Programme plan

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Feb 20, 2013 (4 years and 4 months ago)

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Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

1


Programme plan 2008
-
2009










MicroDrivE










































MicroDrivE




Ethanol fermentation

Ethanol yield

Biomanure

Ethanol


Feed, new products

Methane

CO
2


Sugar beet


Cereal grains


Straw


(energy crops)


(forest products)


Plant nutrients,

hygiene

Digestate

Energy ”lean” storage
storage

Biopreservation

Pretreatment

Hydrolysis, new enzymes

Bioprocessing

Biogas

Methane yield

Spent ”grains”





Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

2


A research programme on sustainable biofuel production 2007
-
09


Overall program objectives




Increase the energy yield of biofuel processes



Improve economic margins and profitability



Minimise environmental impact



Background


MicroDrivE

(Microbially Derived Energy) is a thematic research programme, aiming

towards a holistic understanding of the

biological aspects of sustainable biofuel processes,
extending from farm/forest


to fuel


to farm/forest nutrient recirculation.


The program is funded by the Faculty of Natural resources and Agricultural Sciences, SLU

(50 % funding), the National Ene
rgy Board, the Swedish farmers Research Foundation (SLF),
Medipharm AB, Syngenta Seeds, Tekniska Verken Linköping/ Svensk Biogas AB, Jäst
-
bolaget AB, Chematur Engineering AB, Sala
-
Heby Energi AB and Danisco/Genencor AS.

The participating SLU departments co
ntribute with additional resources to
MicroDrivE
,
while participating companies have substantial development projects connected to programme
research.


The
MicroDrivE

research team is composed of microbiologists and molecular biologists and
biochemists wo
rking within six major projects:
1) Biopreservation of feed stock, 2) Enzymatic hydrolysis of plant polymers,

3) Ethanol fermentation, 4) Bioprocessing of ethanol co
-
products,

5)
Biogas fermentation
, 6
) Biomanure recirculation of biogas digestate.


Programme structure

Research within

MicroDrive

research is organised as three types of projects:


I)

Rapid response to industrial/sectorial requirements


-

20
-
week M SC projects (exjobb) supervised by programme researchers


II)

Long
-
term generic knowledge development


-

4
-
year Ph D student projects supervised by programme researchers


III)

Scientific in
-
depth studies


-

2
-
year post doc projects supervised
by programme researchers


Programme management

A Steering Committee

is appointed to see to that the Program is carried out in accordance
with the intentions in the Program Plan and the policy guidelines for NL
-
faculty thematic
programmes. The NL
-
faculty a
nd each external contributor are entitled to appoint one
Committee

member each. The faculty will also provide the secretary for the meetings. The
right of member appointment includes the right to remove and/or substitute appointed
member.

The Steering Comm
ittee meets regularly, at present four meeting per year are
scheduled.
The Steering Committee has appointed a Program Director, presently professor



Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

3


Johan Schnürer, responsible for directing the day
-
to
-
day work within the Program. The
Program Director has a
ppointed a Program Management Group, responsible for assisting in
managing the program. This group consists of key researchers within
MicroDrivE
, presently
it includes docent Mats Sandgren (hydrolytic enzymes), docent Volkmar Passoth (ethanol
fermentation)

and docent Anna Schnürer (biogas fermentation).


Legal issues

Financing and IPR issues have been handled through a number of agreements as specified
below.

Partner

Project

Type

Date

NL
-
SLU, Medipharm,
Syngenta Seeds,

Tekniska Verken

Jästbolaget,
Chematur
Engin.


Sala Heby Energi


SLF (JS 1,2,3)


STEM(JSt)

STEM (MS)


Libyan goverment



Consortium of

MicroDrivE Researchers


SLU Holding

Financing






Financing


Financing


Financing

Financing


Financing a PhD proj.



MicroDrivErs

for IPR
and MicroDrivE AB


IPR support, etc

Agreement






Agreement


Contract


Contract

Contract


Contract



Agreement



LOI

2007
-
11
-
12






2008
-
02
-
06


2006, 2007


2007

2008


2008
-
01
-
19



2008
-
03
-
20



2008
-
02
-
04




MicroDrivErs

AB

MicroDrivE

has formed a co
-
workers consortium,
MicroDrivErs
, to handle any intellectual
property rights, negotiating licence agreements etc, which might be a result of the work. The
partners in such a body will be the scientists, PhD students and postdocs involved i
n the
centre. The consortium may decide to form a company,
MicroDrivErs AB
, and SLU Holding
have obtained an option to become shareholder. Potential earnings from intellectual properties
will be distributed among inventors participants and a defined port
ion will also be used for
own patent applications or other protections of intellectual property rights.



Communication plan

MicroDrivE

maintains a close direct communication with program contributor/partners,
representing broad areas of the biofuel

sector. The programme also aims for an efficient
communication through standard scientific procedures, e g conference specialist presentations
and peer
-
reviewed journal publications, as well as more general presentations and a home
-
page targetting a broa
der audience (http://microdrive.slu.se).






Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

4



Communication activities
-

resources and deliverables 2008

1. External communication

FTEM

16

Deliverables

Year:Q



Web
-
page maintenance




Lecture and poster preparations, incl.
presentations




Program plan writing
for 2009




Writing of publications




Meetings with industry




Interviews and popular science

0.5




2





0.5



10


0.5


0.5





10OO hits





12 International

presentations




Approved plan





6 publications





Minutes





Press reports

08:4



08:4



08:4



08:4



08:4


08:4



2. Internal communication

FTEM

1.5

Deliverables

Year:Q




Monthly programme meetings




Internal seminars





Preparation of presentations for Steering comm..


0.5


0.5



0.5


Minutes (10)


Hand
-
outs (8)



Reports (3)


08:4


08.4


08:1


08:4


Education

MicroDrivE

scientists are continiously involved in the teaching of future agronomists,
biotechnologists and civil engineers in biofuel related topics. In addition, the programme
offers a series of 20
-
week MSc

projects within bio
-
preservation, enzymatic pre
-
treatments,
ethanol fermentation, bioprocessing of byproducts, biogas production and soil fertility effects
of bioresidues. The projects are supervised by scientists from the Departments of
Microbiology, Mol
ecular Biology, and Chemistry at the Swedish University of Agricultural
Sciences (SLU), Uppsala. Most projects will be industry related, mediating both a wider
perspective, as well as expert knowledge. The projects will be arranged in a comprehensive
MSc P
roject School with common activities, such as lectures within bioenergetics,
microbiology and innovation processes. The
MicroDrivE

MSc project school is intended for
students within the chemistry, microbiology and biotechnology areas, including engineering
,
that are interested in future technologies for biofuel production and environmental concerns.
At SLU, this could include students within the MSc study programmes Biotechnology, Plant
Biology, and Environmental Pollutants and Risk Assessment, as well as w
ithin Agronomy,
with focus on animal husbandry, food science, as well as plant and soils science.


Each autumn and spring term, the
MicroDrivE

program starts 8
-
12 MSc projects of 20
-
weeks
duration. All external contributors to the program are given the opp
ortunity to suggest topics
for the MSc projects.
The NL
-
faculty is funding a four
-
year graduate school (2008
-
2011) in



Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

5


Bioenergy for PhD
-
students (1.1 MSEK per year).

MicroDrivE

scientists will be involved in
planning and teaching of courses, while the PhD
students of
MicroDrivE

will participate.


MicroDrivE

programme deliverables

The programme as such, has a number of overall deliverables, some in common with the
individual projects 1
-
6.


Programme Level


Workpackages, tasks, and deliverables

1.

Staffing,
management and infra structure


Deliverables

Year:Q



Formation of key scientist team




Program management group formed




Recruitment of 5 PhD students






Recruitment of 2 post
-
docs




8 new biorefinery fermentors (ethanol/biogas)

4 additional biogas
fermentors




Advanced sugar analysis intrument










Report to SC


Report to SC


Offical SLU registration




First working day


First fermentor run


-

:
-


First analysis run

08:1


08:1


07:2
07:3

08:1


08:1,3


08:1

08:2

08:2



2.
Communication


Deliverables

Year:Q



MicroDrivE

homepage




General program poster


publi挠捯浭mni捡cion



General program poster


獣sentifi挠捯浭mni捡cion




Press releases and newspaper interviews



Oral presentations for stakeholders


(politicians, farmers, industrialists etc)





Scientific presentations at international symposia





Scientific publications


First external log
-
in


First presentation


First presentation


?

10 presentations




8 oral presentations

5 pos
ters


12 scientific publications


07:2


07:2

07:2


?

08:4




08:4



08:4


3. Education


Deliverables

Year:Q



MicroDrivE

M Sc project school




2 completed PhD projects



3 completed PhD projects



8


12 M S挠the獥s


2 偨䐠the獥s

3 偨䐠the獩s

Annual


10:4

11:2





4. Innovation and development


Deliverables

Year:Q




Research leading to innovations





Industrial partner development process



3 patent applications


2 licence agreements


07,08,09



07:4,
09:2




Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

6






Projects


1. Biopreservation

of crop feed stock


microbial techniques for energy saving storage


Project leader

Docent Karin Jacobsson


Project team

PhD
-
student Matilda Olstorpe (Formas)

MSc
-
project student(s) (Jenny Bohrling, Spring 2008)


Resources

MicroDrivE: 12 FTE months
2007,
18 FTE 2008 and 8

FTE 2009


Objective

To develop biopreservation techniques that will reduce the energy input required for storage
of sugarbeets and grains intended for biofuel production.


Specific goals

-

To isolate bacterial and fungal strains from
sugarbeets to identify and collect relevant
target strains for inhibition studies, as well as putative biocontrol strains.

-

To isolate bacterial and yeast strains from grain storage systems to identify putative
biopreservation strain
s
s.

-

To develop laborator
y scale storage systems for sugarbeets.

-

To develop laboratory scale storage systems for grains.

-

To evaluate of the performance of biopreservation strains under different conditions
such as water activity, temperature variations etc.

-

To evaluate the effects of biopreservation microorganisms on ethanol production.

-

To evaluate the biopreservation efficiency in full scale storage systems.


Material ownership

Syngenta Seeds AB owns the germplasm of sugar beet varieties to be evaluated


Int
ellectual property rights

The MicroDrivEr´s consortium.


Stakeholders

Syngenta Seeds AB,
Swedish Farmers’ Foundation for Agricultural Research

(SLF), the
Federation of Swedish Farmers (LRF) and the sugar industry at large.


Industrial partner

Syngenta Seed
s AB, Box 302, SE
-
261 23 Landskrona, Sweden.
Contact: Mats Levall.


Project strategy and realisation

Sugarbeets




Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

7


From 2008, the project will be focused on storage systems similar to those used today, i.e

where beets are stored in clamps on farms under tarpaulin, and maybe a layer of straw to
protect against the cold, but with the use of biocontrol yeasts. If storage is improved by
oxygen limitation, beets may instead be stored i
n WINLIN bags. Today, the t
ime

for which
the beets can be stored is limited to a few months due to sugar losses as a result of the
inherent respiration of the sugarbeets and to infestation by soil pathogens. In Sweden, storage
problems are usually associated with
Botrytis

and
Fusari
um

species (Persson & Olsson 2006).
Damage to the sugarbeets cannot be avoided due to demands of foliage removal from the
refineries. Growth of
Fusarium

in and a surrounding the top may result in 36% fresh weight
losses in beets stored at 12

C (Persson 2002).

Three putative biocontrol yeasts have been isolated so far and additional strain will be
isolated. New strains and the known biocontrol yeast
Pichia anomala

J121 (se below) will be
evaluated for their ability to inhibit growth of pathoge
ns with focus on
Fusarium culmorum

and
Botrytis cinerae

in the laboratory but under simulated natural conditions, such as
temperature fluctuations and varying air humidity. Also, storage under oxygen limiting
conditions will be compared to non
-
limiting con
ditions.

Candidates will be tested in model scale trials, and finally in on
-
farm sto
rage systems.

Losses of sucrose due to yeast growth will be followed using HPLC (anion
-
exchange chromatography) or NIR
-
based technologies. For beets intended for ethanol or

biogas production, conversion of sucrose into glucose and fructose may not be a problem.
Still, if the techniques developed should be useful also to the sugar industry, sucrose losses
must be considered.

Sugarbeets that have stored well remain to be colle
cted. Such

material

will be
used to compare the microbial communities on sugarbeets that have stored well and those that
have deteriorated with molecular, no
n
-
cultivation based techniques
. If possible, this will also
include a comparison between different
beet cultivars.


Cereals

High moisture grains to be used as animal feed can be stored in airtight silos were the carbon
dioxide produced from the grain respiration inhibits growth of spoilage microorganisms. This
system is sensitive to air leakage, for exa
mple during removal of grains for feeding, as this
can result in heavy mould growth. The biopreservation yeast
Pichia anomala

J121 inhibits
mould growth in airtight systems and has been well characterised (Petersson 1998, Druvefors
2004 ). The mechanism ha
s been shown to be production of ethyl acetate, ethanol and
probably also other metabolites, rather than nutrient limitation or spatial crowding (Druvefors
et al 2005a). The inhibitory activity of a number of other putative biopreservation yeast
species ha
ve been investigated in a mini silos system but no strain with a better inhibitory
capacity than
P. ano
mala

J121 was found (Druvefors 2005b).

Lab scale minisilo systems (test tubes) and pilot scale silos systems for moist grain storage
have been developed

and are described in Druvefors 2004. Work has since been initiated to
develop lab scale systems for storage in bags
made from WINLIN
-
plastic
and this

will
continue in this project
.


A starter culture for airtight storage of grains

Biopreservation of grain

is highly dependent on the water content of the stored material. A
starter culture based on lactic acid bacteria will only be efficient if the water content is high
enough to allow the bacteria to grow (lower than appr. 60% DM) and produce lactic acid.
P.
anomala

J121 can grow at a wateractivity as low as 0.82 (Fredlund et al, 2002) and can be
used to preserve drier grains. It is difficult for the farmer to exactly determine and then



Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

8


control the water content. Thus, the ideal starter culture should work
at both high and low
wateractivities and then contain a mixture of
P. anomala

J121 and a suitable LAB. However,
P. anomala

will utilize the lactic acid produced by LAB as a carbon source resulting in an
increase in pH and growth of spoilage organisms. If a

LAB can be found that can inhibit
growth of
P. anomala

J121, it should be possible to combine the two organisms in a starter
culture where
P. anomala

J121 will dominate in dry grain and LAB in wetter grains and then
simultaneous inhibit the undesired grow
th of
P. anomala
.

The LAB strains collected from grains stored in WINLIN
-
bags at different farms will be
screened for yeast inhibitory properties and suitable strains evaluated in the model that will be
developed.


Common to both crops

For both systems st
udies on the effect on the yeast(s)

or lactic ac on the yeast

used for ethanol
production will be initiated at an early stage in collaboration with MicroDrivE project
3.
Ethanol fermentation
. Biopreservation strains affecting the fermentation negatively wi
ll not
be studied further.


The strain chosen will be subjected to safety assessment in coll
aboration with the DOM
-
program
. Ideally, it should be possible to apply the improved storage techniques also to
sugarbeets to be used for sugar production, and grai
n used as animal feed. Depending on the
use, the legislation will differ, and this has to be dealt with during later stages of the project.


Workpackages, tasks, resources and deliverables

1.

Strain isolation and characterisation (2007)

FTEM

12

Deliverables

Year:Q



Isolation and identification of bacterial and fungal
strains from sugarbeets.





Identification and characterization of bacteria and
yeast from sugarbeets with a biopreservation
activity. Initial inhibitions studies on sugarbe
ets





Isolation of yeast and bacteria from grain storage
systems.



Identification and characterization of bacteria and
yeast with a biopreservation activity

from grains



Community profiling


2




3





2


2


3

Report on identification
and strain identities.

Laboratory work
finished

Report on antifungal
strains

Part 1 completed. Part
2 ongoing


Completed


Delayed until 08


Scientific article


Delayed until 08



07:2




07:4






07:4


07:4



07:4



2. Efficacy Evaluation
and strain

characterisation

(2008)

FTEM

18

Deliverables

Year:Q



Field trial with
P. anomala

in stored grains



Development of laboratory scale storage systems
for sugarbeets

and evaluation of strains
.



Production of material for evaluation in
fermentation with collaboration of ethanol
-
fermentation.

5


3


2



Scientific article




Scientific article




08:2


08:3


08:4






Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

9




Development of a dual LAB
-
yeast for airtight
storage of grains



Development of a laboratory scale plastic bag
-
based storage system for grain




Community profiling on sugarbeets

4



1


3



Scientific article



Protocol


Scientific article

08:4


08:4


08:4



3. Efficacy Evaluation contd and Safety
Evaluation (2009)

FTE
M

8


Deliverables

Year:Q



Evaluation of safety of strains and traceability




Model scale st
orage system and/or field trials


2


6



Final report on safety
assessment

Scientific article

One patent application


09:2


09:2


09:4




GANNT Chart



Tasks

&

Decision points (DP)

2007

Q

2008

Q

2009

Q

1

2

3

4

1

2

3

4

1

2

3

4

1

Strain isolation


sugarbeets

1:1


























2

Strain characterisation


1:2











3

Strain isolation 2


sugarbeets



1:3










4

Community profiling



1:4
























5


Strain isolation
-

grains



1:5
























6

Field trial
-
grain





2:1








7














7

Labscale storage of
sugarbeets

and strain
evaluation





2:2








8














8

Material for ethanol
production evaluation





2:3






















9

Development of dual
starter cultures






2:4





















10

Labscale storage systems
for grain







2:5






11

Community profiling







2:6






12

Strain safety

evaluation









3:1




13

Model scale or f
ield trials










3:2


















References

Druvefors, UÄ. 2004. Yeast biocontrol of grain spoilage moulds


Mode of action of
Pichia anomala
.


Agraria 466. Department of Microbiology, Swedish University of Agricultural Sciences.




Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

10


Druvefors, UÄ, Passoth, V and Schürer, J. 2005a. Nutrient effects on biocontrol of
Penicillium


roqueforti
by
Pichia anomala
J121 during airtight storage.
Appl. Environ. Microbiol.

71: 1865
-


1869.

Druvefors, UÄ and Schnürer, J. 2005b. Mold
-
inhibitory activity of different yeast species during


airtight storage of wheat grain.

FEMS Yeast R
es.

5: 373
-
378.

Fredlund, E., Druvefors, U., Boysen, M.E., Lingsten,
K.
-
J. and Schnürer, J. 2002. Physiological


characteristics of the biocontrol yeast
Pichia anomala

J121.
FEMS Yeast Res
. 2: 395
-
402.

Persson, L. and Olsson, Å. 2006.
Åtgärder mot förluster av svampangrepp i sockerbetor under odling


och lagring.
SBU.

Persson, L. 2002. Inventering av svampsjukdomar i fält och lager.
SBU projektkod: 2002
-
1
-
2
-
408.

Petersson, S. 1998. Yeast/Mould interactions during airtight storage of high
-
moisture feed grain. PhD


thesis. Agraria 97. Department of Microbiology, Swedish
University of Agricultural Sciences.

Ström, K., Sjögren, J., Broberg, A. and Schnürer, J. 2002.
Lactobacillus plantarum

MiLAB393


produces the antifungal cyclic dipeptides cyclo(L
-
Phe
-
L
-
Pro) and cyclo(L
-
Phe
-
trans
-
4
-

OH
-
L
-
Pro)

and 3
-
phenyllactic acid.
App
l. Environ. Microbiol.

8: 4322
-
4327.


2. Hydrolytic enzymes


Project leaders

Docent Jerry Ståhlberg (75%), Dr Mats Sandgren (50%)


Project team (2008)

Researcher Henrik Hansson (mid 2008
-
2009)

Postdoc Evalena Andersson (
-
Jun2008), Mohamed Abdel
-
Aziz (Sept

2008
-

Feb 2010,
scholarship applied from Swedish Institute)

PhD students Jonas Vasur (2008), NN (mid 2008
-
2009), NN (mid 2008
-
2009)

BSc students Hanna Davies, Elin Einarsson, Mia Hertzberg, Emma Jacobsen (Mar
-
Jul 2008)

MSc students Jesper Svedberg (
Jan
-
Jul 2008), Majid Haddad (Jun
-
Dec 2008)


Resources

MicroDrivE:
~50 FTE months 2008, ~50 FTE months 2009, ~25 FTE months 2010.



Objective

Improve enzymatic processes at different stages along the biofuel process chain, with main
focus on enzymes for sac
charification of starch, cellulose and other polysaccharides; in a
shorter perspective for enhanced yields from sugar and starch crops, and in a longer
perspective for the use of cellulosic biomass as the main raw material.


Specific goals

-

Enhance ethano
l yield by enzymatic degradation of cellulose/hemicellulose.

-

Boost biogas production by enzyme supplementation.

-

Compare enzyme mixtures and process conditions to optimise enzymatic saccharification.

-

Structural and functional characterisation of beta
-
glucanases of interest for processing of
cereal beta
-
glucans.

-

Search for superior enzymes in certain wood
-
degrading microbes.

-

Determine molecular structures of key enzymes for biomass saccharification and pursue
protein engineering towards enhanced per
formance.


Material ownership




Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

11


-

Genencor/Danisco will provide certain enzyme preparations. Their use and any components
isolated thereof is regulated by MTAs (Materials Transfer Agreement) with SLU.

-

MTAs need to be negotiated for enzymes, organisms or ra
w material for enzyme preparation
provided by the group of Prof. M. Samejima, University of Tokyo (UT) or other third party.

-

Prepared material should be co
-
owned by MicroDrivE if substantial work has been invested
here in the preparation.

-

The MicroDriv
E consortium shall own material that is entirely produced within the project
(e.g. enzymes isolated from biogas sludge).


Intellectual property rights

-

Previously signed contracts with SLU give Genencor/Dansico a 5 years patent right to any
results emergi
ng from the use of Genencor/Danisco owned material. Current NDAs (Non
-
Disclosure Agreement) give Genencor/Danisco the right to evaluation of patentability for 3
months prior to disclosure/publication of the results.

-

The rights to results emerging from us
e of material provided by the group of Prof Samejima,
UT, or other third party, shall be shared with MicroDrivE.


Stakeholders

The project is co
-
funded by the Swedish Energy Agency (Energimyndigheten) and by
Genencor
-

A Danisco Division.


Industrial
partner

Genencor
-

A Danisco Division, is one of the worlds leading enzyme
-
producing companies.
This company has for many years had extensive research programs with the aim to identify
new enzymes, from many different micro organisms, that potentially coul
d be utilised in
enzymatic pre
-
treatment of different plant materials used in various industrial applications.
The company has also for many years had several research programs with the aim to optimize
the enzyme mixtures used for saccarification of ligno
-
cellulosic plant materials, with the aim
to decrease the total cost for the enzymes used in the process.


Project strategy

Commercial optimised enzyme mixtures for cellulose, hemicelllulose and starch
saccharification will be used initially to evaluate pro
cessing methods of important feed
-
stocks
(e.g. sugar beets, wheat grain, distillation residuals) for enhancing yields in ethanol
fermentation and biogas digestion. The importance of certain individual enzymes will further
be evaluated. Engineering programs

already underway for some key enzymes will be pursued
and complemented with mining for key components produced by biogas microbial
communities and potent wood
-
degrading fungi and protozoa. Enzymes of particular interest
will be subject to structure/functi
on studies to enable modifications towards improved
performance. The MicroDrivE project will also be tied to ongoing structure/function studies
of fungal laminarinases, as potential tools for degradation of cereal beta
-
glucans and for
added
-
value products
from biofuel waste.


Scientific approach and realisation


WP 1. Beta
-
glucanases

-

Collaboration with UT (and Bengt Guss?).

Within the collaboration with UT we study structure and function of one the major
laminarinases, Lam16A, from the wood
-
decaying fung
us
Phanerochaete chrysosporium
. We



Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

12


have solved and published the structure of this enzyme and are currently elucidating
molecular mechanisms for substrate binding and hydrolysis and transglycosylation activity.
The genome of
P. chrysosporium

contains 20 ho
mologous genes belonging to the same
family of glycoside hydrolases as Lam16A. UT are currently trying to express the most
interesting of these. Within MicroDrivE we want to evaluate if Lam16A or any of the
isoenzymes can be useful for degradation of cerea
l beta
-
glucans. Such enzymes may also be
useful for preparation of added
-
value products from biofuel wastes, either through
degradation of beta
-
glucan rich material (predominant component in yeast and fungal cell
walls) or by utilising the transglycosylati
on activity for oligosaccharide synthesis. Certain
beta
-
glucans are known to have immunostimulating and anti
-
tumour effects.


WP 2. Enhanced yield of ethanol from sugar beets and starch crops.

-

Together with Volkmar Passoth and (Karin Jacobsson?).

Applica
tion of enzymes for degradation of cellulose and other polysaccharides will mobilise
further soluble sugar for fermentation and also enhance the yield of sucrose extraction from
the sugar beets and facilitate starch liquefaction with starch crops, both fac
tors contributing to
an enhanced yield of ethanol. Initially an optimised cellulase mixture for cellulose
saccharification will be used in combination with additional carbohydrases (e.g. pectinases,
hemicellulases) in trials with different sugar beet and w
heat grain raw materials. Enzyme
dosage and processing conditions will be evaluated as well as the influence of different
storage conditions, for example frozen vs. fresh sugar beets, and dried wheat grain vs. wet
storage with microbial preservation. Trial
s will also be made with inclusion of the wheat
straw. Further studies may include optimisation of processing conditions, enzyme blend
and/or studies of importance of individual enzymes or further additives, depending on the
outcome of the pilot studies.


WP 3. Enhanced biogas yield by enzyme supplementation.

-

Together with Anna Schnürer.

The microbial communities in biogas communities are able to degrade a large variety of
materials. But when using cellulose
-
rich feedstocks, substantial amounts of the ce
llulose
remain in the residual material. This project aims at evaluating to which extent the utilisation
of cellulose may be improved by supplementary enzyme addition. Optimised cellulase
mixtures will be used with different types of biogas feed
-
stocks (e.
g. distillers spent grain,
wheat grain, silage, household waste) and different methods for enzyme application will be
evaluated, either pre
-
digestion or at various time
-
points during the biogas digestion process.
Mesophilic and thermophilic digestions will

also be compared.


WP 4. Ethanol and biogas from lignocellulose biomass.

-

Together with Volkmar Passoth and Anna Schnürer.

During the first half of 2008 a pilot study will be performed with the aim of establishing
routines for preparation (milling, siev
ing) and thermochemical pretreatment of cellulose
materials as well as enzymatic saccharification and analysis of solubilised sugars and to
devise standard conditions for evaluation of yields from different cellulosic raw materials.
Potential substrates fo
r ethanol production are currently being collected including oat and
wheat straw, hemp, Reed canary grass, pine, spruce, and aspen saw dust, Salix and others.
After estimations of ethanol yields from saccharification under ”standard” conditions, the
influe
nce of various parameters will be examined and optimised for the most interesting
substrates, such as enzyme dose, temperature, pH, ionic strength, and enzyme composition.
Based on the results of theoretical yields, representative substrates will be chosen

for ethanol



Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

13


fermentation studies. Substrates of particular interest will also be selected as feedstocks for
biogas digestion studies in order to evaluate which method would be the most efficient way to
derive biofuel from the biomass material.


WP 5. Stru
ctural characterisation and engineering of biomass degrading enzymes
.

-

Collaborations with Genencor/Danisco and with UT.

Within an ongoing collaboration with Genenor/Danisco we study the structure and function of
biomass degrading enzymes, primarily from

H. jecorina
, and try to engineer key components
towards improved performance in large
-
scale saccharification applications. The aim is to
reduce the cost for enzymes in the process. With cellulosic raw materials the enzyme cost
needs to be reduced to make
bioethanol competitive with fossil fuels. Structures have already
been determined of the major components and engineering programs are well underway. But
there are also several genes that are induced of which less is known. We want to elucidate
their role
in the degradation machinery, and characterise these proteins structurally and
biochemically, in order to enable engineering of proteins that turn out to be of interest in
cellulose saccharification or other biotechnical applications. Some of the enzymes c
urrently
under study will be tested for ethanol and biogas yield enhancement. A similar approach will
be applied for enzymes of interest within the collaboration with UT.


WP 6. Enzymes from potent wood degraders.

-

Collaboration with UT and others.

Industrially produced cellulases come almost exclusively from ascomycete fungi, with
Hypocrea jecorina

(also known as
Trichoderma reesei
) being the major workhorse today. In
nature, however, basidiomycete fungi play a dominating role in wood degradation an
d ligno
-
cellulose recycling. Among them there may exist superior enzymes for certain applications.
Rather few comparative studies of basidiomycete cellulases have been made though, in large
part because the key cellulases notoriously refuse to be heterolog
ously expressed. We
collaborate with the group of Prof. M. Samejima, University of Tokyo, who study molecular
biology and enzymology of wood
-
degrading basidiomycetes. They have already identified
several enzymes homologous to known key enzymes for cellulos
e degradation and are
searching for further enzymes. We want to compare the performance of these enzymes with
their counterparts in
H. jecorina
. Interesting cellulase genes have also been found in certain
protozoa that live as symbionts in the hindgut of t
ermites. We will analyse the sequences from
a structural
-
functional point of view in order to identify targets of interest. The Tokyo group
will try to express those, either in
Pichia pastoris
or in a basidiomycete expression system
currently under develop
ment. Through another collaboration we expect to get access to
enzymes from the root
-
rot fungus
Heterobasidion annosum
, a major pathogen on spruce that
causes severe economical loss in the Swedish forest industry. It is an efficient wood degrader
and we
expect that it will have enzymes with highly interesting properties. The fungus has
been chosen as target for a genome
-
sequencing project at the Joint Genome Institute, JGI,
after an initiative by Prof. Jan Stenlid, Dept. Forest Mycology and Pathology, SLU
.



Work packages, tasks, resources and deliverables


1. Beta
-
glucanases

FTEM

33

Deliverables

Year:Q



Structure studies of reaction mechanism in Pc
Lam16A

9



3 publications

08:3



Studies of hydrolysis and transglycosylation in
9



1 publication

08:4




Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

14


Lam16A wt and engineered
mutants



Finalise structure/function analysis of Pc Lam16A

15



1 PhD thesis

09:1


2.

Enhanced yield of ethanol from sugar beets
and starch crops

FTEM

35

Deliverables

Year:Q



Publication of results from pilot study of ethanol
yields
from wheat grain, +/
-

preservation/drying

2



1 publication

08:4



Pilot study of ethanol yields from sugar beets, +/
-
preservation, +/
-

enzymes

9



Report

08:4



Ethanol yields with various starch crops, +/
-

cellulose, +/
-

enzymes

9



Manuscript

09:2



Follow
-
up study. Process optimisation

15



Processing protocol



Manuscript

10:2


3.

Enhanced biogas yield by enzyme
supplementation

FTEM

29

Deliverables

Year:Q



Pilot study with cellulosic feed
-
stocks +/
-

enzyme,
e.g. silage, house
-
hold waste, saw dust,
Salix

9



Report

08:2



Studies of enzyme dosage and optimisation of
processing conditions. Cost/benefit evaluation.

20



Processing protocol



Manuscript

10:2


4. Ethanol and biogas from lignocellulose

FTEM

64

Deliverables

Year:Q



Pilot study: Routines for analysis of fermentable
sugars from lignocellulose saccharification

9



MSc report

08:3



Yields of fermentable sugars from various cellulose
substrates under “standard” conditions

10



MSc report



1 publication

09:1

09:2



Optimisation

of saccharification conditions for
selected cellulose substrates

10



MSc report



1 publication

09:3

09:4



Ethanol yields from selected cellulose substrates

10



Report

09:4



Biogas yields from selected cellulose substrates

10



Report

09:4



Follow
-
up study and
compilation of results

15



1
-
2 publications

10:2



5. Structural characterisation and engineering of
biomass
-
degrading enzymes

FTEM

36

Deliverables

Year:Q



Engineering of
H. jecorina

key cellulases

11

11

11




One improved enzyme
component per year

08:2

09:2

10:2



Test the influence on ethanol and biogas yield of
other individual
H. jecorina

enzyme components
(other than the two key cellulases)

1

1

1



Performance of one
component per year

08:2

09:2

10:1


6. Enzymes from potent wood
-
degraders

FTEM

30

Deliverables

Year:Q



Isolate Cel7 isoenzymes from

P. chrysosporium

and compare activity with
H. jecorina

bench
-
marks

6



BSc report

08:3



Sequence analysis of protist cellulase genes and
selection of interesting targets

2



Report

08:4



Search for new enzymes in
H. annosum, P.
chrysosporium

and other basidiomycetes

6



MSc report

09:1



Cloning and expression of selected cellulases and
comparison with H. jecorina bench
-
marks

10



Report, manuscript

09:4



Determine structure? Initiate
engineering program

11



Report

10:2





Tema
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forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

15


GANNT Chart



Tasks


2008

Q

2009

Q

2010

Q

1

2

3

4

1

2

3

4

1

2

1:1

Structure studies of Pc
Lam16A























1:2

Studies of Lam16A
transglycosylation























1:3

Finalize analysis of Pc
Lam16A



































2:1

Publication of results
from pilot study























2:2

Pilot study: Ethanol
yields from sugar beets























2:3

Ethanol yields with
various starch crops























2:3

Follow
-
up study.
Process optimizations























3:1

Pilot study: Biogas
from cellulosic mater.























3:2

Enzyme dosage and
process optimizations























4:1

Pilot study: Routines
for sugar analysis























4:2

Yields of fermentable
sugars from dif
substrates























4:3

Saccharification
optimizations























4:4

Ethanol yields from
selected substrates























4:5

Biogas yield from
selected substrates























4:6

Follow
-
up study and
comp. of experiments























5:1

Engineering of
cellulases























5:2

Test the influence on
ethanol and biogas
yield























6:1

Isolation of P.c.
Cel7A enzymes























6:2

Seq. of identified
cellulase proteins























6:3

New enzymes from
H. annosum























6:4

Cloning of identify
new cellulases























6:4

Structure













Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

16


determination of new
cellulases














Integrated evaluation





























3. Ethanol fermentation


Project leader

Docent Volkmar Passoth


Project team

Doktorand Johanna Blomqvist

Postdoc André Förster

Four master students (Anna Eriksson, Madeleine Nilsson, Helena Jansson, Johan
Hägglund)


Resources

MicroDrivE:

36 FTE months (J. Blomqvist, 30 FTE months (V. Passoth), 24 FTE months
(Postdoc), 15 FTE months (master students), 1 FTE months (J. Rejholt, Jästbolaget for co
-
supervision of a master student, work package 6), 2 FTE months
(J. Ståhlberg, M. Sandgren
for supervision of a master student, work package 5, 6)


Objective

To improve ethanol production by identifying new production strains. To investigate the
impact of alternative raw material storage/ new raw materials on the ferme
ntation process

Specific goals

1.

To determine the potential of
Dekkera bruxellensis

for industrial ethanol production

2.

To determine the role of the lactic acid bacteria in ethanol production processes

3.

To investigate the microbial flora in a variety of industr
ial ethanol fermentations to
identify production strains with a high potential

4.

To investigate the competition between different production and infection strains to
find conditions for stable ethanol fermentation

5.

To test ethanol production from airtight (wi
thout drying) stored grain

6.

To test the impact of using new enzymes for poly
-
glucose (starch, cellulose)
degradation on ethanol production

Material ownership

-

not applicable ?


Intellectual property rights

The
MicroDrivErs

consortium


Stakeholders


Industr
ial partner

Jästbolaget, Chematur AB, Medipharm AB





Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

17


Project strategy, scientific approach and realisation

The core task of the project will be the establishment of standard fermentation procedures in
the laboratory scale. Fermentations will be run under de
fined conditions and the relevant
parameters (specific substrate consumption rate, specific biomass and ethanol production
rates, yield coefficients) will be determined. This creates the possibility to compare the results
of the different approaches with e
ach other; thus, the best possible strain or co
-
cultivation
regime will be identified. The potential of
D. bruxellensis

for ethanol production and its
interaction with the lactic acid bacterium (LAB)
Lactobacillus vini

(Passoth et al. 2007) and
other yeast
s will be investigated in a Ph.D. project. As the physiology of the non
-
conventional
yeast
D. bruxellensis
is poorly investigated yet, we will run a postdoc
-
project (interacting
with the Ph.D. project), which is specifically dedicated to the physiology of
this yeast. Partial
projects involving enzymatic degradation before fermentation will be run in collaboration
with the group of
J. Ståhlberg, and M. Sandgren. In this part, several Masters’ projects will be
involved. We will also test the impact of alterna
tive storage of grain (Passoth and Schnürer
2003) on the ethanol productivity.

Fermentation characteristics of
Dekkera bruxellensis

and
Lactobacillus vini
: A defined
cultivation system will be established. Cultivation will be done in continuous fermentation,
either with or without recirculation of the organisms in the fermenter. External conditions like
dilution rate, temperature, pH, and oxygen supply will

be regulated. Cells (yeasts, bacteria
and the consortium) will be cultivated in minimal medium with glucose as sole carbon source
or in medium provided by Reppe AB or other ethanol producing companies. In these systems,
we will identify ethanol and biomas
s yields and productivities under the tested environmental
conditions.

Co
-
cultivation of
D. bruxellensis
and
L. vini
: The yeast and the bacterium will be co
-
cultivated in batch and continuous cultivations with different substrates and the influence of
the
co
-
cultivation on cell viabilities and growth rates will be determined. If we can find a
significant effect we will also test the influence of other lactic acid bacteria on yeast growth
and viability. Finally it will be tested whether there are similar eff
ects of lactic acid bacteria
on the growth of
S. cerevisiae
. For this project, methods for real time PCR quantification of
the involved microorganisms and living cell counting by flow cytometry will be developed.

Characterisation of yeast
-

lactic acid bac
teria consortia in other industrial ethanol
fermentations: Samples will be taken from industrial ethanol plants and the microorganisms
will be spread on medium selective for either yeasts or lactic acid bacteria. The cells will be
quantified and identified

using PCR
-
fingerprint and rDNA
-
sequencing. We have already
contacts to several ethanol
-
producing companies and will establish new contacts with the help
of one partner in our project, who is worldwide selling fermentation equipments. This will
give a gene
ral survey about industrial yeast strains and the role of their interactions with lactic
acid bacteria in the production process.

D. bruxellensis

or the consortium of
D. bruxellensis
and
L. vini

will be cultivated in
continuous cultivation with either gluc
ose or the industrial substrate under several different
culture conditions (dilution rates, temperatures, pH, with and without yeast recycling). The
consortium will be challenged by the addition of high cell numbers of other organisms. These
organisms will

include the yeasts
S. cerevisiae

and
Pichia anomala

and other yeasts and lactic
acid bacteria that have been isolated from other ethanol production processes. By this, we will
determine the stability of the process and under which circumstances one strain

can
outcompete another one.

Fermentation of poly
-
glucose: We will test the ability of several commercially available
enzymes to degrade soluble starch, with special consideration of the degradation velocity at
conditions at which bakers’ yeast can grow (3
0
-
32ºC, pH 5
-
6). Based on these results, a



Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

18


model cultivation (based on a simple shake
-
flask culture) will be established, where bakers’
yeast is grown in the presence of starch and the starch degrading enzymes with the highest
degradation capacity. We will

estimate the basic growth parameters, like specific growth
rates, biomass yields and maximum final biomass concentration dependent on the amount of
substrate and added enzyme. If this process functions, we will further analyse it using the
above
-
mentioned

controlled cultivation system. We will later test cellulose material together
with according enzymes in the same way.

Testing of alternative storage systems: Grain will be stored in mini
-
silos, simulating airtight
storage. According biocontrol yeasts and
lactic acid bacteria will be added to the minisilos.
After 2
-
3 weeks storage, the starch in the grain will be degraded to mono
-

and disaccharides
using according enzymes and the sugar will be fermented to ethanol in model fermentations.
These fermentations

will be compared to control cultivations, where dried grain is used
instead. The parameters of the fermentations will be determined.


Work packages, tasks, resources and deliverables

1.
Potential of
D. bruxellensis

for
industrial ethanol production

FTEM

2
7

Deliverables

Year: Q



Creating defined cultivation
systems (fermentation) for
D.
bruxellensis

6

Fermentation protocols for batch
and continuous cultivations

Batch protocol completed,
Optimisation ongoing
,
continuous culture started,
extension necessary

07: 4



Fermentation characters (specific
production rates, yield
coefficients) of
D. bruxellensis

8

One publication

Ongoing, eventually necessary to
extend to 08: 3

08: 2



Metabolic flux analysis in
D.
bruxellensis

8

One publication, lab
-
scale
protocol to cultivate
D.
bruxellensis
to reach maximum
productivity

09: 2



Pilot scale fermentation with
yeast recycling (at Chemature)

5

Middle scale protocol for ethanol
production with
D. bruxellensis

09: 2


2. Role of lactic

acid bacteria (LAB)
in ethanol fermentation

FTEM
22

Deliverables

Year: Q



Creating defined cultivation
systems for
L. vini

6

Fermentation protocols for batch
and continuous cultivations

Ongoing
,
necessary to extend

07: 4



Fermentation characters (product
spectra, specific production rates,
yield coefficients) from different
carbon sources of
L. vini

4

One publication

Ongoing, necessary to extend

08: 2



Co
-
cultivation of
D. bruxellensis

and
L. vini
: Effects on cell
viability

and fermentation
parameters

6

One publication, protocol for co
-
cultivation, establishing a method
for viable cell count by flow
cytometry, one master thesis

09: 2



Co
-
cultivation of
D. bruxellensis

and
S. cerevisiae

and other LAB
(isolated from ethanol
6

One publication, one protocol for
real time PCR quantification of
L. vini

and other LAB

09: 4




Tema
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forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

19


fe
rmentations): Effects on cell
viability and fermentation
parameters, competition between
different LAB
-
strains


3. Investigation of the microbial flora
in
industrial ethanol fermentations

FTEM
10

Deliverables

Year: Q



Isolation of yeast and LAB from
industrial fermentations
(Chematur customers and others)

5

A collection of yeast and LAB
strains from ethanol productions

Ongoing

09: 4



Monitoring the
population in
selected ethanol fermentations

5

One publication

09: 4


4. Competition between production
and infection strains

FTEM
10

Deliverables

Year: Q



Challenging of
D. bruxellensis

and
D. bruxellensis
/
L. vini

with a
high number of microorganisms
(isolates from industrial
fermenters and other highly
competitive strains)

5

Cultivation protocol for stable
non
-
sterile ethanol production
based on
D. bruxellensis
, one
publication

09: 4



Challenging a
S. cerevisiae

cultivation with
D. bruxellensis

and other microorganisms (see
above)

5

Cultivation protocol for stable
non
-
sterile ethanol production
based on
S. cerevisiae
, one
publication

09: 4


5. Ethanol production from airtight
stored grain

FTEM
6

Deliverables

Yea
r: Q



Airtight storage of grain in model
ensilations with biocontrol
organisms (Druvefors et al.
2005), enzymatic pretreatment
and model fermentation

6

Estimation of the impact of
biocontrol on subsequent steps
(starch degradation and ethanol
production),
one master thesis,
one publication
Experiments and
thesis completed, publication
under writing

07: 4


6. New enzymes for polyglucose
-

degradation

FTEM
14

Deliverables

Year: Q



Degradation of cellulose/ starch
materials, model fermentations,
estimating the

ethanol yield from
the materials

14

Protocols for the degradation of
polysaccharides from different
origins, two master thesis, one to
two publications

Starch completed, one Master
thesis completed, patent
application under consideration,
cellulose postpo
ned

07: 4






Tema
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forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

20







GANNT chart


Tasks

&

Decision points (DP)

2007

Q

2008

Q

2009

Q

1

2

3

4

1

2

3

4

1

2

3

4

1

Creating

defined cultivation
systems (fermentation) for
D.
bruxellensis

1:1












2

Specific production rates,
yield coefficients of
D.
bruxellensis



1:2










3

Metabolic flux analysis in
D.
bruxellensis




1:3









4

Pilot scale fermentation with
yeast recycling









1:4




5

Creating defined cultivation
systems for
L. vini



2:1










6

Fermentation characters of
L.
vini




2:2









7

Co
-
cultivation of
D.
bruxellensis

and
L. vini






2:3







8

Co
-
cultivation of
D.
bruxellensis

and
S. cerevisiae

and other LAB






2:4







9

Isolation of yeast and LAB
from industrial fermentations

3:1












10

Monitoring the population in
selected ethanol fermentations




3:2









11

Challenging of
D. bruxellensis

and
D. bruxellensis
/
L. vini

with a high number of
microorganisms









4:1




12

Challenging a
S. cerevisiae

cultivation with
D.
bruxellensis

and other
microorganisms









4:2




13

Airtight storage of grain in
model ensilations with
biocontrol organisms,
enzymatic pretreatment and
model fermentation



5:1










14

Degradation of cellulose/
starch materials







6:1







References




Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

21


Druvefors UÄ, Passoth V, Schnürer J. 2005. Nutrient effects on biocontrol of
Penicillium
roqueforti

by
Pichia anomala

J121 during airtight storage of wheat. Appl Environ Microbiol
71:
1865
-
1869

Passoth V, Schnürer J. 2003. Non
-
conventional yeasts in
antifungal application.
In de Winde,
J.H. (ed.)
Functional genetics of industrial yeasts.
Springer Verlag Berlin Heidelberg. 297
-
329

Passoth V, Blomqvist J, Schnürer J. 2007.
Dekkera bruxellensis

and
Lactobacillus vini

form a
stable ethanol
-
producing conso
rtium in a commercial alcohol production process. Appl
Environ Microbiol 73: 4354
-
4356


4. Bioprocessing of ethanol fermentation by
-
products


Project leader


Professor Bengt Guss


Project team

Docent Sebastian Håkansson, Dr Thomas Eberhard, Docent Hans Jon
sson


Resources

MicroDrivE:

6 FTE months 2007, 10,5 FTE months 2008, 11 FTE months 2009


Objective

To make bio
-
ethanol production a sound economical as well as environmental alternative to
fossil fuels, profitable use of process waste and by
-
products must

be considered.

Increased bio
-
ethanol production will also increase the availability of the by
-
product
distillers’ waste (sv. “drank”) that today mostly is used as animal feed. The objective of the
present project is to develop novel “ green “ biotechnolog
ical products/ applications of
distiller’s waste.


Specific goals

Goal 1. Basic characterization of distillers’ waste from various sources.

Goal 2. New fermentation substrates from distillers’ waste.

Goal 3. Distillers’ waste as microbial formulation
support.

Goal 4. The use of filamentous fungi grown on distillers’ waste as source for developing
novel biomedical products.

Goal 5.
Stabilisation and refinement of distillers’ waste with selected LAB starters



Material ownership

?

Intellectual property
rights

The MicroDrivEr´s consortium


Stakeholders

?


Industrial partner

XX AB, Medipharm (LAB starters, novel growth media etc)


Project strategy




Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

22


The project is divided into five subprojects further described in WP1
-
5. The different
subproject will collabo
rate and support each other and will be run mainly by master thesis
students.


Scientific approach and realisation

We have characterized wheat based distillers’ waste with respect to carbohydrates, amino
acids, C/N/P, and trace elements. The analysis of co
mmon soluble carbohydrates, soluble
amino acids, C/N/P, and trace elements, as well as analysis of the insoluble part, was
performed by a commercial laboratory. Analysis of a few selected soluble substances was
performed at the Department of Chemistry, SLU
, using NMR spectroscopy, mass
spectrometry and chromatography, supplemented by chemical derivatization techniques when
necessary. Currently we are investigating sources of non grain based distillers’ waste (sugar
beets and potatoes) to also characterize t
hese products if feasible.


We will characterize whole or fractionated distillers’ waste for its function in supporting
growth of certain model microorganisms representing gram
-
positive and gram
-
negative
bacteria as well as yeast and fungi. Distillers’

waste with or without supplement of
carbon/nitrogen/phosphorous/salt sources will be used. The reverse experiments of using
distillers’ waste as carbon/nitrogen/phosphorous supplements to defined laboratory media will
also be performed. In addition, the u
sefulness of the non
-
soluble fraction of distillers’ waste
will be tested as solid support/micro
-
carrier material for model microorganisms that require
solid surfaces for growth. Physical features such as the possibility to measure optical density,
foaming

characteristics as well as sedimentation of non
-
soluble particles will be evaluated.

In addition to evaluating distillers’ waste as a component in new industrial medias, we will
also investigate whether soluble components therein, such as carbohydrates, a
re of use as
drying protectants in dry formulations (e.g. freeze
-

and vacuum drying) of viable
microorganisms. The non
-
soluble fraction of distillers’ waste will also be tested for the
usefulness as carrier or bulk material in convectional air
-
drying (flui
dised bed drying) of
viable microorganisms.


Distillers’ waste will also be evaluated as a substrate for growth of filamentous fungi.
In
the present project we will explore the possibilities of obtaining chitin, a natural
polysaccharide, from filament
ous fungi as an alternative source of industrial chitin
production. The purified chitin will be chemical modified into a biotechnical important form,
called chitosan, a deacetylated derivative of chitin. Chitosan is biocompatible, biodegradable,
show low t
oxicity and can be used in numerous of biotechnical and biomedical applications.
In the present subproject we will start to study if the obtained product(s) have a potential in
future biomedical applications. These studies will be performed in collaboratio
n with another
research group at Lund University.


Today d
istillers' waste is the fermentation residue of ethanol production from cereal grains
and is extensively used in the wet or the dried form as an animal feed worldwide. In the wet
form a second f
ermentation step often occurs spontaneously by lactic acid bacteria (LAB)
resulting in a stable and hygienic product. The population of LAB varies between different
distillers’ grain and is probably largely dependent on physical parameters such as
temperat
ure. Thus a possibility to control the LAB fermentation exists. We have examined the
LAB and yeast populations that develop naturally in different distillers’ waste preparations.
We have also started to examine the fermentation of distillers´grain at diffe
rent temperatures
and with the addition of different strains of LAB. The nutritional and hygienic properties of
distillers’ waste fermented with mixtures of selected strains will be evaluated.






Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

23


Workpackages, tasks, resources and deliverables


WP1.

Basic characterization

FTEM

2.5

Deliverables

Year:Q



1:1Analysis of soluble carbohydrates



1:2 Analysis of soluble proteins, C/N/P and
trace elements



1:3 Analysis of insoluble fraction



1:4 Microbial evaluation



1:5 Physical characteristics relevant to
fermentation



1.6 Setup of fermentors

1




0.5



0.5


0.5







Technical report

07:3


07:3


08:2


08:3


08:3


07:3
-

08:1




WP2. New fermentation substrates

FTEM

5

Deliverables

Year:Q



2:1 Evaluation of the usefulness of distillers’
waste as the main
component for new
industrial media



2:2 Evaluation of the usefulness of distillers’
waste as a media supplement.



2:3 Evaluation of the usefulness of distillers’
waste non
-
soluble fraction as micro
-
carrier in
fermentation

2


2




1



Master thesis 1



08:4



08:4




09:1


WP3. Distillers’ waste as microbial formulation
support

FTEM

5

Deliverables

Year:Q



3:1 Evaluation of soluble substances from
distillers’ waste used as drying protectant in
microbial formulations



3:2 Evaluation of non
-
soluble substances
from

distillers’ waste used as carrier material
in air
-
dried microbial formulations

3



2





Master thesis 2


09:2




WP4.
The use of filamentous fungi grown on
distillers’ waste as source for developing novel
biomedical products.

FTEM

5

Deliverables

Year:Q



4:1 Evaluation of the growth of filamentous
fungi

in various preparations of distillers’ waste

4:2 Preparation of chitin/chitosan from fungi

4:3 Chemical analysis of the chitosan

4:4 Start evaluation of the activity of the
chitosan preparation in various b
ioassays

2






1



1




1



Master thesis 3


09:2





Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

24









WP5. Stabilisation and refinement of distillers’
waste (DW) with selected LAB starters

FTEM

10

Deliverables

Year:Q



5:1 Description of LAB populations in DW
of different origin



5:2
Fermentation and hygienic evaluation
of DW with different mixtures of LAB



5:3 Nutritional content of fermented DW


5



5

Master thesis 4

Delayed until 08:1


Master thesis 5








07:4


08:2



08:4



GANNT Chart 080205



Tasks

&

Decision
points (DP)

2006

Q

2007

Q

2008

Q

2009

Q

2010

Q

3

4

1

2

3

4

1

2

3

4

1

2

3

4

1

2

WP1

1:1





X

X

X











1:2








X










1:3









X









1:4









X

DP








1:5









X









1:6





X

X

X










WP2

2:1









X

X

DP

























2:2









X

X

DP

























2:3










X

X

DP























WP3

3:1











X

X

DP























3:2











X

X

DP






















WP4

4:1










X

X

X

DP























4:2











X


DP























4:3












X

DP























4:4












X

DP






















WP5

5:1






X

X































Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

25



5:2








X










5:3










X
















5. The biogas process





Project leader


Docent Anna Schnürer


Project team

Reseacher Lotta Levén (Formas)

PhD
-
student

Postdoc

Msc
-
project students



Resources

MicroDrivE:


12 FTE months 2007, 32 FTE

months 2008, 32 FTE months 2009

Formas: 8 FTE months 2007, 3 FTE months 2008, 5 FTE months 2009


Objective

The main objective is to optimise biogas systems operating with materials of plant origin and
at high levels of ammonia.


Specific goals



To enhan
ce biogas yield by enzymatic degradation of cellulose rich materials
(coordinated with MicroDrivE part project 2 “Hydrolytic Enzymes”)



To increase the microbial knowledge base of biogas production at high levels of
ammonia.



To optimise

biogas production of protein
-
rich materials



To investigate risks of spreading plant pathogens when using biogas residues as
fertilizing agent (coordinated with MicroDrivE part project “X “).



Optimisation of phenol degradation in biogas processes (Formas)



Material ownership

?

Intellectual property rights

The MicroDrivE consortium


Stakeholders




Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

26


Tekniska Verken in Linköping and Swedish Farmers’ Foundation for Agricultural Research
(SLF)


Industrial partner

Tekniska Verken in Linköping


Project strategy and realisation


Enzymatic pre
-
treatment

To obtain a complete anaerobic degradation of the organic matter and the production of
biogas, a complex process involving a number of steps and many different microorganisms
with different metaboli
c capacities are required. The efficiency and the biogas yield of this
process are dependent on several parameters including for example the character of the
substrate and the operational parameters of the biogas plant. One bottle neck for the
efficiency
of the biogas process is the fist degradation step, the hydrolyis. Generally the
hydrolysis rate of biomass containing plant material is slow as compared to for example
protein rich materials such as slaughter house wastes. This limited digestibility is ca
used by
shielding effects by lignin on otherwise digestible cellulosic material and the need of
microbial production of extra
-
cellular enzymes. Agricultural crop and waste represent a big
biogas source but in order to make biogas production from these mat
erials economically
defendable, in particular as production cost has to be taken into account, the biogas yields
optimally should be increased. In order to increase the biodegradability (and the biogas yield)
from plant materials, different pre
-
treatment m
ethods such as mechanical pre
-
treatment
(ultrasonic ref), steam pressure and chemical solubilization treatment have been investigated.
Another new and interesting pre
-
treatment step is enzymatic pre
-
treatment. As the cost in
enzymes during the last decade
has decreased several
-
fold, this pre
-
treatment technique is
today highly interesting. To investigate the potential of enzymatic pre
-
treatment for improved
biogas production rates and yields the following part projects are planned;



Enzymatic pre
-
treatment o
f different agricultural wastes, including distillers waste,
and evaluation in biogas batch tests.



Evaluation and optimisation of pre
-
treatment conditions and enzymes


Biogas production at high levels of ammonia

Anaerobic digestion of organic material is a

complex microbiological process requiring the
combined activity of several groups of micro
-
organisms with different metabolic capacities
(Zinder 1984). To obtain a stable biogas process, all these conversion steps must work in a
synchronised manner. The k
ey organisms in this process are the methanogenic bacteria,
producing methane from acetate or hydrogen in the terminal step of the anaerobic food chain.
The acetate
-
utilising methanogens are important since they are the main methane producing
bacteria, res
ponsible for as much as 70
-
80% of the methane produced in a biogas digester.
The hydrogen
-
utilising methanogens are essential for providing a low partial pressure of
hydrogen. Without these bacteria several different critical degradation steps cannot prece
de,
e.g. conversion of fatty acids and different aromatic compounds. These degradation steps are
thermodynamically unfavourable at high levels of hydrogen. Inhibition of the methanogenic
bacteria will result in a decreased degradation rate of the whole pro
cess, which ultimately can
lead to process failure.





Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

27


These important methanogens are strongly negatively affected ammonia, released during the
degradation of different protein
-
rich material, e.g. slaughterhouse waste, manure and distiller
waste. Ammonia i
s inhibitory at rather low levels (2 g NH
4
+
-
N/L) but methanogenesis can
occur at ammonium levels above 2 g NH
4
+
-
N/L. However, to obtain such a process, long
adaptation periods are often required and even after adaptation a lower very methane
producing capa
city is usually obtained compared to an low ammonium process. To avoid
problems with inhibitory levels of ammonium, protein rich waste has to be diluted before
anaerobic treatment, leading to high handling and storing costs. On the other hand, a high
conce
ntration (> 3 g/L) of ammonium
-
nitrogen significantly increases the value of the sludge
as a fertiliser. Furthermore, protein
-
rich materials are energy rich and generally have a high
biogas potential.


Microbiological studies in our research group have r
evealed that an alternative mechanism for
methane production can be developed at high levels of ammonium (>5 g NH
4
+
-
N/L);
syntrophic acetate oxidation (SAO).

This process of methane production requires two micro
organisms instead of one, one methanogen

and one acetate oxidizing bacterium. This process
occurs if the process is allowed to slowly acclimatize to increasing ammonium
concentrations. These organisms offer a possibility to run stable biogas processes at high
levels of ammonia. However to use an
d optimize this methane producing pathway in large
-
scale biogas processes more knowledge of the responsible organisms are needed. At present
not much information concerning this mechanism and the responsible organisms is known.
Information concerning oper
ation of large
-
scale processes with this methane producing
pathway is also lacking. Our research group has isolated several ammonia tolerant
methanogens and acetate oxidizing bacteria these isolates are awaiting characterisation and
studies. To increase t
he knowledge base concerning biogas production at high levels of
ammonia and to facilitate the optimisation of such processes the following part projects are
planned.




To develop molecular methods in order to specifically analyse acetate/hydrogen
producing
/consuming communities in biogas processes.



To characterize microbial communities and isolates of importance during biogas
production at high levels of ammonia.



To facilitate stable operation of biogas processes at high ammonia levels by use of
isolated am
monium tolerant methanogenic strains or SAO consortium.


Optimisation of phenolic compounds in biogas processes (Formas)

Phenolic compounds constitute a very important group of contaminating compounds. They
originate from a large variety of natural, agricu
ltural and industrial sources (pesticides, plant
material, swine manure etc.) and appears in biogas processes both as components of the in
-
going substrate, and as intermediates during degradation of different complex organic
materials. Our previous studies

demonstrated high concentrations of phenols in digestates
from several Swedish large
-

scale biogas processes. The amount of phenols found after
application of these digestates as fertilizing agent was just below the Swedish Environmental
Protection Agency
s´ guideline value for contaminated soil (4

g/g d.w). Furthermore,
applications of these digestates were shown to have strong inhibitory effects on soil microbial
activity, implicating long
-
term negative effect on soil fertility.

Previous results from our

research group showed that phenols can be degraded in biogas
processes, resulting in residues with low phenol content. However, the phenol degrading
capacity varied greatly between different processes with different management. This suggests



Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

28


that the phen
ol content in anaerobic digestates likely can be controlled and reduced by the use
of optimized process management strategies. The described project aims at finding conditions
for optimised phenol degradation in biogas processes. The knowledge generated ca
n be used
for the development of recommendations for process management strategies towards
production of digestates with low phenol content. Furthermore, optimization of phenol
degradation can also facilitate the degradation of other aromatic compounds pre
sent in the
biogas process.


Plant pathogens in biogas processes

During biogas production of different organic materials a nutrient rich residual is also
produced. This residual, also named biomanure or digestate, can be used as a fertilizing agent
on arab
le soil. By application of this digestate on soil a recirculation of nutrients is achieved,
thereby supporting a sustainable development. For the digestate to be accepted as a fertilizing
agent a good quality is required. The nutrient content and the conce
ntrations of heavy metals
as well as the concentration of certain pathogenic organisms are secured by a voluntary
certification protocol. This protocol is updated on a regular basis in line with new knowledge
arising. At present the pathogens included in t
his protocol includes only species infecting
humans and animal. As the larger part of biogas in the future is believed to be of agricultural
source, including also different types of energy crops, it is important to also include different
plant pathogens i
n the certification protocol. The knowledgebase concerning plant pathogens
is presently very limited. To increase the knowledge concerning fate and content of plant
pathogens in biogas processes and digestates the following part studied are planed;




Isola
tion and identification of plant pathogens in digestates.



Survival of different plant pathogens during production of biogas from grain and sugar
beats.



Analysis of the biogas potential from grain and sugar beats of “low” quality.

When energy crops are us
ed for biogas production the substrate has to be storied
during a long period of time. For some crops this will not be a problem but for others a
decomposition process will occur. Will this decomposition process influence the
biogas potential and the quali
ty of the produced digestate?



Workpackages, tasks, resources and deliverables

1. Enzymatic pre
-
treatment

FTEM

14

Deliverables

Year:Q




Set
-
up of batch systems for tests with
enzymatically pre
-
treated substrates




Biogas potential of enzymatically

pre
-
treated
wheat 1




Biogas potential of enzymatically pre
-
treated
cellulose rich substrates, other then wheat




Enxymatic pre
-
treatment of cellulose rich
materials as substrates for biogas production



2



5.5



5.5



1







Set
-
up description

(
In
preparation
)




Master thesis (1)

(Delayed until 08:2)




Master thesis (2)





Manuscript



07:3



08:1



09.1



09.2





Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

29


2. Biogas production at high levels of ammonia

FTEM

42

Deliverables

Year:Q




Characterisation of isolated bacterial strains
critical f
or
high ammonia biogas processes




Set
-
up and start of 4 laboratory
-
scale biogas
processes for digestion of distillers waste





Stabilization of the laboratory biogas
processes




Inoculation of ammonium tolerant
methanogenic

strains in laboratory biogas
processes with preceding evaluation of
process performance and microbial analysis




Use of ammonium tolerant methanogenic
strains for running stable biogas processes




Development of method for community
structure analysis of th
e acetate/hydrogen
utilizing population in ammonia enriched
biogas processes




Community structure analysis of the
acetate/hydrogen utilizing population in
ammonia enriched biogas processes





6



1



4


6





1



12




12





Manuscript





Technical
specification





Management
protocol 1




Management
protocol 2







Manuscript





Method protocol






Manuscript




08:2



07:4



08:3


09:2





09.4



08.4




09.4


3. Degradation of phenolic compounds

FTEM

24

Deliverables

Year:Q




Characterisation of phenol

degrading
communities




Characterisation of phenol degrading
communities




Set
-
up and running of continuous lab
-
scale
biogas processes for optimisation of phenol
degradation





SIP
-
analysis of phenol degrading
microorganism in biogas processes




3


1


12




8




Sequence data




Manuscript




Biogas management
protocol, including
phenol analysis




Manuscript


08.2


08:3


09:2




09:4



4. Survival of plant pathogens

FTEM

15.5

Deliverables

Year:Q




Isolation of plant pathogens from anaerobic
digestates.






Survival of plant pathogens during anaerobic
digestion 1


1





4.5





List of species

(isolation finalized,
identification in progress)




Master thesis 1

(Finalized)



07:3





08:1





Tema
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forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

30






Survival of plant pathogens during anaerobic
digestion 2




Plant pathogens during anaerobic digestion




Ammonia hygienisation in biogas processes 1





Survival of pathogens in high ammonia
biogas
processes





5.5



1


1



5.5




Master thesis 2





Manuscript




Manuscript

(Article published)




Master thesis



08:2



08:4


08.1



09.2






GANNT Chart


Tasks

&

Decision points (DP)

2007

Q

2008

Q

2009

Q

1

2

3

4

1


2

3

4

1

2

3

4

1

Set
-
up of batch
system



1.1
























2

Biogas potential





1.2




1.3


















3

Enzymatic pre
-
treatment











1.4
















4

Characterization of
bacterial strains






2.1





















5

Set
-
up of lab
-
scale
biogas reactors




2.2























6

Stabilization of lab
-
scale biogas reactors







2.3




















7

Inoculation with
ammonium

tolerant
stains










2.4

















8

Use of ammonium
tolerant strains












2.5















9

Development of
method for
community analysis








2.6



















10

Community
structure analysis












2.7















11

Characterisation of
phenol degrading
communities


3.1





3.2




















12

Set
-
up of continuous
lab
-
scale biogas








3.3







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Programme plan 2008
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2009

31


processes

13

SIP
-
analysis












3.4

14

Isolation of plant
pathogens



4.1










15

Survival of plant
pathogens





4.2

4.3


4.4





16

Plant pathogens
during anaerobic
digestion













17

Ammonia
hygienisation in
biogas processes




4.5









18

Survival of
pathogens in high
ammonia biogas
processes









4.6






6. Biomanure



recirculation of biogas digestate


Project leader

Docent Mikael Pell


Project team

FD Veronica Arthurson

Docent Anna Schnürer

Dr Lotta Levén

PhD Student Jamal Abubaker

MSc Project Student (
Kajsa Johansson
)


Resources

MicroDrivE:

2

FTE months 2007;
16

FTE months 2008;
18,5

FTE months 2009;
5.5

FTE
months 2010


Objective

The overall objective is to evaluate fertilizing effects, changes

in microbial soil quality,
rates of green house gas emissions

and occurrence and survival of plant and human
pathogens

after application of organic residue
(biofertilizers)
from biogas and ethanol
production processes based on arable crops
.


Specific goals

Specific goals are two evaluate some new types of biofertilizers with respect to their:



Potential to provide a crop sy
stem with the necessary nutrients to produce fast growing
and healthy plants.



Ability to sustain and improve an active microbial soil ecosystem as indicated by
potential ammonium oxidation and potential nitrogen mineralization
activity.



Influence on green
house gas emissions with focus on methane and nitrous oxide.



C
ontent

of
plant

and

human
pathogens
, and their survival in the soil ecosystem
.




Tema
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forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

32



Material ownership

?


Intellectual property rights

MicroDrivE consortium


Stakeholders

Stiftelsen
Lantbruksforskning (SLF)


The Swedish Farmers’ Foundation for Agricultural
Research


is the Swedish agricultural industry’s organization for funding research and
development. The purpose of the foundation is to strengthen the competitive ability of
Swedi
sh agriculture. Swedish agriculture faces the challenge of increasing its efficiency per
unit (i.e. per hectare or per animal), while at the same time society is demanding
improvements in quality. Consumers are also demanding that consideration be given in

the
production process to the environment and to ethics.


Industrial partner

The work will be performed in cooperation with Tekniska Verken, a regional utility company
based in Linköping, Sweden, with energy and environment as the cornerstones for busines
s.
Teknisk Verken is the biggest producer of biogas in Europe and is world leading in
incineration of waste.


Project strategy

In our future society it will be increasingly important to recycle plant nutrients from our
activities. The project will screen

through a number of different new organic wastes and
evaluate them for use as biofertilizers. The results will be compared with those from
conventional organic fertilizers. The use of biofertilizers that meet quality criteria for plant
nutrients, hygiene
and environmental standards will contribute to lessen our future
dependence on fossil fuels.


Scientific approach and realisation

Animal manure and slurry are well
-
known sources of plant nutrients for crop production,
while the use of biogas residues from
treatment of organic waste is less well documented.
During anaerobic digestion a large part of the energy contained in the organic waste is
transformed into methane. At the same time, the nitrogen is conserved in the biogas residue,
predominantly as ammoni
um, which when added to soil is immediately available to plants. In
addition phosphorus, potassium and magnesium, as well as trace elements essential to the
plant, are preserved in the residue.


Obviously, an increased recirculation of biogas residues to a
rable soils has several
environmental benefits. In addition, residues from the ethanol production process may also
have the potential for being biofertilizers. The rapid development within the area of
microbially derived energy sources (ethanol and

biogas
)

regarding both technologies and raw
material used results in new organic residues that have to be evaluated before large
-
scale use
as soil conditioners and biofertilizers. This calls for reliable tests to assess changes in soil
quality as well as effects
on crop and risks for spreading plant pathogens. In addition, the
residue has high water content which makes it expensive to handle and to spread in the field.
Handling and spreading may also pose an environmental risk, not only due to leakage of
nitrate t
o recipient waters but also due to substantial gaseous losses of ammonia and the



Tema
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MicroDrivE Microbially Derived Energy

Programme plan 2008
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2009

33


greenhouse gas nitrous oxide. On the other hand, upon drying, as much as 90% of the
ammonium might be lost as ammonia.


Our work will include pot experiments with plants to ev
aluate the fertilizing effect and to
assess short
-
term changes in
potential ammonium oxidation

and potential
nitrogen
mineralization

rates.
Changes in functions will be related to the microbial community
structure as assessed by nucleic acid based techniqu
es. 16S rRNA genes will be targeted in
PCRs, followed by visualization of bacterial community profiles using T
-
RFLP
.

Rates of
nitrous oxide emission is measured after addition of biogas residue to soil cores collected in
the field and incubated in gastight

chambers in the lab.
As a complement to the emission data,
functional genes (eg. nitrification/denitrification genes)
will

be assessed by T
-
RFLP, enabling
correlation of functional bacterial community structures and their effects on the soil
ecosystem fun
ctioning.
The biogas and ethanol residue will be screened for content of
potential bacterial plant pathogens.
In

spiked biofertilizer experiment, the pathogens will be
tagged with marker genes (e
.
g. GFP, RFP) prior to biofertilizer application, and
their f
ate in
soil
monitored at different time intervals
.

In all experiments
conventional pig slurry and
biofertilizer from municipal biogas plants fed with source separated house hold waste will be
used

a
s reference material.


Work packages, tasks, resources and

deliverables


WP1

Plant growth and microbial soil quality
(2007/2008)

FTEM

11

Deliverables

Year:Q

1.1

Establishment of method for measurements of
potential ammonium oxidation (PAO)



0.5

R
epo
rt on method for
PAO

Completed

08:1

1.2

Establishment of
method for measurements of
potential nitrogen mineralization (PNM)


0.5

On
-
going

08:2

1.3

Measurements of PAO and PNM in a long
-
term
field experiment (ORC) to evaluate effects of
organic fertilizers


2

Research paper ORC
-
field; Manuscript

On
-
going

08:2

1.4

Literature study on biofertilizers and pot
experimental techniques


2

On
-
going

08:2

1.5

Setting up experimental design and protocol to
follow crop growth: pot size, soil selection,
experimental crop, light regimes and climate
parameters, duration, met
hods for soil sampling
and handling. Selection of model biofertilizers


2

On
-
going

08:2

1.6

Establichment of TRFLP method to study the
overall bacterial community structure


2


08:2

1.
7

C
heck of
protocol and performance of

main
crop
and growth experiment
. Soil microbial analyses:
PAO, PNA and TRFLP.


3

MSc report on
literature
study an
crop
experiment; Manuscript


08:3








WP2

Green house

gas
(GHG)
emission
(2008/2009)

FTEM

10

Deliverables

Year:Q

2.1

GHG emission measurements in ongoing filed
experiment study

3

Report on
GHG

emissions; Manuscript

08:3




Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

34




2.2

Laboratory study on GHG emissions from soil
cores in controlled environment

3

MSc report on
GHG
study an
crop
experiment; Manuscript


08:4

2.3

Development of a modified pot growth
experimental set up to include measurements of
methane and nitrous oxide emissions


2


0
9
:
1

2.4

Establismnemt of techniques to follow
nitrification (Nit) and denitrification (Den) genes
structures in soil.


2


09:1

2.
5

Collection of biofertilizers

and p
e
rform
ence of

green house gas emission experiment
. Analyses
of GHG emissions and Nit and Den gene profiles.


2

Report on green house
gas emissions;
Manuscript


09:
2








WP3

Hygiene and plant pathogens

(2009/2010)

FTEM

12
.5

Deliverables

Year:Q

3.1

Literature study on hygienic quality and
occurrence of plant pathogens in biofertilizers


choose organisms to trace


3

Report on hygiene and
pathogens

09:3

3.2

Set up protocol for screening selected hygienic
indicator organism


3


09:3

3.3

Set up
protocol for screening of selected plant
pathogens


3


09:4

3.4

Collection of biofertilizer
and p
erform
ance of

screening

3.5

MSc report on screening
experiment

09
:
4

3.5

GFP tagging of selected patogens and
performance
spiked biofertilizer experiment


3.5


10:1








WP4

Exit phase

(2010)

FTEM

2

Deliverables

Year:Q

4.1

Integrated evaluation of
biogas residues as
biofertilizers


2

Final report 2007
-
2010


10:2



GANNT chart


WP

Tasks

&

Decision points (DP)

2007

Q

2008

Q

2009

Q

2010

Q


3

4

1

2

3

4

1

2

3

4

1

2

1















Method for PAO


1:1


























Method for PNM



1:2

























Measurements of PAO
and PNM in field
experiment




1:3


























Tema
-
forskningsprogram

MicroDrivE Microbially Derived Energy

Programme plan 2008
-
2009

35



Literature study




1:4
























Development of pot
experiment technique




1:5
























Establichment of TRFLP
method




1:6
























Main crop experiment




1:7


DP







2















GHG study field





2:1























GHG from soil
cores in lab






2:2






















Modified pot experimental
set up








2:
3





















Establismnemt of Nit and
Den genes
techniques







2:
4





















G
HG

main
experiment








2:5

DP




3















Literature
hygiene and
plant pat
h
ogens









3:1



















Protocol indicator
organisms









3:2



















Protocol plant pathogens










3:3


















Collection
of
biofertilizers
and s
creening










3:4

DP


















GFP tagging experiment











3:5


4















Integrated evaluation











4:1


















References

Johansson M., Pell M. & Stenström J. 1998.
Kinetics of substrate induced respiration (SIR) and
denitrification
: Applications to a soil amended with silver. Ambio. 27:40
-
44.


Odlare M. 2005. Organic residues


A resource in Arable Soils. Doctoral diss. Dept. of Microbiology,
SLU. Acta Universitatis agriculturae Sueciae. Agraria vol. 71.

Odlare M., Pell M. & Svensso
n K. 2008.
Changes in soil chemical and microbiological properties
during 4 years of application of various organic residues. Waste Management

(2007), doi:10.1016/j.wasman.2007.06.005


Pell M., Stenberg B. & Torstensson M. 1998. Potential denitrification a
nd nitrification tests for
evaluation of pesticide effects in soil. Ambio 27:24
-
28.


Svensson K. 2002. Microbial Indicators of Fertility in Arable Land. Doctoral diss. Dept. of
Microbiology, SLU. Acta Universitatis agriculturae Sueciae. Agraria vol. 330.


Svensson K., Odlare M. & Pell M. 2004. The fertilising effect of compost and biogas residues from
source separated household waste. Journal of Agricultural Science 142:461
-
46
7.