PAH, while other combinations of fungal-

bacterial degraders did not.

Figure 2. The possible

synergistic effects within a

fungal-bacterial consortium

leading to enhanced

biodegradation of the

pesticide. Bacteria often

perform best in water

saturated systems were the

accessibility to the pesticide

is high. Filamentous fungi

are well adapted to

heterogeneous environments

like soil as the hyphae may

for instance grow through

air-filled gaps. As described

in the text interactions in a

fungal-bacteria consortium

can lead to superior

degradation efficiency.

In Figure 2 the most important synergistic effects within a fungal-bacterial consortium leading

to enhanced biodegradation are illustrated. Above all is the ability of the organisms to co-exist as it

is the foundation of any synergistic relationship. If one of the organisms in a consortium severely

inhibits the growth or activity of the other, then the benefit of having them together will be

eliminated. In Manuscript III, for instance, the presence of A. globiformis D47 inhibited the

growth of the two Mortierella strains. However, this effect was somewhat alleviated in the three-

28

member consortium Variovorax sp. SRS16, A. globiformis D47 and Mortierella sp. LEJ702

possibly contributing to enhanced degradation by this consortium.

From the results obtained in Manuscript III and the studies discussed above it is clear that

consortia of fungal-bacterial degraders can yield more efficient degradation of pollutants.

Nevertheless, these studies also show that care, consideration and research are needed to construct

and investigate these consortia as the organisms employed must be compatible in several ways.

29

Conclusions and perspectives

This thesis has through the results obtained and presented in the enclosed manuscripts contributed

with new knowledge on the degradation of pesticides by fungi. In Manuscript I it was established

that Mortierella does not utilize the pesticide diuron as a carbon or nitrogen source, and it was

therefore concluded that the degradation occurs as a co-metabolic process. Furthermore, it was

shown that there, under the experimental conditions of this study, was a phylogenetic relationship

between the diuron degrading strains. Lastly, a previously unknown diuron metabolite, one that

might be specific for fungal degradation, was found and identified.

Future research should focus on examining the enzymatic activity of the fungal pesticide

degrades; seeking to elucidate the key enzyme(s) for the degradation. The activity of these enzymes

may be linked to certain phylogenetic groups as suggested in Manuscript I, and studying this could

bring us one step closer to discovering the genes responsible for the degradation.

Differences in the fungal degradation by individual strains (e.g. LEJ702 or LEJ703) in

Manuscript I, II and III points to that abiotic factors like temperature, moisture and growth matrix

influence fungal pesticide degradation. This is not surprising but may also be an area of further

study.

The other parts of the work focused on creating and investigating consortia for bioremediation

of pesticides. Successful construction of fungal-bacterial consortia for degradation of diuron and

BAM was achieved and presented in Manuscript II and III, respectively. In addition, the

hypothesis that these consortia would be more efficient than single strains for biodegradation in

unsaturated systems was confirmed, though this depended greatly on which organisms where

included in the specific consortium. It therefore also proved useful to examine the interactions

between the fungal and bacterial strains within the consortia to elucidate which mechanism(s)

where responsible for the differences in degradation efficiency (e.g. for consortia with Mortierella

sp. LEJ702 or LEJ703). For instance, Mortierella sp. LEJ702 mediated the transport not only of the

BAM-degrading Aminobacter sp. MSH1, but also of the diuron-degrading Variovorax sp. SRS16

and Arthrobacter globiformis D47, whereas no bacterial transport where found in consortia with

Mortierella sp. LEJ703. In addition, the growth of these two Mortierella strains was not equally

affected by the presence of the bacteria in the three-member consortia with Variovorax sp. SRS16

and Arthrobacter globiformis D47. Therefore, this underlines the importance of careful strain

30

selection when constructing consortia for bioremediation, as there are clearly differences between

strains, even within the same genus, with regard to the degradation efficiency and the extent of

interactions with the other members of the consortium. That Mortierella sp. LEJ702 worked well in

consortia for both diuron and BAM degradation suggests this as a superior candidate strain for

further research on fungal-bacterial consortia for biodegradation of pesticides.

The BAM degrading consortium and the most efficient diuron degrading consortium both

have potential for use for bioremediation of soils contaminated with these compounds. Indeed it is

possible that the synergistic effect may be even greater in soil where the pesticides are

heterogeneously distributed and sorbed to soil particles. Here the fungal mediated transport of

degrader bacteria could prove essential and the extent of this transport should be monitored along

with the survival of the introduced strains and the pesticide degradation.

A challenge when moving towards applying consortia such as these for soil bioremediation

may be that the heterogeneity of organic compounds and microbes in the soil environment might

destabilize the consortium, possibly leading to poor degradation and/or accumulation of toxic

metabolites. The interactions with indigenous soil microorganisms should therefore be thoroughly

investigated together with performance of the consortium in soil mesocosms with regard to its

degradation efficiency, before establishing its applicability for in situ bioremediation.

Formulation of the consortia in carriers could be a possible means to achieve successful

introduction and stabile growth of the consortia in the soil matrix. The microorganisms would be

embedded in synthetic beads supplemented with carbon and nitrogen sources supporting them in the

first phase of growth after introduction into the soil environment. The microorganisms may in this

way reach a sizeable biomass and be better fit for competition with indigenous organisms. Since the

strains applied in these consortia are isolated from soil there is a real possibility that they can in fact

be successfully re-inoculated into the soil matrix.

31

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Manuscript I

DEGRADATION OF THE PHENYLUREA HERBICIDE DIURON BY SOIL FUNGI:

DIFFERENT DEGRADATION POTENTIALS OF FIVE MORTIERELLA STRAINS

Lea Ellegaard-Jensen, Jens Aamand, Birthe B. Kragelund, Anders H. Johnsen and Søren Rosendahl

In revision for Biodegradation

43

Degradation of the phenylurea herbicide diuron by soil

fungi: different degradation potentials of five Mortierella

strains

Lea Ellegaard-Jensena,b *, Jens Aamandb, Birthe B. Kragelundc, Anders H. Johnsend and Søren Rosendahla.

a Department of Biology, Copenhagen University, Universitetsparken 15, DK-2100 Copenhagen Ø,

Denmark. b Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Øster

voldgade 10, DK-1350 Copenhagen K, Denmark.c Structural Biology and NMR Laboratory, Department of

Biology, University of Copenhagen, Ole Maaloes Vej 5, DK-2200 Copenhagen N, Denmark. d Department of

Clinical Biochemistry, Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhahagen Ø, Denmark

* Corresponding author: Tel.: 45 3532 2257; Fax: 45 3532 2321; E-mail: Leael@bio.ku.dk

Abstract Microbial pesticide degradation studies have until now mainly focused on bacteria, although fungi

have also been shown to degrade pesticides. In this study we clarify the background for the ability of the

common soil fungus Mortierella to degrade the phenylurea herbicide diuron. Diuron degradation potentials

of five Mortierella strains were compared, and the role of carbon and nitrogen for the degradation process

was investigated. Results showed that strains able to degrade diuron constituted a closely related group,

indicating that diuron degradation was not a general feature, but limited to a phylogenetic group within

Mortierella. Degradation of diuron was fastest in carbon and nitrogen rich media while suboptimal nutrient

levels restricted degradation, making it unlikely that Mortierella utilize diuron as carbon or nitrogen sources.

Degradation kinetics showed that diuron degradation was followed by formation of the metabolites 1-(3,4-

dichlorophenyl)-3-methylurea, 1-(3,4-dichlorophenyl)urea and an hitherto unknown metabolite suggested to

be 1-(3,4-dichlorophenyl)-3-methylideneurea.

Keywords Fungal biodegradation, Phenylurea herbicide, Diuron, Fungal genetics, Phylogenetic

relationships

44

Introduction

Pesticide pollution is one of the major concerns regarding contamination of the environment.

Pesticides are mainly used on agricultural land, but also in private gardens, along railways and other

public areas. Herbicides account for the largest part of overall pesticide use worldwide (Grube et al.

2011). Among these are the phenylurea herbicides which are frequently found as environmental

pollutants (Eriksson et al. 2007; Lapworth and Gooddy 2006; Struger et al. 2011). The phenylurea

herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethyl-urea] is on the EU Water Framework

Directive´s list of priority substances (European Parliament 2008) and as it is often found in

groundwater at concentrations exceeding the EU limit of 0.1µg l-1

(Torstensson 2001; Lapworth and

Gooddy 2006) biodegradation of this compound is a central issue.

The majority of studies on microbial pesticide degradation have focused on bacteria (e.g. El-

Bestawy and Albrechtsen 2007; El-Deeb et al. 2000; Sørensen et al. 2008; Simonsen et al. 2006),

whereas fewer studies examine the role of fungi (Harms et al. 2011). Several fungi are known to

produce enzymes that are able to degrade aromatic herbicides and have therefore been suggested as

candidates for bioremediation (Entry et al. 1996).

Fungi can account for up to 75% of soil microbial biomass (Harms et al. 2011) and their

hyphae can grow to a length of 102-10

4 m g

-1 soil (Ritz and Young 2004). Fungi may play an

important role in bioremediation of soil as the fungal hyphae can grow into micropores between soil

aggregates and through air filled gaps (Wösten et al. 1999) and in this way gain a better access to

the compound to be degraded.

Earlier studies have shown that certain fungal species/strains have the ability to degrade

diuron (Badawi et al. 2009; Khadrani et al. 1999; Tixier et al. 2000; Tixier et al. 2001; Vroumsia et

al. 1996). These studies have given insight into formation of different metabolites as well as toxicity

of the parent compound and the metabolites. The studies usually include a number of species and

consequently compare differences in diuron degradation potential among fungal species. Such

differences may likely reflect differences in enzymatic abilities and adaptation to different habitats,

but little is known about the variation within fungal genera or species concerning their ability to

degrade pesticides.

The common soil fungus Mortierella is one of the fungi that are capable of degrading

phenylurea herbicides e.g. isoproturon (Rønhede et al. 2005) and diuron (Tixier et al. 2001; Tixier

et al. 2000; Vroumsia et al. 1996). Nevertheless, it is not known whether this ability is a general

characteristic of the genus or the tested isolates were unique in this respect. In the present study we

45

therefore compare Mortierella isolates, originating from the same agricultural field, to investigate if

the ability to degrade diuron is restricted to a specific phylogenetic group or it is a general feature in

the genus.

Though many microorganisms have been shown to degrade pesticides, the physiological

significance of the degradation remains unclear. For some pesticide degrading bacteria it has been

shown that they are able to utilize the compound as a carbon or a nitrogen source (e.g. El-Deeb et

al. 2000). Whether the same could be the case for fungal degradation remains unclear.

Alternatively, degradation could also be a co-metabolic detoxification process, as some compounds

may share characteristics with natural compounds. To answer these questions we investigate if

Mortierella can utilize diuron as a carbon and a nitrogen source, or whether degradation is caused

by co-metabolism.

Materials and methods

Chemicals and media

Analytical-grade standards were purchased from Dr. Ehrenstorfer GmbH (Augsburg, Germany):

Diuron [N-(3,4-dichlorophenyl)-N,N-dimethyl-urea] (CAS no. 330-54-1; 97.5% purity), DCPMU

[1-(3,4-dichlorophenyl)-3-methylurea] (CAS no. 3567-62-2; 97.5% purity), DCPU [1- (3,4-

dichlorophenyl) urea] (CAS no. 2327-02-8; 99% purity), and 3,4-DCA [3,4-dichloroaniline] (CAS

no. 95-76-1; 99% purity). 3,4-DCAA [3,4-dichloroacetanilide] (CAS no. 2150-93-8) was purchased

from Sigma-Aldrich (St. Louis, Missouri). Acetonitrile-D3 was from Eurisotop (99.8% D).

For fungal isolation the following growth media were used: corn meal agar (CMA), synthetic

nutrient deficient agar (SNA), and potato dextrose agar (PDA) all autoclaved and supplied with

ampicillin (5ml l-1

) and streptomycin (5ml l-1

). In addition modified Melin Norkans medium

(MMN), with the fungicide Benomyl (trade name: Benlate, DuPont), was synthesised according to

Kjøller and Bruns (2003). After isolation all fungi were maintained on PDA plates. Liquid glucose-

mineral media used for degradation experiments were made according to Badawi et al. (2009).

46

Isolation and characterization of fungi

Fungi were isolated from an agricultural field in Græse, Denmark (55° 51' 51 N, 12° 5' 44 E) with a

previous history of phenylurea (isoproturon) application. For isolation CMA, SNA, PDA and MMN

media all with ampicillin and streptomycin were used to select for different fungal strains with

different growth requirements.

To characterize the isolates, DNA was extracted from fungal mycelia using the following

protocol: 20µl Chelex, 40µl TE buffer and mycelia heated to 95oC for 2 min followed by

centrifugation at 13.000g for 2 min, DNA now in the supernatant was transferred to clean tubes and

frozen (-18 oC). PCR amplification of the internal transcribed spacer (ITS) region was performed as

described by Gardes and Bruns (1996) with the following modifications: Amplification for 35

cycles consisting of denaturation for 35 s at 94°C, annealing for 55 s at 55°C and extension at 72°C

for 45 s rising with 4 s per cycle. 10 × diluted fungal DNA was amplified with primers ITS 1F

(Gardes and Bruns 1993) and ITS4 (White et al. 1990). PCR products were sequenced by Macrogen

(Seuol, Korea) and the sequences aligned in MEGA version 5 (www.megasoftware.net/index.php).

The most similar sequences were found by BLAST search in Genbank. The phylogenetic

relationship between the isolates and related sequences from Genbank was made by Maximum

likelihood in MEGA version 5 (Tamura et al. 2011), and the robustness of the phylogenetic tree

tested by 1000 bootstraps.

Fungal degradation potential

Five Mortierella isolates were chosen for further studies (see table 1). The experiment was

conducted to test the potential of the different fungal strains for degradation of diuron. It was

carried out in 100ml screw-cap flasks with Teflon-lined lids. Initially 25µl of stock solution diuron

(5000ppm) dissolved in acetonitrile was added to flasks, acetonitrile was allowed to evaporate, and

25ml of glucose-mineral medium was added to each flask to give a final concentration of 5mg

diuron l-1

. Inoculation for each flask was done with three agar plugs (ø = 4mm) with mycelium cut

from PDA plates. The experiment was carried out in triplicates of each treatment, and with an

abiotic treatment to serve as control.

Fungal cultures were incubated on an orbital shaker at 110 rpm in the dark at 8oC. Repeated

aliquots sampling from flasks were done with 1ml sterile syringes, transferring samples to UPLC

47

vials through a syringe filter (0.2 µm PTFE membrane, 15mm). Samples were frozen (-18oC) until

UPLC analysis.

Diuron degradation by Mortierella sp. LEJ701 at different C and N concentrations

One fungal strain, Mortierella sp. LEJ 701, was used to investigate the effect of different carbon

and nitrogen concentrations on fungal diuron degradation. Experimental setup was done as above

with the following changes: for different glucose treatments three different media were used with 5

g, 0.5 g or 0 g glucose l-1

(only glucose available to the fungi is from the agar plug ≤ 0.045 g l-1

).

For all treatments (three glucose levels and with/without diuron) 12 flasks were prepared for

destructive sampling of triplicates at four pre-determined sampling times. At sampling aliquots

were taken for UPLC analysis as described above.

In addition, effects of different nitrogen concentrations were also studied. Again the setup had

six treatments (three nitrogen levels and with/without diuron). NH4Cl in the media was (l-1

): 1 g,

0.04 g or 0 g (only N available to the fungi is from the agar plug ≤ 0.001 g l-1

). Three additional

flasks were used where fungal mycelia were removed after 10 days to test if extracellular enzymes

would continue the diuron degradation in the media. In both experiments described above fungal

biomasses were collected on filters and dry-weights were measured (80oC for 20h).

UPLC analysis of diuron and metabolites

Ultra performance liquid chromatography (UPLC) was performed for quantitative analysis of

diuron and its metabolites in the culture media. The system used was an Acquity UPLC (Waters,

Milford, MA) equipped with a UV detector (200-225 nm absorbance) and an Acquity BEH C18

column (1.7 µm particle size, 2.1 mm inner diameter, and 100 mm length) set at a constant flow rate

of 0.4 ml min-1

, column temperature of 43oC, and an injection volume of 10µl. The initial mobile

phase was composed of 30% acetonitrile and 70% water with a 5 min gradient ending with 35 %

acetonitrile and 65% water. Hereafter conditions returned to the starting values giving a total

analysis time of 6 min per sample.

Retentions times for the compounds analyzed were: DCPU 2.45 min, DCPMU 3.33 min, 3,4-

DCAA 3.96 min, diuron 4.24 min, and 3,4-DCA 4.72 min. In addition, an unknown metabolite with

a retention time of 2.16 min was also detected.

48

Biosynthesis of the unidentified metabolite

Mortierella sp. LEJ701 was inoculated in flasks containing glucose-mineral medium and diuron (5

mg l-1

), as described above, for large scale production of the unknown metabolite for identification

by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). Flasks were incubated on an

orbital shaker at 110 rpm in dark at 8oC for 20 days. Sampling was done with 1ml sterile syringes,

transferring samples to 2 ml vials through syringe filters (0.2 µm PTFE membrane, 15mm).

Samples were frozen (-18oC) until HPLC analysis.

Preparative HPLC

Reverse phase HPLC was used to purify the unknown metabolite, and the identity was determined

by NMR spectroscopy and MS. The system used was an ÄKTApurifier (GE Healthcare, Munich,

Germany) equipped with a UV detector (210-212 nm absorbance) and a Phenomenex Luna C18

column (5µm, 100Å; 4.6 mm x 100 mm). Preparative HPLC was performed using a gradient

program with a flow rate of 1.0 ml min-1

. The initial mobile phase was composed of 30%

acetonitrile and 70% water with a 30 min gradient ending with 50 % acetonitrile and 50% water.

The injection volume was 1.8 ml. Fractions of 0.5 ml were collected. The identity and elution

positions of the known compounds, diuron, DCPU, DCPMU and 3,4-DCA, were confirmed by

mass spectrometry.

NMR spectroscopy

Fractions containing the unknown metabolite were isolated, pooled, lyophilized and dissolved in

CD3CN (99.80 %D) to a volume of 600 µL and transferred to a 545 NMR tube. A parallel sample

of diuron was prepared from lyophilized powder and analyzed and a comparative sample of the

same volume of HPLC buffer at a matched % of acetonitrile was lyophilized and dissolved in

CD3CN (99.80 %D). For all samples 1D 1H NMR spectra were recorded on a Varian INOVA 750

MHz NMR spectrometer equipped with a 5 mm triple-resonance probe and Z-field gradient at 298

K utilizing 128 (diuron), 80.000 (solvent) or 80.000 (metabolite) transients. The spectra were

49

transformed and analyzed using MesTreNova (Mestrec Research, Spain) and referenced to internal

CD3CN at 1.94 ppm.

MS analyses

Two µl fractions from the preparative HPLC were diluted with 8 µl 0.5 % formic acid and analyzed

by LC-MS. The set-up consisted of an EASY nanoLC (Proxeon, Odense, Denmark) in front of a

micrOTOF Q II (Bruker, Bremen, Germany). HPLC conditions were: A 75 µm x 100 mm 3 µm

C18 column (Thermo Scientific, Copenhagen, Denmark) eluted at 300 nl/min with a 10 min

gradient from 10 – 50 % B (A: 0.5 % HCOOH in H2O, B: 0.5 HCOOH in CH3CN).

Statistics

Statistical analyses were done in SigmaPlot version 11.0 (Systat Software Inc). Results from

different treatments were tested for significance with ANOVA including Tukey Test for pairwise

multiple comparison procedures. Linear regression was performed to test the correlation between

amount of fungal biomass and diuron degradation. Differences are found statistically significant

when p ≤ 0.05. Data are presented as means ± standard error (SE) unless stated otherwise.

Strain deposition and nucleotide sequence accession numbers

The five Mortierella strains LEJ701-LEJ705 are deposited at the Centraalbureau voor

Schimmelcultures under the accession numbers CBS 133175-CBS 133179 in the CBS Collection of

Fungi. In addition, the sequences determined in this study have been deposited in the Genbank

database under accession numbers JX206802-JX206806.

50

Results

Phylogeny of fungal pesticide degraders

The five strains showed very different potential for degradation of diuron (Table 1). The most

efficient diuron degrader Mortierella sp. LEJ701 was able to degrade all diuron in the medium and

the two closely related Mortierella sp. isolates LEJ702 and LEJ704 showed some potential for

diuron degradation removing 33.6% and 9.2% of the diuron, respectively. The two strains that did

not degrade diuron were found to be only distantly related to the diuron degraders (Fig. 1). The

most efficient diuron degrader Mortierella sp. LEJ701 had a sequence similarity of 97% and 96%

with the two other degraders Mortierella sp. LEJ702 and LEJ704, respectively. Whereas

Mortierella sp. LEJ701 only had a sequence similarity of 92% and 91% with the non-degraders

Mortierella sp. LEJ703 and LEJ705, respectively.

Substrate effects on diuron degradation

Mortirella sp. LEJ701 clearly responded to the level of glucose in the medium. Diuron degradation

was faster and more complete in the medium with a high glucose concentration compared to the

degradation in media with intermediate and low glucose concentrations (Fig. 2). The diuron

degradation rate in the low glucose medium was very slow, whereas the degradation rates were

significantly higher for intermediate and high glucose concentration media (p < 0.001) (see Table

3). No significant difference between degradation rates was found for intermediate and high glucose

media (p = 0.154).

Fungal biomass differed significantly between the three glucose treatments at experimental

termination (Table 2, ANOVA Tukey test; all p < 0.001). Diuron degradation was positively

correlated with fungal biomass (p < 0.001, r2

= 0.683; Fig. 3), and the degradation rates per unit of

biomass differed significantly for the three treatments (i.e. carbon concentration) (Table 3).

The results showed no effect of diuron on fungal biomass quantity at experimental

termination (p = 0.607; Table 2), although an alteration in pellet structure was observed. The fungal

pellets in treatments with diuron had long hyphal outgrowths from the otherwise spherical pellets.

The effect of nitrogen on diuron degradation was very similar to the effects observed with

glucose concentrations. Diuron degradation was slow in the treatment with low nitrogen

concentration and significantly higher in the treatments with intermediate nitrogen concentration (p

51

< 0.001). Finally the treatment with high nitrogen concentration showed the fastest degradation rate

(Table 3; p < 0.001).

There was no degradation in the abiotic controls. Furthermore no further degradation was

seen by possible exudates after removal of fungal biomass (data not shown).

Accumulation and identification of diuron metabolites

Diuron degradation and accumulation of the two metabolites DCPMU and DCPU corresponded

with the patterns shown in previous studies (Badawi et al. 2009). The first metabolite DCPMU

accumulated as the diuron concentration decreased and after approximately ten days the second

metabolite DCPU appeared (Fig. 2a). In addition, to these compounds, our results showed an

unknown metabolite with a retention time of 2.16 min observed by UPLC (Fig. 4). When this

metabolite was included we achieved complete recovery of the diuron initially added to the system

(Fig. 2a & b).

To identify the unknown metabolite it was produced in large-scale and analyzed by NMR

spectroscopy and MS analyses. From the analytical MS analyses it was directly apparent that the

unknown metabolite-fraction from the preparative fractionation contained two different species of

molecular mass (m.m.) 204.004 Da and 215.996 Da, (monoisotopc values), corresponding to DCPU

(theoretical m.m. = 203.986 Da) and the unknown metabolite as well as trace amounts of intact

diuron (m.m. = 232.019 Da compared to the theoretical 232.017 Da). Hence any quantitative NMR

analysis would be meaningless. Despite the very low concentration, and hence low signal-to-noise

in the NMR spectrum, it was however immediately apparent that compared to the NMR spectrum of

diuron, several sets of peaks originating from the aromatic ring suggested this to be intact also in the

metabolite with both chloride atoms as well as the amide proton present. The two chloride atoms

were corroborated from the 35

Cl-37

Cl isotope ‖signature‖ in the MS spectra. In addition, from the

molecular mass and isotopic pattern using the software application SmartFormula (Bruker

Daltonics) the composition of the unknown metabolite was suggested to be C8H6ON2 (theoretical

m.m. = 215.986 Da) with high confidence. None of the methyl groups were present in the NMR

spectrum as concluded from lack of these signals in the aliphatic region, suggesting that the fraction

of diuron in the NMR sample was below detection. A small but significant change in the chemical

shifts for all the aromatic protons ortho-positioned relative to the amide group suggested a change

in the chemistry of this side group compared to diuron, although the structure was not elucidated by

52

NMR due to the sparse amount of sample. The shorter retention time on the UPLC excluded the

metabolite as either DCPMU or DCPU, so based on the NMR data, the molecular mass, the

suggested atomic composition of the metabolite, as well as the lack of methyl groups, we suggest

the metabolite to be 1-(3,4-dichlorophenyl)-3-methylideneurea (DCPMDU). Figure 5 depicts the

proposed degradation pathway of diuron, including the most likely position of 1-(3,4-

dichlorophenyl)-3-methylideneurea based the degradation kinetics (Fig. 2). The molecular masses

were calculated as m/z-values – 1.0078 (H+).

Discussion

Our results show that the ability to degrade diuron among the Mortierella strains was linked to their

phylogenetic position. This is in agreement with a phylogenetic analysis by Hussain et al. (2009)

who demonstrated that bacterial strains degrading isoproturon were closely related. The most

efficient degrader Mortierella sp. LEJ701 and the two other degraders Mortierella sp. LEJ702 and

LEJ704 showed 96-97% genetic similarity, suggesting that though they are closely related they may

belong to separate species. Nevertheless these closely related strains share a particular trait enabling

them to degrade diuron by demethylation. Vroumsia et al. (1996) found, among the 90 fungal

strains they screened for degradation potential, that four Mortierella isolates had degraded 19 - 54%

diuron after 7 days incubation at 24oC. These values are within the same range as in the present

study, though we achieved this at a much lower temperature – one comparable to average yearly

temperature in a temperate climate. Furthermore, their work was not supported by molecular data

on the fungal phylogeny, and it is therefore not possible to evaluate the phylogenetic relationship

between the isolates.

The intra- and inter-specific differences in ability to degrade pesticides clearly demonstrate

the need for further studies to clarify the genetic background for this characteristic. Future

functional genetic studies should clarify these differences and may facilitate more focused isolation

strategies for potential pesticide degraders. For biotechnological purposes this will be important as

it may be possible to isolate strains with even higher potential for bioremediation under natural

conditions.

Our results show that it is unlikely that the fungus utilizes diuron as a carbon and/or nitrogen

source, since growth was not stimulated by diuron addition even at the lowest levels of glucose and

NH4Cl where diuron C and N would otherwise contribute significantly to biomass quantity. On the

53

contrary our results showed that Mortierella sp. LEJ 701 required an additional carbon and nitrogen

source to be able to degrade diuron. Likewise, Rønhede et al. (2005) found that isoproturon did not

serve as an energy or nutrient source for the fungi used in their experiment. In contrast, Kulshrestha

and Kumari (2011) found that an Acremonium strain could utilize the organophosphate chlorpyrifos

as a source of both carbon and nitrogen. Since the Mortierella strain cannot utilize diuron as a N or

C source it seems plausible, that the degradation process is co-metabolic mediated by enzymes

excreted by the fungus during growth leading to successive demethylation through the metabolite

DCPMDU.

We did not detect a decrease in fungal biomass quantity in treatments with diuron (Table 2).

This is in contrast to Vroumsia et al. (1996) who reported a minor growth inhibition of Rhizoctonia

solani exposed to a four times higher diuron concentration. However, we did observe an altered

morphology of the fungal pellets during the experiment. The growth response characterized by long

hyphal outgrowths from the pellet is often a response to stress e.g. toxic or lack of nutrients

(Fomina et al. 2003). Toxicity of diuron or its metabolites towards the fungus can therefore not be

excluded, and the co-metabolic degradation process could be a detoxification mechanism.

Ecotoxicity studies by Tixier et al. (2000) have shown that the metabolites DCPU and

DCPMU are three times more toxic than diuron. It is consequently of concern that the degradation

of diuron by common soil fungi leads to accumulation of these compounds (see Fig. 2). We have no

information on the toxicity of the new metabolite since only small amounts could be synthesized,

but seen in a regulatory context all these metabolites should be included in monitoring programs as

well as in approval of pesticides on the basis of their toxicity and potential accumulation in the

environment.

In conclusion, we have shed light on the phylogenetic relationship within the Mortierella

genus in respect to diuron degradation potential. Also we have shown that diuron degradation by

Mortierella is a co-metabolic process resulting in accumulation of its metabolites. Further research

should include degradation of these metabolites. We suggest a possible way of achieving fast diuron

degradation beyond these intermediate metabolites could be by developing consortia incorporating

both fungal and bacterial strains.

Acknowledgments The authors thank Nora Badawi and Spire Kiersgaard for guidance on the UPLC method and Signe

Sjørup and Allan Kastrup for expert technical assistance. The work was supported by the MIRESOWA project funded

by the Danish Council for Strategic Research (grant number 2104-08-0012).

54

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56

Table 1 Fungal strains isolated from soil and tested for diuron degradation potential

Strain

Most related organisma

GenBank

nr.

Genetic

similarity

(%)

Diuron

degradation

(%)b

LEJ701 Mortierella sp. (strain CBS 118520) AJ890432.1 99 100.0 (±0.00)

LEJ702 Mortierella elongata (isolate RR 171) AJ878504.1 100 33.6 (±2.29)

LEJ703 Mortierella alpina (strain CBS 224.37) AJ271630.1 99 Nd

LEJ704 Mortierella sp. 68 EU877758.1 99 9.2 (±5.88)

LEJ705 Mortierella sarnyensis (CBS 121162) FJ161927.1 97 Nd a BLAST search in GenBank of PCR amplified DNA (ITS) sequence.

b Diuron degradation experiment

conducted in liquid media at 8○C for 43 days. Nd: no degradation. Initial diuron concentration 5 mg l

-

1.

Table 2 Mortierella sp. LEJ 701 biomass at different carbon and nitrogen

concentrations

Treatment

Diuron

Biomass (mg) Control

Biomass (mg)

Low C

22.7 ± 1.1 22.4 ± 3.3

Intermediate C

31.1 ± 1.3 32.5 ± 0.8

High C

47.6 ± 0.1 43.8 ± 2.4

Low N

23.5 ± 1.3 26.6 ± 2.9

Intermediate N

38.2 ± 2.9 39.9 ± 1.5

High N

53.8 ± 3.9 58.1 ± 12.9

Biomasses at termination of experiments (day 33-34) with diuron (5

mg l-1

) or without diuron (control). Low C: ≤ 0.045g glucose l-1

,

Intermediate C: 0.5g glucose l-1

, and High C: 5g glucose l-1

. Low N:

≤ 0.001g NH4Cl l-1

, Intermediate N: 0.04g NH4Cl l-1

, and High N: 1g

NH4Cl l-1

. Numbers are means ± SE.

Table 3 Diuron degradation rates for Mortierella sp. LEJ 701 at different carbon and nitrogen

concentrations

Degradation rate

(µM day-1

)

Degradation rate per unit

biomass

(µM day-1

g-1

)

Low C¤

0.02 ± 0.02 a

1.16 ± 1.10 a

Intermediate C¤

0.53 ± 0.04 b

17.17 ± 1.76 c

High C¤

0.61 ± 0.01 b

12.82 ± 0.14 b

Low N#

0.07 ± 0.05 a

3.10 ± 2.18 a

Intermediate N#

0.40 ± 0.01 b

10.54 ± 1.05 b

High N#

0.78 ± 0.03 c

14.68 ± 0.75 b

¤ degradation rate calculated day 6-34.

# degradation rate calculated day 4-33. Numbers given are

means ± SE. Initial diuron concentration 5mg l-1

. Low C: ≤ 0.045g glucose l-1

, Intermediate C: 0.5g

glucose l-1

, and High C: 5g glucose l-1

. Low N: ≤ 0.001g NH4Cl l-1

, Intermediate N: 0.04g NH4Cl l-1

,

and High N: 1g NH4Cl l-1

. Different letters show significant difference between treatments –

comparing Low, Intermediate and High C or N, respectively.

57

Fig. 1 Maximum likelihood phylogenetic tree showing the five Mortirella isolates and chosen

isolates from Genbank. The weight of the branches corresponds to their bootstrap values. Only

lines with bootstrap above 90 are shown

58

(a)

Days

0 10 20 30

µm

ol l-

1

0

5

10

15

20

Are

a

0

10000

20000

30000

40000

(b)

Days

0 10 20 30

µm

ol l-

1

0

5

10

15

20A

rea

0

10000

20000

30000

40000

(c)

Days

0 10 20 30

µm

ol l-1

0

5

10

15

20

Are

a

0

10000

20000

30000

40000

Diuron

DCPMU

DCPU

Unknown

Total

Fig. 2 Diuron degradation kinetics by Mortierella sp. LEJ 701 at (a) high, (b) intermediate and (c)

low glucose concentrations. Diuron: [N-(3,4-dichlorophenyl)-N,N-dimethyl-urea], DCPMU: [1-

(3,4-dichlorophenyl)-3-methylurea], DCPU: [1-(3,4-dichlorophenyl) urea], and unknown metabolite

(RT 2.16min)

59

Biomass (mg)

0 10 20 30 40 50 60

Diu

ron d

eg

rade

d (

%)

0

20

40

60

80

100

Low C

Intermediate C

High C

Fig. 3 Multiple scatter for all data points day 6-34. Diuron degradation shown as percent degraded

of initial concentration. Low C: ≤ 0.045g glucose l-1

, Intermediate C: 0.5g glucose l-1

, and High C:

5g glucose l-1

.

Fig. 4 UPLC chromatogram showing an unknown metabolite. Retention time (RT): Unknown 2.16

min, DCPU 2.44 min, DCPMU 3.33 min, Diuron 4.24.

51

Time0.00 1.00 2.00 3.00 4.00 5.00 6.00

AU

0.0

1.0

2.0

3.0

4.0

5.0

030211-14 Diode Array Range: 5.7310.53

0.58

3.33

0.69

2.16

2.444.24

60

Fig. 5 Proposed degradation pathway of diuron. Diuron: [N-(3,4-dichlorophenyl)-N,N-dimethyl-

urea], DCPMU: [1-(3,4-dichlorophenyl)-3-methylurea], DCPU: [1-(3,4-dichlorophenyl) urea], 3,4-

DCA: [3,4-dichloroaniline] and DCPMDU: [1-(3,4-dichlorophenyl)-3-methylideneurea]. 3,4- DCA

was only found as trace amounts by MS. The structure and placement of DCPMDU, in brackets, are

suggested based on the composition C8H6ON2 and the degradation kinetics.

Manuscript II

TRANSPORT OF DEGRADER BACTERIA VIA FUNGAL HYPHAE INCREASES

DEGRADATION OF 2,6-DICHLOROBENZAMIDE

Berith E. Knudsen, Lea Ellegaard-Jensen, Christian N. Albers, Søren Rosendahl and Jens Aamand

Submitted to Environmental Pollution

63

Transport of degrader bacteria via fungal hyphae increases degradation of 2,6-

dichlorobenzamide

Berith Elkær Knudsena,b,§,*

, Lea Ellegaard-Jensena,b,§

, Christian Nyrop Albersa, Søren Rosendahl

b, and Jens Aamand

a

a) Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Øster Voldgade 10, DK-1350

Copenhagen K, Denmark. b) Department of Biology, Copenhagen University, Universitetsparken 15, DK-2100

Copenhagen Ø, Denmark.

§) Joint first author

*) Corresponding author: bekn@geus.dk, Phone: +45 38142316, Fax: +45 38142050

Abstract

Introduction of specific degrading microorganisms into polluted soil or aquifers is a promising

remediation technology provided that the organisms survive and spread in the environment. We

suggest that consortia, rather than single strains, may be better suited to overcome these challenges.

Here we introduced a fungal-bacterial consortium consisting of Mortierella sp. LEJ702 and the

2,6-dichlorobenzamide (BAM)-degrading Aminobacter sp. MSH1 into small sand columns. A more

rapid mineralisation of BAM was obtained by the consortium compared to MSH1 alone especially

at lower moisture contents. Results from quantitative real-time polymerase chain reaction (qPCR)

demonstrated better spreading of Aminobacter when Mortierella was present suggesting that fungal

hyphae are transport routes for the bacteria. Extraction and analysis of BAM showed that also the

compound was transported by the fungal hyphae in the sand. This suggests that fungal-bacterial

consortia are promising for successful bioremediation of pesticide contamination.

Key words: 2,6-dichlorobenzamide (BAM), consortium, fungal highway, pesticide biodegradation,

fungal-bacterial interactions.

Capsule: This study brings new knowledge to the benefits of applying bacterial-fungal consortia for

bioremediation.

64

1. Introduction

Extensive use of pesticides on agricultural and urban areas poses a threat to groundwater

resources. This is of great concern, especially in countries where the majority of the drinking water

originates from groundwater. The most frequently detected pollutant in Danish groundwater is 2,6-

dichlorobenzamide (BAM), a degradation product of the herbicide dichlobenil, the use of which has

been banned in Denmark since 1997 (Thorling et al., 2010). Several water supply wells have been

closed due to detection of BAM at concentrations exceeding the threshold value of 0.1 µg l-1

. The

closing of wells, however, does not solve the problem, as the pollutants still remain in the water

resources.

Bioremediation, especially bioaugmentation, has been suggested as a possible means of

cleaning pesticide-polluted sites (Thompson et al., 2005). Various bacterial strains capable of

degrading different pesticides have been identified, e.g. the BAM-degrading Aminobacter sp. strain

MSH1 (Sørensen et al., 2007). There are, however, still major challenges that need to be overcome

before bioaugmentation can be efficient (El Fantroussi & Agathos, 2005; Thompson et al., 2005).

These challenges include: i) securing the survival of the introduced strains, ii) development of

efficient tools for spreading degrader organisms in the polluted environment and iii) providing

access for the microorganisms to the pollutants sorbed to the sediment and organic matter or

trapped in micropores, e.g. small cracks or voids on the particle surfaces. It is difficult to ensure

survival of the introduced strains (van Veen et al., 1997; Pepper et al., 2002), since various factors

like temperature, moisture content and nutrient availability in the new environment may differ from

their original habitat (Vogel, 1996). Furthermore, studies have shown that bacteria applied to soil

surfaces rarely are transported more than 5 cm into the soil without the help of a transport agent,

such as percolating water (Edmonds, 1976; Madsen and Alexander, 1982). Even if the strains are

successfully added to the environment, the pollutants are often only present at low concentrations

and are not easily accessible for the degrader bacteria (Harms and Bosma, 1997; Wick et al., 2007).

It has been suggested that fungal hyphae may function as a transport vector for bacteria capable of

degrading hydrophobic PAHs (Furuno et al., 2010). Also, many bacteria can be found in the

hyphosphere; thus they have better access to nutrients from fungal exudates, ensuring their survival

in the environment (Boersma et al., 2010; de Boer et al., 2005). Therefore, adding fungal-bacterial

consortia instead of single bacterial strains may be a way to overcome these challenges. Previous

studies by Kohlmeier et al. (2005) and Wick et al. (2007) have moreover shown that fungal hyphae

may facilitate transport of PAH-degrading bacteria in soil. The water film produced by some fungi

65

around their hyphae allows a better transport of the bacteria present in the hyphosphere. Motile

bacteria can utilise the water film as a transport vector enabling transport along the hyphae − a

transport mechanism termed the fungal highway (Kohlmeier et al., 2005). Thus fungal growth may

enable the bacteria to access areas and thereby potentially access pesticides they otherwise would

not be able to reach. The water film may also facilitate transport of the pollutant. However, the

above mentioned studies have focused on hydrophobic compounds like PAHs and it has therefore

not been examined whether bacteria degrading hydrophilic compounds like pesticides are

transported in the same way. The transport of hydrophilic compounds along the fungal hyphae will

also potentially differ from the transport of the hydrophobic PAHs.

In the present study we used the common soil fungus Mortierella and the BAM-degrading

bacterium Aminobacter sp. MSH1. Mortierella is known to produce hydrophilic mycelia (Chau et

al., 2010), a feature which has been shown to facilitate transport of motile bacteria (Kohlmeier et

al., 2005). The aim of this study was to examine whether the presence of Mortierella sp. LEJ702

affected BAM degradation and mineralization by the motile Aminobacter sp. MSH1 in sand

columns. Moreover, it is investigated whether BAM would be transported along the hyphae of

Mortierella. Our hypothesis was that presence of fungal hyphae facilitates transport of both bacteria

and BAM, thereby increasing the accessibility of BAM to the degrader bacteria, leading to an

increased degradation.

2. Materials and Methods

2.1. Chemical and media.

[Ring-U-14

C]- 2,6-dichlorobenzamide (BAM) (25.2 mCi mmol-1

) was purchased from Izotop,

(Institute of Isotopes Co., Ltd., Hungary). At the time of use, the standard had a radiochemical

purity >99% as determined by Thin Layer Chromatography (TLC). A stock solution of radiolabeled

BAM (3,000,000 DPM ml-1

) was prepared in acetonitrile.

Potato Dextrose Agar (PDA) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) was

used for cultivation of Mortierella sp. LEJ702. Minimal medium (MSNC) containing 0.2 g L-1

Na-

succinate (CAS 6106-21-4), 0.476 g L-1

(NH4)2SO4, 0.1 g L-1

KNO3 and 0.08% glycerol (Sørensen

and Aamand 2003) was used for growing Aminobacter sp. MSH1.

66

The liquid medium used for the experimental setup was buffered MilliQ water. The buffer

contained 136 g L-1

KH2PO4 and 178 g L-1

Na2HPO4.2H2O. The buffer was mixed 1:99 with MilliQ

water, giving a final phosphate concentration in the medium of 6 mM (pH 7).

Agar and media were sterilized by autoclaving at 121oC for 30 minutes.

2.2. Strains/organisms

The BAM-degrading bacterial strain used in the experiments was the motile Aminobacter sp.

MSH1 isolated and described by Sørensen et al. (2007). The strain was pre-grown from a -80oC

stock culture in MSNC medium at 20oC on an orbital shaker (125 rpm). The cells were harvested in

the exponential growth phase, determined by OD600nm measurements. The cells were harvested by

centrifugation at 8000 x g for 10 minutes and washed twice in buffered MilliQ water.

The fungus Mortierella sp. LEJ702 was isolated by Ellegaard-Jensen et al. (in preparation). It

was grown and maintained at 8oC on PDA plates.

2.3. Sand

The sand used was oven-dried quartz sand purchased from Saint Gobain Weber A/S (Optiroc,

Randers, Denmark) with grain sizes between 0.3 and 1 mm. However, before experimental use it

was sieved to remove particles between 0.3 and 0.6 mm. Thus, the sand used had a grain size

between 0.6 and 1 mm giving a less densely packed matrix in the experimental setup. The sand was

sterilized by autoclaving at 121oC for 30 minutes. The water holding capacity (WHC) was

determined (28.7%) according to Danish Standards; DS/ISO 14238-1.

2.4. Mineralization experiment

Mineralization experiments were set up in glass vials (24 mm Ø, 95 mm h) containing 500 μl

PDA agar. Then either Aminobacter sp. MSH1, Mortierella sp. LEJ702, or the fungal-bacterial

consortium was added to the agar. Mortierella was added as an agar plug (Ø = 4mm) and the

bacteria were added to reach 105 cells g

-1 sand. Vials without microorganisms served as abiotic

controls. The sand was weighed into portions of 10 g and added [ring-U-14

C]-BAM dissolved in

acetonitrile, giving a concentration of 100 μg kg-1

. The sand was left for a few minutes in order for

the acetonitrile to evaporate before adding buffered MilliQ water. The amount of water added in the

treatments corresponded to 10%, 5%, 1.7% and 0% of WHC, respectively. A small glass tube

containing 1 ml 1M NaOH was added to each vial to trap evolved 14

CO2. The experiment was

67

incubated at 20oC. The NaOH was replaced approximately once a week and mixed (1:5) with

Optiphase ‗hisafe‘ 3 scintillation liquid (PerkinElmer Inc., Waltham, Massachusetts, USA). The

amount of radioactivity in the alkaline solution was measured on a Wallac 1409 DSA Liquid

scintillation Counter (PerkinElmer Inc., Waltham, Massachusetts, USA). All experiments were

carried out in triplicate. At termination of the experiments, the columns were analysed for BAM

residues using TLC (see below). To follow the removal of BAM and accumulation of degradation

products a parallel series of vials was set up with the same inocula, but at a WHC of 1.7% only.

Vials were harvested once a week for TLC analysis. The vials were harvested by removing the

CO2-trap and adding 6 ml MeOH-MilliQ water (50:50). The vials were then shaken vigorously for

two hours, followed by centrifugation for 10 min at 200 x g. From the supernatant, 2 ml was

transferred to Eppendorf tubes and centrifuged for 2 min at 13000 x g. The supernatant was used for

scintillation counting (500 μl) and for TLC analysis (40 μl).

2.5. DNA extraction and qPCR

At termination of the mineralization experiment, DNA was extracted from the top of the sand

layer (0.25 g) to investigate whether there had been transport of Aminobacter cells up through the

sand. DNA extractions were performed using the PowerLyzer PowerSoil DNA isolation kit (Mobio

Laboratories Inc., Carlsbad, CA, USA). Aminobacter specific primers described by Sjøholm et al.

(2010) were used for quantitative real-time polymerase chain reaction (qPCR). The amplifications

were performed in volumes of 20 μl, containing 2 μl BSA, 10 μl SiBir master mix (Bioron GmbH,

Ludwigshafen, Germany) and 0.4 μM of each primer as described by Sjøholm et al. (2010). The

conditions for the reactions were as follows: an initial denaturation step at 95 oC for 5 min. fifty

cycles at 95oC for 30 s, 53

oC for 30 s and 72

oC for 30 s, followed by an elongation step at 72

oC for

6 min and 71 cycles at 60oC for 30 s with a 0.5

oC increase per cycle. Final step was at 72

oC for 45 s.

Results of the qPCR were processed using the Bio-Rad iQ5 program (www.discover.bio-rad.com).

2.6. Transport of BAM

An experiment was set up to study whether Mortierella sp. LEJ702 facilitated the transport of

BAM through the sand. The experiment was set up in 20 ml sterile plastic syringes (Terumo

Corporation, Belgium). The pistons were set at the 20 ml marking, 500 μl PDA and a Mortierella

sp. LEJ702 plug (Ø = 4 mm) were added to the syringes. Five grams quartz sand was mixed with

buffered MilliQ-water and [Ring-U-14

C]-BAM giving a concentration of 100 μg kg-1

as described

68

above and added to the syringes followed by 5 g of unspiked quartz sand mixed with buffered

MilliQ-water. The experiment was carried out at the same moisture conditions as in the

mineralization experiment. The syringes were sealed with rubber plugs and wrapped in parafilm and

incubated at 20oC. Once a week the syringes were opened for aeration. At termination of the

experiment the sand was pushed to the top of the syringe using the piston. The unspiked sand was

divided into two equal size fractions, while the 14

C-spiked portion was kept as one portion.

Radiolabeled carbon was extracted from the top fraction of the unspiked sand as described above

with the modification that 3 ml MeOH:MilliQ-water was used for the extraction. After

centrifugation, 500 μl of the supernatant was mixed with 3 ml Optiphase ‗hisafe‘ 3 scintillation

liquid (PerkinElmer Inc., Waltham, Massachusetts, USA) and measured on a Liquid Scintillation

Analyzer, Tri-Carb 2810 TR (PerkinElmer Inc., Waltham, Massachusetts, USA). Subsamples of the

remaining extract were used for TLC analysis.

2.7. Thin Layer Chromatography

A total of 40 µL of each sample was spotted onto a 10x20 cm silica gel 60 normal phase TLC

plate (Merck, Darmstadt, Germany) and eluted with CH3CN:H2O:CH2Cl2;HCOOH (70:20:5:5) in a

CAMAG Horizontal Developing Chamber (CAMAG, Berlin, Germany). After air-drying, the plates

were developed on phosphor imaging screens and analyzed autoradiographically on a Cyclone

Scanner (Packard Instrument Company, Meriden, CT). The chromatograms obtained were analyzed

using the OptiQuant Image Analysis software (Packard Instrument Company) to integrate the

different bands. Rf-values were: BAM (0.95), 2,6-dichlorobenzoic acid (0.88) and unidentified

degradation products (0.37, 0.61, 0.80 and 0.99). [ring-U-14

C]-BAM and [Carboxy-14

C]-2,6-DCBA

(Moravek Biochemicals Inc., Brea, Ca) were used as analytical standards and analyzed on the first

line of each TLC plate.

3. Results

3.1. Mineralization of BAM

The most rapid mineralization of BAM was by the consortium with Aminobacter sp. MSH1 and

Mortierella sp. LEJ702 at all moisture contents. In the vials with moisture content of 10% of WHC

(fig. 1A) about 29% of the added BAM was mineralized by the consortium after 14 days, whereas

MSH1 alone had only mineralized 19%. No BAM was mineralized by Mortierella alone or in the

69

abiotic controls at any of the moisture contents. The stimulating effect on mineralization by

growing MSH1 with the fungus was even more pronounced at 5% of WHC (see fig. 1B) where

more than 50% of the added BAM was recovered as CO2. MSH1 alone only mineralized 16% of the

BAM at this WHC. Moreover, the mineralization rate was significantly increased by the consortium

compared to MSH1 alone.

A stimulation of the mineralization by the consortia was also seen at the lower moisture

contents of 1.7% and 0% of WHC (fig. 1C and D), although at an overall lower level with a

maximum of 35% and 27% recovered as CO2, respectively.

3.2. Transport of Aminobacter

The qPCR analysis revealed that Aminobacter sp. MSH1 had been transported to the very top of

the sand layer in all vials (fig. 2). For all moisture contents the greatest transport of the bacterium

through the sand was seen when grown with the fungus. The difference was most pronounced at a

moisture content of 5% of WHC, where the number of MSH1 detected in the top layer was more

than 2000 times higher in the consortium vials than in vials containing MSH1 alone. In the vials

with MSH1 alone the numbers of bacteria detected were approximately at the detection limit of 400

cells g-1

. At the highest moisture content, the MSH1 count for the consortium was 600 times higher

than for MSH1 alone.

3.3. Transport of BAM

The pesticide residue BAM was likewise transported up through the sand layer in both

experiments with Mortierella and in the abiotic controls (see fig. 3). Even in the syringes with a

moisture content of 0% of WHC, BAM was detected in the top layer of the sand, though at lower

levels than in syringes with higher moisture contents. More BAM appears to be transported to the

top sand in the syringes with Mortierella at 5%, 1.7% and 0% of WHC, though the difference is

only statistically significant at 0% of WHC. Thus, the ability of the Mortierella to facilitate

transport of BAM becomes increasingly pronounced the lower the moisture content. At the driest

condition (0% of WHC) about 10% of the added BAM was found in the top sand with Mortierella

compared to only 5.7% in the abiotic control.

70

3.4. Determination of degradation products

The results of the TLC analyses confirmed the results of the mineralization experiments, as the

most rapid dissipation of BAM was seen in vials containing both the bacterium and the fungus (fig.

4). Furthermore, the results show that degradation of BAM is a biological process as BAM was not

degraded in the abiotic controls.

The TLC analysis at termination of the mineralization experiment enabled elaboration of the

results, making it possible to account for the fraction of BAM that was not mineralized (fig. 5). At

5% of WHC a great deal more 14

C was recovered as BAM when MSH1 was grown alone compared

to the consortium (fig. 5B). The TLC analysis revealed that there was a significantly larger amount

of BAM remaining in the MSH1 treatment and that there had been a very limited partial

degradation of BAM. Similar to the results at the highest moisture contents, a small fraction of the

radiolabelled compound was detected as 2,6-dichlorobenzoic acid (2,6-DCBA).

The vials containing Mortierella alone were included in the TLC analyses at 1.7% moisture

content (fig. 5C). Results showed that approximately 80% of the initially added BAM could be

recovered in the vials with Mortierella. The TLC analysis also revealed the presence of an unknown

compound not present at the higher moisture content. The compound was only detected in the vials

containing Mortierella, indicating that it could be a fungal metabolite (designated ―F‖ in figure 5).

This potential fungal metabolite was also detected in the TLC analysis of the weekly harvested vials

in concentrations up to 5% of the added BAM (data not shown). More BAM remained in the

consortium vials at this moisture content than at higher moisture contents.

The distribution pattern at 0% moisture content was similar to that at 1.7% with less BAM

having been mineralized and more remaining as BAM. The unknown compound only detected in

the Mortierella vials was also detected here.

4. Discussion

One of the abiotic factors that has proven to strongly influence survival and ability to degrade

pollutants is moisture content (Cattaneo et al., 1997; Ronen et al., 2000). Ronen et al. (2000)

showed that low water contents led to poor bacterial survival, and at a water content of 10%

population size declined rapidly. This does not seem to be the case for Aminobacter sp. MSH1. In

fact, our results indicate that MSH1 was growing, as a high number could be detected at the top of

the sand layer at the end of the experiment (fig. 2). Growth of MSH1 seemed to be enhanced by the

71

presence of Mortierella sp. LEJ702, probably because of the exudates produced by the fungus being

utilised by the bacteria.

The mineralization results for MSH1 showed high activity by MSH1 at 10% of WHC (fig. 1A)

and an increase in activity at the lower moisture contents in the presence of Mortierella. Results by

Cattaneo et al. (1997) showed that moisture contents of between 30% and 90% of WHC were

required in order to have optimal biodegradation. The high activity of MSH1 even at low moisture

contents will likely increase the chance of successful bioremediation in natural heterogeneous

environments.

A fungal presence may not just increase activity. It has previously been demonstrated that fungi

produce exudates that in some cases can be utilised by bacteria and thereby increase their chance of

survival (de Boer et al., 2005; Boersma et al., 2010; Furuno et al., 2010). This could prove

especially important under stressful conditions such as drought or starvation. Fungi require less

moisture, and results by Cattaneo et al. (1997) showed that fungi dominate the microbial population

under dry conditions. Moreover, the ability of fungi to transport nutrients through the mycelium,

thus supporting hyphal growth in otherwise low-nutrient environments, renders the fungi less

affected by conditions that are stressful for other organisms, for example bacteria (Furuno et al.,

2010). This also seems to be the case in our experiments. Less BAM is mineralized by MSH1 when

the moisture content is reduced (fig. 1). The presence of the fungus reduces this effect. However,

detection of unknown compounds at the two lowest moisture contents and only in the presence of

Mortierella may indicate that the fungus was itself also affected at the two lower moisture contents

(fig. 5). Thus these compounds could be the result of a fungal response to increased stress.

Besides potentially providing nutrients, the fungal hyphae can function as transport vectors for

bacteria. Several experiments have shown that bacteria need the help of a transporting agent in

order to move through the soil (Madsen and Alexander, 1982; Goldstein et al., 1985; Kohlmeier et

al., 2005). The fungi-mediated transport may help the introduced bacteria survive as it enables

relocation, potentially to areas with better nutrient supply or less competition. Hydrophilic hyphae

provide a network of liquid pathways capable of bridging air-filled gaps, thus allowing dispersion

of motile bacteria to otherwise unavailable areas (Ritz and Young, 2004; Wick et al., 2007; Furuno

et al., 2010).

We have shown that moisture content plays an important role in the relocation of the BAM-

degrading MSH1 bacteria, as this declined with decreasing moisture content. We also showed that

bacterial transport was strongly increased in the presence of Mortierella, to some degree alleviating

72

the effects of the driest conditions, which corresponds well with previous findings. Furuno et al.

(2010), for example, found that both bacteria and chemicals could be translocated via the fungal

hyphae.

Studies on hydrophobic compounds have shown that fungal hyphae increase the access to

compounds (Kohlmeier et al., 2005; Wick et al., 2007). Other studies have shown that spatial

variability in pesticide degradation is correlated with heterogenous distribution of degraders rather

than with soil properties (Gonod et al., 2003; Gonod et al., 2006; Sjøholm et al. 2010). Our results

obtained with MSH1 also indicate that the presence of fungal hyphae increases the bioavailability of

BAM as greater rate of mineralization and degradation takes place with the consortium. These

findings support the hypothesis that accessibility to the pollutant is the main limitation for

successful removal of the compound and that a fungal-bacterial consortium is better equipped to

overcome this challenge. Hence, for bioremediation of pesticides and their metabolites fungal-

bacterial consortia may prove more efficient and future research should focus on investigating

effects of biotic as well as abiotic factors on these consortia.

Acknowledgements

The work was supported by the ―Microbial Remediation of Contaminated Soil and Water Resources

(MIRESOWA)‖ project funded by the Danish Council for Strategic Research (grant number 2104-

08-0012). The authors wish to thank Pia Bach Jakobsen for help with the qPCR method.

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Biochemistry 32, 1643-1650.

Sjøholm, O. R., Aamand, J., Sørensen, J. and Nybroe, O., 2010. Degrader density determines spatial

variability of 2,6-dichlorobenzamide mineralization in soil. Environmental Pollution 158,

292-298.

Sørensen, S. R., Holtze, M. S., Simonsen, A. and Aamand, J., 2007. Degradation and mineralization

of nanomolar concentrations of the herbicide dichlobenil and its persistent metabolite 2,6-

dichlorobenzamide by Aminobacter spp. isolated from dichlobenil-treated soils. Applied and

Environmental Microbiology 73 (2), 399-406.

Thompson, I. P., van der Gast, C. J., Ciric, L. and Singer A. C., 2005. Bioaugmentation for

bioremediation: the challenge of strain selection. Environmental Microbiology 7(7), 909-915.

Thorling, L., Hansen, B., Langtofte, C., Brüsch, W., Møller, R. R., Mielby, S. and Højberg, A. L.,

2010. Grundvand. Status og udvikling 1989 – 2009. Teknisk rapport, GEUS 2010.

Van Veen, J. A., van Overbeek, L. S. and van Elsas, J. D., 1997. Fate and activity of

microorganisms introduced into soil. Microbiology and Molecular Biology Reviews 61 (2),

121-135.

Vogel, T. M., 1996. Bioaugmentation as a soil remediation approach. Current Opinion in

Biotechnology 7, 311-316.

Wick, L. Y., Remer, R., Würz, B., Reichenbach, J., Braun, S., Schäfer, F. and Harms, H., 2007.

Effect of fungal hyphae on the access of Bacteria to phenanthrene in soil. Environmental

Science and Technology 41, 500-505.

75

Figure 1. Results of the mineralization experiment at different moisture contents: A) 10% of WHC,

B) 5% of WHC, C) 1.7% of WHC and D) 0% of WHC. : MSH1 + Mortierella consortium, :

Aminobacter sp. MSH1, : Mortierella sp. LEJ702 and : Abiotic control.

76

Figure 2. Bacterial cell numbers in the top sand layer at the different moisture contents. M+M:

Aminobacter sp. MSH1 + Mortierella sp. LEJ702 consortium, and MSH1: Aminobacter sp.

MSH1. Numbers are averages.

Moisture content (% of WHC)

10 5 1.7 0

% o

f a

dd

ed

14C

-BA

M

0

5

10

15

20

25

Mortierella

abiotic

Figure 3. Percent of initially added 14

C-BAM transported to the top fraction of the sand columns.

Data are presented as means ± SE.

77

Days

0 10 20 30 40 50 60

% o

f in

itia

lly a

dde

d 1

4C

-BA

M

0

20

40

60

80

100

Figure 4. Data from the TLC analysis of the weekly harvested vials. Moisture contents: 1.7% of

WHC. : Aminobacter sp. strain MSH1 + Mortierella consortium (n=3), : Aminobacter sp. strain

MSH1 (n=3), and : Abiotic control (n=1). Data are means ± SE.

78

Figure 5. Results of the TLC analysis after terminating the mineralization experiment. A) 10% of

WHC, B) 5% of WHC, C) 1.7% of WHC and D) 0% of WHC. Abbreviations: BAM: 2,6-

dichlorobenzamide, 2,6-DCBA: 2,6-dichlorobenzoic acid, U: unknown polar and apolar

compounds, F: fungal-produced metabolite. Data are means ± SE (n=3). Note that the treatment

containing Mortierella alone was analyzed only at 1.7% WHC.

Manuscript III

FUNGAL-BACTERIAL CONSORTIUM OVERCOMES DRY PATCHES AND

INCREASES DIURON DEGRADATION

Lea Ellegaard-Jensen, Berith E. Knudsen, Anders Johansen,

Christian Nyrop Albers, Jens Aamand and Søren Rosendahl

In prep. for submission to Science of the Total Environment

81

Fungal-bacterial consortium overcomes dry patches and increases diuron

degradation

Lea Ellegaard-Jensena,b,§,*

, Berith Elkær Knudsena,b,§

, Anders Johansenc, Christian Nyrop Albers

b,

Jens Aamandb, and Søren Rosendahl

a.

aDepartment of Biology, Copenhagen University, Universitetsparken 15, DK-2100 Copenhagen Ø,

Denmark. bDepartment of Geochemistry, Geological Survey of Denmark and Greenland (GEUS),

Øster Voldgade 10, DK-1350 Copenhagen K, Denmark. cDepartment of Environmental Science,

Aarhus University, Frederiksborgvej 399, DK-4000, Roskilde, Denmark.

§Joint first authors

*Corresponding author: Lea Ellegaard-Jensen

Phone: +45 38142304, Email: Leael@bio.ku.dk

Abstract

Consortia of fungal-bacterial strains could prove more efficient for bioremediation of

pesticide contaminations than individual strains alone. This may be due to co-operative catabolism

along the degradation pathway. The strains should, however, also be able to survive and preferably

spread into the environmental matrix e.g. soil. Fungal hyphae can act as transport vectors for

degrading bacteria. This may lead to a higher accessibility to the pollutant and ultimately to a more

efficient degradation.

To test this we chose the phenylurea herbicide diuron as our model compound. We

constructed a small column system in which diuron spiked sand and organisms were divided by a

layer of sterile glass beads for spatial separation. Fungal-bacterial consortia were created by

different combinations of the bacterial strains Sphingomonas sp. SRS2, Variovorax sp. SRS16 and

Arthrobacter globiformis D47 and the fungal strains Mortierella sp. LEJ702 and LEJ703.

Results showed that, of the consortia tested for degradation of 14

C-labeled diuron, the three-

member consortium LEJ702/SRS16/D47 achieved the overall highest mineralization measured as 14

CO2. In addition, production of diuron metabolites by this consortium was minimal. Molecular

results suggested that bacteria were transported more efficiently by LEJ702 than by LEJ703.

Finally, it was found that the fungal growth differed for LEJ702 and LEJ703 in the three-member

consortia. This study demonstrates new possibilities for creating efficient fungal-bacterial consortia

for bioremediation.

Keywords: Phenylurea herbicide, microbial consortia, synergistic interactions, fungal highway,

pesticide, biodegradation.

82

1. Introduction

The phenylurea herbicide diuron [N-(3,4-dichlorophenyl)-N,N-dimethyl-urea] is mainly used

for control of broad-leaved weeds on agricultural land, but also for weed control on non-cultivated

areas like roads, railways and parks. As a consequence of its use diuron is found in surface- and

groundwater at concentrations above the EU threshold limit of 0.1 µg l-1

(Lapworth and Gooddy,

2006; Struger et al., 2011; Torstensson, 2001). In addition, diuron has adverse effects on the

environment and presumably also on human health (Cox, 2003; Giacomazzi and Cochet, 2004), and

diuron is therefore on the EU Water Framework Directive´s list of priority substances (European

Parliament, 2008). In order to alleviate persistent diuron contamination in terrestrial environments,

bioremediation has been suggested as a possible means to remove diuron from soils.

Bioaugmentation, i.e. introduction of specific degrading microorganisms into the

environment, may be used as a technology to remediate diuron polluted soils. Finding strains

suitable for bioremediation is, however, challenging (Thompson et al., 2005). Not only should the

strains be able to degrade the contaminant, but they must also survive, proliferate and preferably be

spread into the soil matrix to obtain higher accessibility to the pollutants and ultimately a more

efficient degradation. It has been suggested that fungal-bacterial consortia might perform better than

single degrader strains in a heterogeneous soil matrix (Bennett et al., 2007; Mikesková et al., 2012);

this has been demonstrated for degradation of both hydrophobic (Wick et al., 2007) and hydrophilic

compounds (Knudsen et al., submitted). This synergistic effect has partly been explained by an

increased dispersal of the degrader bacteria along the hyphae of the fungal partner – a mechanism

referred to as the ´Fungal Highway´ (Furuno et al., 2010; Kohlmeier et al., 2005; Warmink and van

Elsas, 2009). The fungal hyphae may act exclusively as physical vectors for bacterial transport

enabling the bacteria to cross air-filled gaps in the matrix and in this way access otherwise

inaccessible contaminants. Certain fungi, however, also have potentials for degradation of

pollutants like phenylurea herbicides (Badawi et al., 2009; Ellegaard-Jensen et al., submitted;

Rønhede et al., 2005; Tixier et al., 2000) and may therefore, besides mediating bacterial transport,

also directly contribute to the degradation process.

We propose that consortia, consisting of both bacterial and fungal pesticide degraders might

lead to faster and more complete pesticide degradation in spatial heterogeneous systems, compared

to single strains. A synergistic effect on degradation by consortia may partly be due to the

individual degrader strains being metabolically active at different parts of the degradation pathway.

Recently, Sørensen et al. (2008) showed a synergistic effect on diuron degradation by a two-

83

member bacterial consortium consisting of Variovorax sp. SRS16 and Arthrobacter globiformis

D47.

Though microorganisms may exhibit mutual growth stimulating effects that may lead to

enhanced pesticide degradation, fungi and bacteria are also known to be antagonists (Höppener-

Ogawa et al., 2008; Mille-Lindblom and Tranvik, 2003; Trifonova et al., 2009). Clearly, knowledge

of such effects is necessary for evaluating the function of constructed consortia.

In this study we construct consortia of fungi and bacteria using different combinations of five

diuron-degrading organisms – the bacterial strains Sphingomonas sp. SRS2, Variovorax sp. SRS16

and Arthrobacter globiformis D47 and the fungal strains Mortierella sp. LEJ702 and LEJ703 to

elucidate the controlling mechanisms leading to enhanced degradation. The following mechanisms

will be examined: fungal mediated bacterial transport, co-operative catabolism i.e. the organisms

complementing each other along the metabolic degradation pathway, and physical interactions

between the organisms effecting growth.

2. Materials and methods

2.1. Chemicals and media

[Ring-U-14

C] diuron (36.3 mCi mmol-1

) was purchased from Izotop, (Institute of Isotopes Co.,

Ltd., Hungary) and had a radiochemical purity of 98.9%. A stock solution of radio labeled diuron

(1,700,000 DPM ml-1

) was prepared in acetonitrile.

Analytical-grade diuron [3-(3,4-dichlorophenyl)-1,1-dimethyl-urea] (CAS no. 330-54-1;

97.5% purity), DCPMU [1-(3,4-dichlorophenyl)-3-methylurea] (CAS no. 3567-62-2; 97.5% purity),

DCPU [1- (3,4-dichlorophenyl) urea] (CAS no. 2327-02-8; 99% purity), 3,4-DCA [3,4-

dichloroaniline] (CAS no. 95-76-1; 99% purity), and linuron [3-(3,4-dichlorophenyl)-1-methoxy-1-

methylurea] (CAS no. 330-55-2; 99.5% purity) were purchased from Dr. Ehrenstorfer GmbH

(Augsburg, Germany). 3,4-DCAA [3,4-dichloroacetanilide] (CAS no. 2150-93-8) was purchased

from Sigma-Aldrich (St. Louis, Missouri).

Growth media: Potato Dextrose Agar (PDA) (Difco, BD Diagnostics, Sparks, MD), Luria-

Bertani broth (LB) (Difco, BD Diagnostics, Sparks, MD), and R2A-based broth (R2B) prepared

according to (Reasoner and Geldreich, 1985).

84

MilliQ water with a phosphate-buffer (136 g l-1

KH2PO4; 178 g l-1

Na2HPO4.2H2O), giving a

final phosphate concentration of 6mM (pH 7), was used as liquid medium in experimental set-up.

Clean quartz sand (grain size 0.8-1.4 mm, 99.0% SiO2) was used as a growth matrix in the

experiments (DKI A/S, Denmark). The water holding capacity (WHC) was determined according to

Danish Standards; DS/ISO 14238-1 to be 25.2%. All media and sand were sterilized by autoclaving

at 121oC for 30 minutes before use.

2.2. Microorganisms

The diuron degrading fungal strains Mortierella sp. LEJ702 and Mortierella sp. LEJ703 were

isolated and described by Ellegaard-Jensen et al. (submitted). Both strains were maintained on PDA

at 8oC.

The diuron degrading bacterial strains Sphingomonas sp. SRS2 and Variovorax sp. SRS16

were both isolated and characterized in our laboratory (Sørensen et al., 2005; Sørensen et al., 2001),

while the diuron degrading Arthrobacter globiformis D47 (Turnbull et al., 2001) was kindly

provided by A. Walker. All strains were stored in 40% glycerol stock solutions at -80 °C.

Prior to the experiments, each bacterial strain was thawed and grown as follows: SRS2 in R2B,

SRS16 in R2B supplemented with 25 mg l-1

linuron, and D47 in LB supplemented with 25 mg l-1

diuron. Immediately before used for experiments, the strains were washed twice in buffered MilliQ

water.

2.3. Experiment 1: degradation of diuron

Degradation of diuron was studied using a setup described by Knudsen et al. (submitted) with

modifications (See Fig. S1). In short, the experiment was set up in sterile glass vials (Ø= 24 mm, h=

95 mm). PDA (500 μl) was added to each vial and surface-inoculated with either single strains, two-

member consortia or three-member consortia (See Table 1).

The fungi were added as agar plugs with mycelia (Ø = 4 mm). The bacteria were added in

suspensions corresponding to approximately 5x106 cells g

-1 sand. Vials without microorganisms

served as abiotic controls. A layer of sterile glass beads (2.97g; Ø = 1 mm, Assistent, Rhön,

Germany) was added on top of the inoculated PDA to separate the microbes from the contaminated

sand, simulating air-filled spaces in soil.

[Ring-U-14

C]-diuron dissolved in acetonitrile was added to 10 grams of quartz sand, giving a

concentration of 100 μg kg-1

. After evaporation of the acetonitrile, the sand was added buffered

85

MilliQ (corresponding to 10% of quartz sand WHC) and transferred to the vials. To trap the 14

CO2

evolved from the mineralization of the labeled diuron one small glass tube (Ø = 10.5 mm, h = 40

mm) with 1 ml 1M NaOH was added to each vial. The NaOH was replaced at regular intervals,

mixed with 5 ml scintillation liquid (Optiphase ‗hisafe‘ 3, PerkinElmer Inc, Waltham,

Massachusetts, USA), and the radioactivity in the solution was measured for 10 min on a Liquid

Scintillation Analyzer (Tri-Carb 2810 TR, PerkinElmer Inc., Waltham, Massachusetts, USA). All

treatments were carried out in triplicates. The experiment was incubated in the dark at 20o C.

At experimental termination sand from the top layer in each vial was gently mixed and 0.25 g

of this was used for DNA extraction (see Section 2.6). Subsequently, chemical extractions were

made from the vials to quantify diuron residues and metabolites using thin-layer chromatography

(TLC). All the vials were added 7ml MeOH:MilliQ (75:25), shaken for 2 hours (200rpm) and

centrifuged at 200 x g for 10 min. The supernatant was transferred to Eppendorf tubes and

centrifuged at 13 000 x g for 2 min. One ml of the supernatant was used for TLC (see Section 2.5)

and 500 µl was mixed with 5 ml Optiphase ‗hisafe‘ 3 scintillation liquid (PerkinElmer Inc) for

measurement of radioactivity in the solution.

2.4. Experiment 2: effect of bacteria on fungal growth

Growth of the fungal mycelium in the presence of the bacteria was examined in columns of

similar composition as described in experiment 1 (Section 2.3). Since the difference between the

three-member consortia LEJ702/SRS16/D47 and LEJ703/SRS16/D47 resides in the choice of

fungal strain, we wished to investigate the growth of these fungal strains within these consortia. The

experiment also included two-member consortia with the fungi and either of the bacterial strains

(see Table 1).

The experiment was set up in 20 ml sterile plastic syringes (Terumo Corporation, Leuven,

Belgium) with the front end of the barrel cut off. Syringes standing in a vertical position with the

cut-off end upwards had the pistons set at the 20-ml marking. PDA was added to each syringe and

inoculated with microorganisms (see Table 1). As above a layer of sterile glass beads (2.97g; Ø = 1

mm, Assistent, Rhön, Germany) was added on top of this for spatial separation of the microbes

from the diuron-spiked sand. Ten grams of quartz sand with a concentration of 100μg kg-1

[Ring-U-

14C]-diuron and a moisture content of 10% of WHC was then added to the syringes. Syringes were

sealed with rubber stoppers and parafilm. The experiment was incubated in the dark at 20oC. Twice

a week the syringes were opened for aeration in a sterile flow bench.

86

After 14 and 24 days triplicates from each treatment were harvested destructively. The sand

was pushed upwards using the piston, and the sand was divided into two fractions (i.e. top and

bottom) of approximately equal size. The fractions were instantly placed on ice and kept frozen

until phospholipid fatty acid (PLFA) analysis.

2.5. Thin-layer chromatography

Analysis of diuron and its degradation products were performed using TLC as previously

described (Sørensen et al., 2008). Briefly, 30 µl of centrifuged extract (see Section 2.3) was spotted

on a 10x20 cm Silica Gel 60 thin-layer chromatography plate (Merck, Darmstadt, Germany) and

eluted with a mixture of dichloromethane and methanol (95:5, vol/vol) in a CAMAG horizontal

developing chamber (CAMAG, Berlin, Germany). After air drying, the plates were developed on

phosphor imaging screens and analyzed autoradiographically on a Cyclone Scanner (Packard

Instrument Company, Meriden, CT). The chromatograms obtained were analyzed using the

OptiQuant Image Analysis software (Packard Instrument Company) to integrate the different bands.

Rf-values of identified compounds were: DCPU, 0.24; DCPMU, 0.30; 3,4-DCAA, 0.35; diuron,

0.37 and 3,4-DCA, 0.61.

2.6. Detection of bacteria with specific primers

DNA from 0.25 g top-sand from each vial (only experiment 1) was extracted using the

PowerLyzer PowerSoil® Isolation Kit (Mobio Laboratories Inc, Carlsbad, CA, USA) according to

the manufacturer‘s instructions. For detection of the bacteria the following specific primer sets were

used: (i) Sphingomonas, Sphingo108f - Sphingo420r (Leys et al., 2004), (ii) Variovorax, VarF –

VarR (Bers et al., 2011), and (iii) Actinobacteria/Arthrobacter, Com2xf – Ac1186r (Schäfer et al.,

2010). The PCR mixture and protocol (modified to 35 cycles) described by Leys et al. (2004) were

used with the Sphingo108f - Sphingo 420r primer pair to detect Sphingomonas sp. SRS2. Likewise,

for detection of Variovorax sp. SRS16 the PCR mixture and protocol described by Bers et al. (2011)

were used with the VarF – VarR primer set. For Arthrobacter globiformis D47 the PCR mixture

contained 1U Taq DNA polymerase, 62.5 pmol of each primer (Com2xf – Ac1186r), 25 nmol

dNTP, and 1 x PCR buffer in a final volume of 20 µl. The Arthrobacter PCR protocol was as

follows: an initial denaturation step at 95 o

C for 5 min, followed by 35 cycles consisting of

denaturation for 30 sec at 95 o

C, annealing for 30 sec at 55 o C and extension at 72

o C for 30 sec,

87

and finalized by an elongation step at 72 o C for 6 min. DreamTaq DNA polymerase and buffer

(Fermentas, St. Leon-Rot, Germany) were used in all PCR reactions.

2.7. Phospholipid fatty acid analysis (PLFA)

The procedure for extraction of phospholipid fatty acids was as described by Frostegård et al.

(1993) and modified by Johansen and Olsson (2005). Approximately 5 g of quartz-sand sample was

placed in Teflon centrifuge tubes (Oak Ridge, Nalge Nunc Int., US) and extracted in 10 ml of

dichloromethane/methanol/citrate buffer (0.15 M; pH4.0; 1:2:0.8,vol:vol:vol). Supernatants from

two repeated extractions were pooled and split into two phases by the addition of dichloromethane

and citrate buffer. Polar lipids from the lower phase were purified and derivatized according to

Joner et al. (2001). Samples were analyzed on an Agilant 7890 GC (Agilant, CA, USA) equipped

with an autoinjector (splitless mode), a flame ionization detector, and a 60-m HP5 column, using H2

as carrier (2 mL min-1

). The initial oven temperature was 80 °C (5 min), increased at 20 °C min-1

to

160 °C and at 5 °C min-1

to 270 °C (maintained for 5 min). Inlet and detector temperatures were

230 and 270 °C, respectively. In addition, PLFAs of the bacterial and fungal pure strains were

extracted (as described above) and the individual fatty acids identified using the Midi Sherlock

system (Mansfeld-Giese et al., 2002).

2.8. Statistics

Statistical analysis of PLFA data was performed using SYSTAT v. 9.0 using the GLM

procedure to test for main effects of consortium type and time of incubation (14 and 24 days) as

well as interactions. Bonferroni Tukey multiple comparison analysis was used to determine

significance of differences (presented as P values) between consortia at day 24.

3. Results

3.1. Mineralization and transformation by consortia

The LEJ702/SRS16/D47 consortium mineralized 32.2 ± 5.7 % of the total amount of diuron

after 54 days (Fig. 1), where the single strains or other consortia reached no more than 10 %

mineralization (Fig. 1 and Table 1). Low mineralization rates does not imply that diuron was not

degraded by some of the stains or consortia as metabolites (DCPMU, DCPU and 3,4-DCA) were

found in several treatments (Fig. 2 and Fig. S2). In the abiotic treatments, no degradation products

88

were detected. The degradation pathway of diuron is illustrated in Fig. 3, where the steps performed

by either fungal or bacterial strains are also seen.

Both Mortierella strains degraded diuron to DCPMU and DCPU (Fig. 2A and Fig. S2D),

whereas the bacterial single strains and consortia with solely bacterial strains degraded only

minimal amounts of diuron (Fig. S2). With the LEJ702/SRS16 consortium, a small amount of the

diuron was mineralized while the two demethylated metabolites were also present (Fig. 2B).

Similarly, in the LEJ702/D47 consortium DCPMU and DCPU were present, in addition 3,4-DCA

and 3,4-DCAA (Fig. 2C) were found. The three-member LEJ702/SRS16/D47 consortium was the

most effective combination of microorganisms as only small amounts of intermediary metabolites

could be detected and one third of the diuron was mineralized to CO2 (Fig. 2D).

Some 14

C did not migrate on the TLC plate and was therefore termed non-extractable fraction

(NEF). This fraction varied in the different treatments and was generally largest in treatments with

LEJ703 where it constituted as much as 50% of the 14

C originating from diuron compared to 10-

25% in treatments without this strain and 0% in the abiotic control (Fig. S2). Part of the NEF

fraction (50-80%) was found to be able to move in a more polar solvent (MeOH:CH2Cl2 ratio

reversed from 5:95 to 95:5). This method could not be used for quantification since diuron and its

metabolites were not chromatographically separated, but it indicates that the NEF includes one or

more unidentified polar degradation products rather than being 14

C bound in non-dissolvable

organic macro structures as observed in a soil system by (Sørensen et al., 2008).

3.2. Transport of bacteria

PCR analysis with Variovorax-specific primers revealed that Variovorax sp. SRS16 could be

detected in the top sand layer, but only in the vials containing the three-member consortium

LEJ702/SRS16/D47. Likewise, Arthrobacter globiformis D47 was only detected in samples from

the top sand layer of the vials containing the two-member consortium LEJ702/D47 when analyzed

with Actinobacteria/Arthrobacter-specific primers (data not shown).

These observations indicate that bacteria were only transported to the top of the sand column

when Mortierella sp. LEJ702 was present. Interestingly, in the vials with Mortierella sp. LEJ703 no

bacteria could be detected in the top sand independent of the treatments. Analysis with the

Sphingomonas-specific primers did not detect Sphingomonas sp. SRS2 in the top sand layer in any

of the treatments.

89

3.3. Growth of Mortierella

PLFAs from Mortierella sp. LEJ702 and LEJ703 were measured in high amounts in the

presence of Variovorax sp. SRS16, with significantly more in the uppermost sand layer than in the

bottom (Fig. 4A, B; main effect top vs. bottom P<0.001), but with no statistically significant

difference between day 14 and 24 for any of the consortia. In contrast, the sand content of PLFAs

indicative of both the Mortierella strains were much lower when A. globiformis D47 was present,

both in the top and bottom of the sand column (Fig. 4; main effect of consortia P<0.001). For

Mortierella sp. LEJ703 this D47-induced decrease in growth was still very pronounced in the three-

member consortium. In comparison, the inhibition effect on the biomass of Mortierella sp. LEJ702

was lessened in the three-member consortium at day 24 (Fig. 4B; P<0.022). This indicates that the

presence of SRS16 alleviates the growth inhibition that D47 causes on Mortierella sp. LEJ702. The

PLFAs indicative of Variovorax sp. SRS16 and A. globiformis D47 could not be detected in

significant proportions (above detection level) in any of the treatment (data not shown).

4. Discussion

Reports of consortia including both fungal and bacterial degraders are scarce, and our finding

that a fungal-bacterial three-member consortium can indeed increase pesticide degradation has to

our knowledge not been reported previously. The three-member consortium LEJ702/SRS16/D47

was mineralizing three times more diuron than either of the single strains or other consortia tested.

The two-member consortium SRS16/D47 did not mineralize more than 8.3 ± 2.6 % of the added

diuron in our experiment. This is in contrast to previous studies where the two bacterial strains in

co-culture have been reported to enhance degradation of diuron with approximately 60 % of the

added 14

C-diuron mineralized in liquid cultures (Sørensen et al., 2008). This suggests that the

bacteria might not have been able to cross the air-filled gap created by the glass-bead-layer and

reach the diuron in the sand in the present study.

The fungal hyphae may have acted as transport vector for the degrading bacteria enabling the

bacteria to cross air-filled gaps to reach the diuron in the sand. Such hyphal mediated bacterial

transport, known as ´the fungal highway´(Kohlmeier et al., 2005), has been demonstrated by other

authors previously, where transport of bacteria was shown to occur in the thin water film around

the fungal hyphae (Furuno et al., 2010; Wick et al., 2010).

90

In heterogeneous and water-unsaturated environments, e.g. soil, bacteria are almost

completely immobilized and can, unlike fungi, not cross over air-filled gaps without a transport

vector (Banitz et al., 2011a; Banitz et al., 2011b). Therefore, bioavailability has been suggested to

be a critical factor for bioremediation success (Bosma et al., 1996) - a challenge which may be

overcome by applying fungal-bacterial consortia.

In agreement with our findings that fungal transport of bacterial degraders increase

degradation of diuron, Wick et al. (2007) found that transport of phenanthrene degrading bacteria

via fungal hyphae increased the degradation of this compound. Likewise, Knudsen et al. (submitted)

showed for the pesticide metabolite 2,6-dichlorobenzamide (BAM) that transport of the degrader

bacterium also increased BAM mineralization. Transport of bacteria was only detected in consortia

with Mortierella sp. LEJ702, and not in consortia with Mortierella sp. LEJ703. Whether this is due

to morphological or physiological differences between the two fungal strains is not known.

Kohlmeier et al. (2005) showed that fungal transport of Achromobacter sp. SK1 strongly depended

on the surface hydrophobicity of the fungal hyphae, with enhanced transport along the hydrophilic

hyphae of Fusarium oxysporum. It could be speculated that this could also be the principle cause in

the present experiment.

A co-operative catabolism may be expected when inspecting the degradation pathway of

diuron (Fig. 3). Here it is seen that Sphingomonas sp. SRS2 and Mortierella degrade diuron by

demethylation (Badawi et al., 2009; Ellegaard-Jensen et al., submitted; Sørensen and Aamand,

submittet), while Variovorax sp. SRS16 and A. globiformis D47 degrades diuron directly to 3,4-

dichloroaniline (Sørensen et al., 2008).

The single bacterial strains degraded only limited amounts of diuron in the present study (Fig.

S2 A-C). Low concentrations of 3,4-DCA were detected with D47 alone and in the two-member

consortium LEJ702/D47. However, in the two-member consortium the demethylated metabolites

DCPU and DCPMU along with the metabolite 3,4-DCAA were also present (Fig. 2C). This can be

seen as a clear indication of co-operative catabolism since this metabolite is created from 3,4-DCA

by the fungus (Fig. 3). This process was also demonstrated by Tixier et al. (2002) testing the ability

of four fungal strains to degrade synthesized 3,4-DCA in pure cultures. Though the process does not

lead to complete mineralization, it still leads to detoxification as 3,4-DCAA is less toxic than 3,4-

DCA (Tixier et al., 2002). Variovorax sp. SRS16 is the organism with the highest potential for

complete mineralization of diuron among the tested strains (Sørensen et al., 2008). Although some

mineralization was seen in all treatments with SRS16 (Table 1), it was only in the three-member

91

consortium LEJ702/SRS16/D47 that the mineralization was markedly increased (Fig. 1). This

suggests that there were other distinctive interactions between these three organisms, besides

cooperative catabolism and bacterial transport, which enabled them to perform superior compared

to the other consortia.

The two Mortierella strains differed in their growth response in the consortia. In the three-

member consortia with SRS16 and D47, the growth of LEJ703 was clearly restricted compared to

that of LEJ702 (Fig. 4). This outcome seemed to be caused by a D47-inhibitory effect on both

fungi, which was alleviated by SRS16, although only for LEJ702. This may, at least partly, explain

the increased diuron mineralization in the three-member consortium LEJ702/SRS16/D47 compared

to the consortium LEJ703/SRS16/D47 (Table 1).

The two bacteria SRS16 and D47 exerted different effects on the growth of the fungi. Bacteria

are known to potentially inhibit fungal growth. Trifonova et al. (2009) found that four out of seven

bacterial species changed the mycelium morphology and reduced colony size of the ascomycete

Coniochaeta ligniaria grown on agar plates. We did, however, not detect any inhibitory effects on

fungal morphology and growth on agar plates with the bacterial and fungal strains used in the

present work (data not shown). The PLFA method enabled us to follow the growth dynamics of

fungi in the constructed consortia and it has, to our knowledge, not been used in this way before.

The PLFA profiles of the two fungal strains were identical and very different from those of the

bacterial strains. PLFAs from the bacterial partners were not quantifiable in any of the consortia.

From the PCR analysis we know that Variovorax sp. SRS16 and, Arthrobacter globiformis D47

were present in the sand compartment, although this must have been in proportions of biomass

which were far less than the fungi. Hence, the present results indicate that efficient degradation of

contaminants by microbial consortia can proceed even with large differences in amount of biomass

between the microbial partners involved in the degradation.

Fungal exudates of Mortierella sp. LEJ702 might also have contributed to the synergistic

effect on diuron degradation. Exudates have been shown to be an important source of carbon for

bacteria in the hyphosphere (Boersma et al., 2010; de Boer et al., 2005; Nazir et al., 2010;

Trifonova et al., 2009), and since diuron was the only added carbon source available for the

bacteria in the sand they were likely to depend on the fungal hyphae for transport and release of

water and nutrients. Previously, it has been demonstrated that the mineralization of diuron by

SRS16 only occurred when it was supplemented with appropriate growth substrates or in co-culture

with D47 (Sørensen et al., 2008).

92

Our study underlines the importance of careful strain selection in constructions of consortia

for bioremediation. Fungal-bacterial consortia for degradation of PAHs have been created with

varying success; either showing no positive effect (Arun and Eyini, 2011) or enhanced PAH

degradation by specific consortia (Boersma et al., 2010; Machín-Ramírez et al., 2010). The last case

supports our findings that fungal-bacterial consortia may be useful for the remediation of polluting

compounds, but also that more research is needed to unravel the complex interactions within the

consortia. It is valuable to gain knowledge about these interactions if the ultimate aim is

bioaugmentation of soil where competition with indigenous microorganisms will add to the

complexity. The interactions with indigenous soil microorganisms should therefore be thoroughly

investigated including performance of the consortium in soil mesocosms with regard to its

degradation efficiency, before establishing its applicability for in situ bioremediation.

In conclusion, the present study shows that microbial consortia may be much more efficient in

degrading chemical contaminants compared to the consortium members individually. However, it

also shows that the traits of the member organisms are important to obtain the synergism necessary

for optimal remediation in a heterogeneous environment like soil. Our results also show that a

fungal partner is crucial for spatial distribution of the bacterial degraders.

Acknowledgments

This work was supported by grant 2104-08-0012 from the Danish Council for Strategic Research

given through the MIRESOWA project. Anders Johansen was supported by the SOILEFFECTS

project funded by the Norwegian Research Council. We thank Dr. Sebastian R. Sørensen for

providing the strains SRS2 and SRS16 and Pia B. Jakobsen, Pernille Stockmarr and Spire

Kiersgaard for skillful technical assistance in the laboratory.

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95

Table 1. Accumulated 14

CO2 produced from 14

C-labeled

diuron during 54 days incubation.

Treatment

Mineralization

(% of added 14

C diuron ± SE)

Sphingomonas sp. SRS2 2.2 ± 0.4 %

Variovorax sp. SRS16 4.4 ± 0.8 %

Arthrobactor sp. D47 4.8 ± 1.0 %

Mortierella sp. LEJ702 0.4 ± 0.1 %

Mortierella sp. LEJ703 1.6 ± 0.2 %

Two member consortia

SRS2/SRS16 7.3 ± 1.3 %

D47/SRS16* 8.3 ± 2.6 %

LEJ702/SRS2 0.6 ± 0.2 %

LEJ702/SRS16* 7.3 ± 2.8 %

LEJ702/D47* 1.3 ± 0.4 %

LEJ703/SRS2 1.1 ± 0.2 %

LEJ703/SRS16* 10.0 ± 3.5 %

LEJ703/D47* 0.9 ± 0.1 %

Three member consortia

LEJ702/SRS16/SRS2 7.0 ± 1.9 %

LEJ702/SRS16/D47* 32.2 ± 5.7 %

LEJ703/SRS16/SRS2 9.1 ± 4.1 %

LEJ703/SRS16/D47* 7.2 ± 1.4 %

* treatments also used in experiment 2

96

Days

0 10 20 30 40 50

% 1

4C

O2 o

f added 1

4C

-diu

ron

0

10

20

30

40

SRS16

D47

LEJ702

SRS16&D47

LEJ702&SRS16

LEJ702&D47

LEJ702&SRS16&D47

Figure 1. Mineralization of 14

C-labeled diuron by single strains and consortia. 14

C-diuron

mineralization by (●) Variovorax sp. SRS16, (○) Arthrobacter globiformis D47, (▼) Mortierella

sp. LEJ702, (∆) consortium SRS16/D47, (■) consortium LEJ702/SRS16, (□) consortium

LEJ702/D47, and (♦) consortium LEJ702/SRS16/D47. Data are presented as means ± SE.

97

(A)

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%)

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%)

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100

Figure 2. Distribution of 14

C residues

from 14

C-labeled diuron in the sand

determined by thin-layer

chromatography at termination of the

mineralization experiment. (A)

Mortierella sp. LEJ702, (B) consortium

LEJ702/SRS16, (C) consortium

LEJ702/D47, and (D) consortium

LEJ702/SRS16/D47. DCPMU: [1-(3,4-

dichlorophenyl)-3-methylurea], DCPU:

[1-(3,4-dichlorophenyl) urea], 3,4-DCA:

[3,4-dichloroaniline], 3,4-DCAA: [3,4-

dichloroacetanilide], and NEF: Non-

extractable fraction. Radioactivity

measured from the abiotic control

served as total on the y-axis (values are

shown as % of this). Data are presented

as means ± SE.

98

Figure 3. Degradation pathway of diuron showing organisms performing the different

transformations. DCPMU: [1-(3,4-dichlorophenyl)-3-methylurea], DCPU: [1-(3,4-dichlorophenyl)

urea], 3,4-DCA: [3,4-dichloroaniline], and 3,4-DCAA: [3,4-dichloroacetanilide].

99

Mort

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lla s

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nm

ol g

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47

LE

J703/S

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47

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RS

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47

LE

J703/S

RS

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47

none

0

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nd

(A)

(B)

Figure 4. Amount of PLFAs measures in bottom and top layers of sand columns added diuron. The

species compositions of the consortia are given in Table 1. The two Mortierella strains contained

the same types of PLFAs, which serve as an indicator of their respective biomasses at incubation

time 14d (A) and 24d (B) (n=3, bars indicate SE, nd= not determined).

100

Fig. S1. Illustration of mineralization experiment

setup. See Section 2 for written description.

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102

Fig S2. Distribution of 14

C residues from 14

C-labeled diuron in the sand determined by thin-layer

chromatography at terminating the mineralization experiment. (A) Sphingomonas sp. SRS2, (B)

Variovorax sp. SRS16, (C) Arthrobacter globiformis D47, (D) Mortierella sp. LEJ703, (E)

consortium SRS2/SRS16, (F) consortium SRS16/D47, (G) consortium LEJ702/SRS2, (H)

consortium LEJ703/SRS2, (I) consortium LEJ703/SRS16, (J) consortium LEJ703/D47, (K)

(I)D

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103

consortium LEJ702/SRS2/SRS16, (L) consortium LEJ703/SRS2/SRS16, (M) consortium

LEJ703/SRS16/D47, and (N) abiotic treatment.

DCPMU: [1-(3,4-dichlorophenyl)-3-methylurea], DCPU: [1-(3,4-dichlorophenyl) urea], 3,4-DCA:

[3,4-dichloroaniline], 3,4-DCAA: [3,4-dichloroacetanilide], and NEF: Non-extractable fraction.

Data are presented as means ± SE.

104

Appendix

Table X. Physical-chemical properties of diuron and its most frequently reported metabolites

Diuron

DCPMU DCPU

3,4-DCA

Type

Phenylurea herbicide –

photosynthetic electron

transport inhibitor1

Metabolite Metabolite Metabolite

IUPAC Name2

3-(3,4-dichlorophenyl)-

1,1-dimethylurea

1-(3,4-

dichlorophenyl)-3-

methylurea

3,4-dichlorophenyl

urea 3,4-dichloroaniline

Structure

Use

Broad spectrum active

against mosses, algae,

broadleaf and grassy

weeds1

- - -

Molecular mass2

(g mol-1

) 233.09 219.07 205.04 162.02

Properties

Solubility in water

20oC (mg l

-1)

2

35.6

490 940 580

Stability in soil

DT502 75.5-89 days - - 33.4 days

Log Kow2

2.87 - 2.35 2.69

Toxicity

Mammals – acute

oral LD50 (mg kg-1

)2 437 - - 545

Fish – acute 96 hour

LC50 (mg l-1

)2 6.7 - - 1.94

Microtox test - EC50

(mg l-1

) 68

3 18.53

15.03

0.484

S. teres – 24 hour

LC50 (mg l-1

) 40

3 6.23

10.23

-

1Active ingredient factsheet (http://www.pan-uk.org/pestnews/Actives/Diuron.htm accessed September 2012)

2 Pesticide Properties DataBase (http://sitem.herts.ac.uk/aeru/footprint/en/index.htm accessed September 2012)

3 Tixier et al. (2000) Pest Manag Sci

4 Tixier et al. (2001) Environ Toxicol Chem

105

Table X. Physical-chemical properties of dichlobenil and its most frequently reported metabolites

Dichlobenil

BAM 2,6-DCBA OBAM

Type

Benzonitrile herbicide

– cellulose biosynthesis

inhibitor1

Metabolite Metabolite Metabolite

IUPAC Name2

2,6-

dichlorobenzonitrile

2,6-

dichlorobenzamide

2,6-

dichlorobenzoic acid 2-chlorobenzamide

Structure3

Use

To control annual and

perennial weeds e.g. in

fruits and other crops1

- - -

Molecular mass2

(g mol-1

) 172.01 190.03 190.00 155.58

Properties

Solubility in water

20oC (mg l

-1)

2 21.2 1830 14100 -

Stability in soil

DT502 5.4-70 days 137.7 days 660 days -

Log Kow2

2.7 0.38 2.23 1.12

Toxicity

Mammals – acute

oral LD50 (mg kg-1

)2 > 2000 500 316 1250

Fish – acute 96 hour

LC50 (mg l-1

)2 7.2 240 130 -

1 Tomlin (1997) British Crop Protection Council

2 Pesticide Properties DataBase (http://sitem.herts.ac.uk/aeru/footprint/en/index.htm accessed September 2012)

3 Holtze et al. (2007) Environ Pollut