Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
Programme plan 2008-2009
MicroDrivE
Bioprocessing
Ethanol fermentation Feed, new products
CO2
Ethanol
Biogas
Methane
Spent ”grains”
Pretreatment
Methane yield
Ethanol yield
Hydrolysis, new enzymes
Digestate
MicroDrivE
Biomanure
Plant nutrients,
hygiene
Biopreservation
Energy ”lean” storage
storage
Sugar beet
Cereal grains
Straw
(energy crops)
(forest products)
1
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 Energy Board, the Swedish farmers Research Foundation (SLF),
Medipharm AB, Syngenta Seeds, Tekniska Verken Linköping/ Svensk Biogas AB, Jästbolaget AB, Chematur Engineering AB, Sala-Heby Energi AB and Danisco/Genencor AS.
The participating SLU departments contribute 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 working 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 and 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 Committee meets regularly, at present four meeting per year are
scheduled. The Steering Committee has appointed a Program Director, presently professor
2
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
Johan Schnürer, responsible for directing the day-to-day work within the Program. The
Program Director has appointed 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
NL-SLU, Medipharm,
Syngenta Seeds,
Tekniska Verken
Jästbolaget, Chematur
Engin.
Project
Financing
Type
Agreement
Date
2007-11-12
Sala Heby Energi
Financing
Agreement
2008-02-06
SLF (JS 1,2,3)
Financing
Contract
2006, 2007
STEM(JSt)
STEM (MS)
Financing
Financing
Contract
Contract
2007
2008
Libyan goverment
Financing a PhD proj.
Contract
2008-01-19
Consortium of
MicroDrivE Researchers
MicroDrivErs for IPR
and MicroDrivE AB
Agreement
2008-03-20
SLU Holding
IPR support, etc
LOI
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 in 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 portion 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 homepage targetting a broader audience (http://microdrive.slu.se).
3
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
Communication activities - resources and deliverables 2008
1. External communication
 Web-page maintenance
 Lecture and poster preparations, incl.
presentations
FTEM
16
0.5
2
Deliverables
Year:Q
 10OO hits
08:4
 12 International
presentations
08:4
▪ Approved plan
08:4
 Program plan writing for 2009
0.5
 Writing of publications
10
▪ 6 publications
08:4
 Meetings with industry
0.5
▪ Minutes
08:4
0.5
▪ Press reports
08:4
FTEM
1.5
Deliverables
Year:Q
▪ Monthly programme meetings
0.5
Minutes (10)
08:4
▪ Internal seminars
0.5
Hand-outs (8)
08.4
▪ Preparation of presentations for Steering comm..
0.5
Reports (3)
08:1 –
08:4
 Interviews and popular science
2. Internal communication
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, Molecular 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 Project 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 within 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 opportunity to suggest topics
for the MSc projects. The NL-faculty is funding a four-year graduate school (2008-2011) in
4
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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
▪
Formation of key scientist team
Report to SC
Year:Q
08:1
▪
Program management group formed
Report to SC
08:1
▪
Recruitment of 5 PhD students
Offical SLU registration
07:2
07:3
08:1
▪
Recruitment of 2 post-docs
First working day
08:1,3
▪
8 new biorefinery fermentors (ethanol/biogas)
4 additional biogas fermentors
First fermentor run
-:-
08:1
08:2
08:2
▪
Advanced sugar analysis intrument
First analysis run
2. Communication
Deliverables
Year:Q
07:2
▪
MicroDrivE homepage
First external log-in
▪
▪
General program poster – public communication
General program poster – scientific communication
First presentation
First presentation
07:2
07:2
▪
▪
Press releases and newspaper interviews
Oral presentations for stakeholders
(politicians, farmers, industrialists etc)
?
10 presentations
?
08:4
▪
Scientific presentations at international symposia
8 oral presentations
5 posters
08:4
▪
Scientific publications
12 scientific publications
08:4
3. Education
Deliverables
▪
MicroDrivE M Sc project school
8 – 12 M Sc theses
▪
▪
2 completed PhD projects
3 completed PhD projects
2 PhD theses
3 PhD thesis
4. Innovation and development
▪
Research leading to innovations
▪
Industrial partner development process
Deliverables
3 patent applications
Year:Q
Annual
10:4
11:2
Year:Q
07,08,09
2 licence agreements
07:4,
09:2
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Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 strainss.
- To develop laboratory 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
Intellectual 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 Seeds AB, Box 302, SE-261 23 Landskrona, Sweden. Contact: Mats Levall.
Project strategy and realisation
Sugarbeets
6
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 in WINLIN bags. Today, the time 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 Fusarium 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 pathogens 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 conditions.
Candidates will be tested in model scale trials, and finally in on-farm storage systems.
Losses of sucrose due to yeast growth will be followed using HPLC (anionexchange 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 collected. Such material will be
used to compare the microbial communities on sugarbeets that have stored well and those that
have deteriorated with molecular, non-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 example 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 has 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 have been investigated in a mini silos system but no strain with a better inhibitory
capacity than P. anomala 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
7
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 growth 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 studies 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 will not
be studied further.
The strain chosen will be subjected to safety assessment in collaboration with the DOMprogram. Ideally, it should be possible to apply the improved storage techniques also to
sugarbeets to be used for sugar production, and grain 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.
2
Report on identification
and strain identities.
Laboratory work
finished
▪
Identification and characterization of bacteria and
yeast from sugarbeets with a biopreservation
activity. Initial inhibitions studies on sugarbeets
3
Report on antifungal
strains
Part 1 completed. Part
2 ongoing
07:4
▪
Isolation of yeast and bacteria from grain storage
systems.
Completed
07:4
▪
Identification and characterization of bacteria and
yeast with a biopreservation activity from grains
2
Delayed until 08
07:4
▪
Community profiling
3
Scientific article
Delayed until 08
07:4
2. Efficacy Evaluation and strain characterisation
(2008)
2
FTEM
18
Deliverables
07:2
Year:Q
▪
Field trial with P. anomala in stored grains
5
Scientific article
08:2
▪
Development of laboratory scale storage systems
for sugarbeets and evaluation of strains.
3
Scientific article
08:3
▪
Production of material for evaluation in
fermentation with collaboration of ethanolfermentation.
2
08:4
8
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
▪
Development of a dual LAB-yeast for airtight
storage of grains
4
▪
Development of a laboratory scale plastic bagbased storage system for grain
1
▪
Community profiling on sugarbeets
3
3. Efficacy Evaluation contd and Safety
Evaluation (2009)
FTEM
8
Scientific article
08:4
08:4
Protocol
08:4
Scientific article
Deliverables
Year:Q
▪
Evaluation of safety of strains and traceability
2
Final report on safety
assessment
09:2
▪
Model scale storage system and/or field trials
6
Scientific article
09:2
One patent application
09:4
GANNT Chart
Tasks
&
Decision points (DP)
1
2007
Q
2
3
4
1
2008
Q
2
3
4
1
2009
Q
2
3
4
1 Strain isolation –
1:1
sugarbeets
2 Strain characterisation
1:2
3 Strain
isolation
2–
1:3
sugarbeets
4 Community profiling
1:4
5
1:5
Strain isolation - grains
6 Field trial -grain
77 Labscale storage of
sugarbeets and strain
evaluation
88 Material for ethanol
production evaluation
9 Development of dual
starter cultures
10 Labscale storage systems
for grain
11 Community profiling
12 Strain safety evaluation
13 Model scale or field trials
2:1
2:2
2:3
2:4
2:5
2:6
3:1
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.
9
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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: 18651869.
Druvefors, UÄ and Schnürer, J. 2005b. Mold-inhibitory activity of different yeast species during
airtight storage of wheat grain. FEMS Yeast Res. 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-LPro) and 3-phenyllactic acid. Appl. 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 saccharification 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 ethanol 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 performance.
Material ownership
10
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
- 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 raw 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 MicroDrivE 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 emerging from the use of Genencor/Danisco owned material. Current NDAs (NonDisclosure 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 use 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 could 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 processing 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/function 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 fungus Phanerochaete chrysosporium. We
11
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 homologous 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 cereal 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 transglycosylation 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?).
Application 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 factors 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 wheat 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. Trials 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 cellulose
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, sieving) 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 for 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
influence 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
12
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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. Structural 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 currently
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 and lignocellulose 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 heterologously 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 cellulose 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 termites. 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 development. 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
Deliverables
33
▪
▪
Structure studies of reaction mechanism in Pc
Lam16A
Studies of hydrolysis and transglycosylation in
Year:Q
9
 3 publications
08:3
9
 1 publication
08:4
13
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
Lam16A wt and engineered mutants
▪
Finalise structure/function analysis of Pc Lam16A
15
2. Enhanced yield of ethanol from sugar beets
and starch crops
▪ Publication of results from pilot study of ethanol
yields from wheat grain, +/- preservation/drying
▪ Pilot study of ethanol yields from sugar beets, +/preservation, +/- enzymes
▪ Ethanol yields with various starch crops, +/cellulose, +/- enzymes
▪ Follow-up study. Process optimisation
FTEM
3. Enhanced biogas yield by enzyme
supplementation
▪ Pilot study with cellulosic feed-stocks +/- enzyme,
e.g. silage, house-hold waste, saw dust, Salix
▪ Studies of enzyme dosage and optimisation of
processing conditions. Cost/benefit evaluation.
FTEM
4. Ethanol and biogas from lignocellulose
FTEM
 1 PhD thesis
Deliverables
35
09:1
Year:Q
2
 1 publication
08:4
9
 Report
08:4
9
 Manuscript
09:2
 Processing protocol
 Manuscript
10:2
15
Deliverables
29
9
20
 Report
08:2
 Processing protocol
 Manuscript
10:2
Deliverables
Year:Q
 MSc report
08:3
64
▪
Pilot study: Routines for analysis of fermentable
sugars from lignocellulose saccharification
Yields of fermentable sugars from various cellulose
substrates under “standard” conditions
Optimisation of saccharification conditions for
selected cellulose substrates
Ethanol yields from selected cellulose substrates
10





▪
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
▪ Engineering of H. jecorina key cellulases
▪
Test the influence on ethanol and biogas yield of
other individual H. jecorina enzyme components
(other than the two key cellulases)
6. Enzymes from potent wood-degraders
9
Year:Q
10
10
FTEM
MSc report
1 publication
MSc report
1 publication
Report
Deliverables
36
11
11
11
1
1
1
FTEM
 One improved enzyme
component per year
 Performance of one
component per year
▪
▪
▪
▪
Isolate Cel7 isoenzymes from P. chrysosporium
and compare activity with H. jecorina bench-marks
Sequence analysis of protist cellulase genes and
selection of interesting targets
Search for new enzymes in H. annosum, P.
chrysosporium and other basidiomycetes
Cloning and expression of selected cellulases and
comparison with H. jecorina bench-marks
Determine structure? Initiate engineering program
Year:Q
08:2
09:2
10:2
08:2
09:2
10:1
Deliverables
Year:Q
6
 BSc report
08:3
2
 Report
08:4
6
 MSc report
09:1
10
 Report, manuscript
09:4
11
 Report
10:2
30
▪
09:1
09:2
09:3
09:4
09:4
14
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
GANNT Chart
Tasks
1
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
2008
Q
2
3
4
1
2009
Q
2
3
4
2010
Q
1
2
15
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 cosupervision 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 fermentation 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 industrial 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 (without 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
Industrial partner
Jästbolaget, Chematur AB, Medipharm AB
16
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 defined 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 each 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 yeasts 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 alternative 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 biomass yields and productivities under the tested environmental
conditions.
Co-cultivation of D. bruxellensis and L. vini: The yeast and the bacterium will be cocultivated 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 effects 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 bacteria 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 general 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 glucose 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 (30-32ºC, pH 5-6). Based on these results, a
17
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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
FTEM Deliverables
industrial ethanol production
27
6
Fermentation protocols for batch
 Creating defined cultivation
and continuous cultivations
systems (fermentation) for D.
Batch protocol completed,
bruxellensis
Optimisation ongoing,
continuous culture started,
extension necessary
One publication
 Fermentation characters (specific 8
Ongoing, eventually necessary to
production rates, yield
extend to 08: 3
coefficients) of D. bruxellensis
8
One publication, lab-scale
 Metabolic flux analysis in D.
protocol to cultivate D.
bruxellensis
bruxellensis to reach maximum
productivity
5
Middle scale protocol for ethanol
 Pilot scale fermentation with
production with D. bruxellensis
yeast recycling (at Chemature)
07: 4
08: 2
09: 2
09: 2
2. Role of lactic acid bacteria (LAB)
in ethanol fermentation
 Creating defined cultivation
systems for L. vini
Year: Q

08: 2


FTEM Deliverables
22
6
Fermentation protocols for batch
and continuous cultivations
Ongoing, necessary to extend
One publication
Fermentation characters (product 4
Ongoing, necessary to extend
spectra, specific production rates,
yield coefficients) from different
carbon sources of L. vini
One publication, protocol for coCo-cultivation of D. bruxellensis 6
cultivation, establishing a method
and L. vini: Effects on cell
for viable cell count by flow
viability and fermentation
cytometry, one master thesis
parameters
One publication, one protocol for
Co-cultivation of D. bruxellensis 6
real time PCR quantification of
and S. cerevisiae and other LAB
L. vini and other LAB
(isolated from ethanol
Year: Q
07: 4
09: 2
09: 4
18
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
fermentations): Effects on cell
viability and fermentation
parameters, competition between
different LAB-strains
3. Investigation of the microbial flora
in industrial ethanol fermentations
 Isolation of yeast and LAB from
industrial fermentations
(Chematur customers and others)
 Monitoring the population in
selected ethanol fermentations
FTEM Deliverables
10
5
A collection of yeast and LAB
strains from ethanol productions
Ongoing
5
One publication
Year: Q
4. Competition between production
and infection strains
 Challenging of D. bruxellensis
and D. bruxellensis/ L. vini with a
high number of microorganisms
(isolates from industrial
fermenters and other highly
competitive strains)
 Challenging a S. cerevisiae
cultivation with D. bruxellensis
and other microorganisms (see
above)
FTEM Deliverables
10
5
Cultivation protocol for stable
non-sterile ethanol production
based on D. bruxellensis, one
publication
Year: Q
5
09: 4
Cultivation protocol for stable
non-sterile ethanol production
based on S. cerevisiae, one
publication
09: 4
09: 4
09: 4
5. Ethanol production from airtight
FTEM Deliverables
stored grain
6
Estimation of the impact of
 Airtight storage of grain in model 6
biocontrol on subsequent steps
ensilations with biocontrol
(starch degradation and ethanol
organisms (Druvefors et al.
production), one master thesis,
2005), enzymatic pretreatment
one publication Experiments and
and model fermentation
thesis completed, publication
under writing
Year: Q
6. New enzymes for polyglucoseFTEM Deliverables
degradation
14
Protocols for the degradation of
 Degradation of cellulose/ starch 14
polysaccharides from different
materials, model fermentations,
origins, two master thesis, one to
estimating the ethanol yield from
two publications
the materials
Starch completed, one Master
thesis completed, patent
application under consideration,
cellulose postponed
Year: Q
07: 4
07: 4
19
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
GANNT chart
Tasks
&
Decision points (DP)
1 Creating defined cultivation
2
3
4
5
6
systems (fermentation) for D.
bruxellensis
Specific production rates,
yield coefficients of D.
bruxellensis
Metabolic flux analysis in D.
bruxellensis
Pilot scale fermentation with
yeast recycling
Creating defined cultivation
systems for L. vini
Fermentation characters of L.
vini
1
2007
Q
2
3
4
8
9
10
4
1
1:3
1:4
2:1
2:2
2:3
2:4
3:1
3:2
4:1
and D. bruxellensis/ L. vini
with a high number of
microorganisms
Challenging a S. cerevisiae
cultivation
with
D.
bruxellensis
and
other
microorganisms
4:2
13 Airtight storage of grain in
14
model ensilations with
biocontrol organisms,
enzymatic pretreatment and
model fermentation
Degradation of cellulose/
starch materials
4
1:2
11 Challenging of D. bruxellensis
12
2009
Q
2
3
1:1
7 Co-cultivation of D.
bruxellensis and L. vini
Co-cultivation of D.
bruxellensis and S. cerevisiae
and other LAB
Isolation of yeast and LAB
from industrial fermentations
Monitoring the population in
selected ethanol fermentations
1
2008
Q
2
3
5:1
6:1
References
20
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 consortium 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 Jonsson
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 “ biotechnological 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
21
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
The project is divided into five subprojects further described in WP1-5. The different
subproject will collaborate 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 common 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 these 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 usefulness 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, are 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 (fluidised 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 filamentous 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 toxicity 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 collaboration with another
research group at Lund University.
Today distillers' 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 fermentation 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
temperature. 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 different 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.
22
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
Workpackages, tasks, resources and deliverables
WP1.
Basic characterization
▪
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
WP2. New fermentation substrates
▪
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
WP3. Distillers’ waste as microbial formulation
support
▪
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
WP4. The use of filamentous fungi grown on
distillers’ waste as source for developing novel
biomedical products.

4:1 Evaluation of the growth of filamentous
fungi
FTEM
2.5
1
Deliverables
Year:Q
07:3
07:3
0.5
0.5
08:2
Technical report
08:3
08:3
0.5
07:308:1
FTEM
5
2
Deliverables
 Master thesis 1
2
5
3
08:4
08:4
09:1
1
FTEM
Year:Q
Deliverables
 Master thesis 2
Year:Q
09:2
2
FTEM
5
2
Deliverables
 Master thesis 3
Year:Q
09:2
in various preparations of distillers’ waste
4:2 Preparation of chitin/chitosan from fungi
1
4:3 Chemical analysis of the chitosan
1
4:4 Start evaluation of the activity of the
chitosan preparation in various bioassays
1
23
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
WP5. Stabilisation and refinement of distillers’
waste (DW) with selected LAB starters
 5:1 Description of LAB populations in DW
of different origin
FTEM
Deliverables
Year:Q
10
5
Master thesis 4
Delayed until 08:1
5
Master thesis 5
07:4
08:2
 5:2 Fermentation and hygienic evaluation
of DW with different mixtures of LAB
08:4
 5:3 Nutritional content of fermented DW
GANNT Chart 080205
Tasks
&
Decision
points (DP)
2006
Q
3 4
2007
Q
2
3
4
1
WP1 1:1
1:2
1:3
1:4
1:5
X
X
X
1:6
X
1
2008
Q
2
3
4
1
2009
Q
2
3
X
X
X DP
X
X
X
WP2 2:1
X X
DP
2:2
X X
X
DP
X
DP
X
X
DP
2:3
WP3 3:1
3:2
WP4 4:1
X
4:2
4:3
X
X
X
X
X
X
X
4:4
WP5 5:1
4
2010
Q
1 2
X
DP
DP
DP
DP
DP
X
24
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
5:2
5:3
X
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 enhance 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
25
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 metabolic 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 caused 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 materials 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 methods 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 of 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 key 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, responsible 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 precede,
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 process, which ultimately can
lead to process failure.
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Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 is inhibitory at rather low levels (2 g NH4+-N/L) but methanogenesis can
occur at ammonium levels above 2 g NH4+-N/L. However, to obtain such a process, long
adaptation periods are often required and even after adaptation a lower very methane
producing capacity 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
concentration (> 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 revealed that an alternative mechanism for
methane production can be developed at high levels of ammonium (>5 g NH4+-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 and optimize this methane producing pathway in largescale 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 operation 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 the 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 ammonium 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, agricultural and industrial sources (pesticides, plant
material, swine manure etc.) and appears in biogas processes both as components of the ingoing 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 Agencys´ 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
27
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
that the phenol 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 can 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 present 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 arable 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 concentrations 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 this 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 in 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;



Isolation 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 used 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 quality of the produced digestate?
Workpackages, tasks, resources and deliverables
1. Enzymatic pre-treatment
FTEM
Deliverables
14
▪
Set-up of batch systems for tests with
enzymatically pre-treated substrates
▪
Biogas potential of enzymatically pre-treated
wheat 1
▪
▪
2
Year:Q
 Set-up description
(In preparation)
07:3
5.5
 Master thesis (1)
(Delayed until 08:2)
08:1
Biogas potential of enzymatically pre-treated
cellulose rich substrates, other then wheat
5.5
 Master thesis (2)
09.1
Enxymatic pre-treatment of cellulose rich
materials as substrates for biogas production
1
 Manuscript
09.2
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Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
2. Biogas production at high levels of ammonia
FTEM
42
Deliverables
Year:Q
▪
Characterisation of isolated bacterial strains
critical for high ammonia biogas processes
6
 Manuscript
08:2
▪
Set-up and start of 4 laboratory-scale biogas
processes for digestion of distillers waste
1
 Technical
specification
07:4
▪
Stabilization of the laboratory biogas
processes
4
▪
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 the acetate/hydrogen
utilizing population in ammonia enriched
biogas processes
6
08:3
 Management
protocol 1
 Management
protocol 2
1
12
▪
▪
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
▪
09.4
 Manuscript
08.4
 Method protocol
12
Community structure analysis of the
acetate/hydrogen utilizing population in
ammonia enriched biogas processes
3. Degradation of phenolic compounds
09:2
09.4
 Manuscript
FTEM
24
Deliverables
Year:Q
3
 Sequence data
08.2
1
 Manuscript
08:3
 Biogas management
protocol, including
phenol analysis
09:2
 Manuscript
09:4
12
8
SIP-analysis of phenol degrading
microorganism in biogas processes
4. Survival of plant pathogens
FTEM
15.5
▪
Isolation of plant pathogens from anaerobic
digestates.
1
▪
Survival of plant pathogens during anaerobic
digestion 1
4.5
Deliverables
 List of species
(isolation finalized,
identification in progress)
 Master thesis 1
(Finalized)
Year:Q
07:3
08:1
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Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
 Master thesis 2
▪
Survival of plant pathogens during anaerobic
digestion 2
▪
Plant pathogens during anaerobic digestion
5.5
08:2
 Manuscript
▪
Ammonia hygienisation in biogas processes 1
▪
Survival of pathogens in high ammonia
biogas processes
1
08:4
 Manuscript
(Article published)
1
08.1
 Master thesis
5.5
09.2
GANNT Chart
Tasks
&
Decision points (DP)
1
2007
Q
2
3
1 Set-up of batch
system
2 Biogas potential
3 Enzymatic pre4
5
6
7
8
9
1
2009
Q
4
1
2
3
4
1.1
1.2
1.3
1.4
treatment
Characterization of
bacterial strains
Set-up of lab-scale
biogas reactors
Stabilization of labscale biogas reactors
Inoculation with
ammonium tolerant
stains
Use of ammonium
tolerant strains
Development of
method for
community analysis
10 Community
structure analysis
11 Characterisation of
phenol degrading
communities
12 Set-up of continuous
lab-scale biogas
4
2008
Q
3
2
2.1
2.2
2.3
2.4
2.5
2.6
2.7
3.1
3.2
3.3
30
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
13
14
15
16
17
18
processes
SIP-analysis
Isolation of plant
pathogens
Survival of plant
pathogens
Plant pathogens
during anaerobic
digestion
Ammonia
hygienisation in
biogas processes
Survival of
pathogens in high
ammonia biogas
processes
3.4
4.1
4.2
4.3
4.4
4.5
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 system 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.
 Content of plant and human pathogens, and their survival in the soil ecosystem.
31
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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
Swedish 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 business.
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 ammonium, 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 arable 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 to recipient waters but also due to substantial gaseous losses of ammonia and the
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Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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 evaluate 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 techniques. 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 functioning. 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 fate 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 as reference material.
Work packages, tasks, resources and deliverables
WP1
Plant growth and microbial soil quality
(2007/2008)
Establishment of method for measurements of
potential ammonium oxidation (PAO)
FTEM
11
0.5
1.2
Establishment of method for measurements of
potential nitrogen mineralization (PNM)
0.5
1.3
Measurements of PAO and PNM in a long-term
field experiment (ORC) to evaluate effects of
organic fertilizers
1.4
1.1
Deliverables
Year:Q
Report on method for
PAO
Completed
08:1
On-going
08:2
2
Research paper ORCfield; Manuscript
On-going
08:2
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, methods 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
1.7
Check of protocol and performance of main crop
and growth experiment. Soil microbial analyses:
PAO, PNA and TRFLP.
3
WP2
Green house gas (GHG) emission
(2008/2009)
GHG emission measurements in ongoing filed
experiment study
2.1
FTEM
10
3
08:2
MSc report on literature
study an crop
experiment; Manuscript
Deliverables
Report on GHG
emissions; Manuscript
08:3
Year:Q
08:3
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Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
2.2
Laboratory study on GHG emissions from soil
cores in controlled environment
3
2.3
Development of a modified pot growth
experimental set up to include measurements of
methane and nitrous oxide emissions
2
09:1
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 performence of
green house gas emission experiment. Analyses
of GHG emissions and Nit and Den gene profiles.
2
WP3
Hygiene and plant pathogens
(2009/2010)
Literature study on hygienic quality and
occurrence of plant pathogens in biofertilizers –
choose organisms to trace
FTEM
12.5
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 performance of
screening
GFP tagging of selected patogens and
performance spiked biofertilizer experiment
3.1
3.5
WP4
4.1
3.5
MSc report on GHG
study an crop
experiment; Manuscript
Report on green house
gas emissions;
Manuscript
Deliverables
09:2
Year:Q
Report on hygiene and
pathogens
MSc report on screening
experiment
3.5
Exit phase
(2010)
Integrated evaluation of biogas residues as
biofertilizers
08:4
09:3
09:4
10:1
FTEM Deliverables
2
2
Final report 2007-2010
Year:Q
10:2
GANNT chart
WP
Tasks
&
Decision points (DP)
2007
Q
3 4
1
2008
Q
2
3
4
1
2009
Q
2
3
4
2010
Q
1
2
1
Method for PAO
Method for PNM
Measurements of PAO
and PNM in field
experiment
1:1
1:2
1:3
34
Tema-forskningsprogram
MicroDrivE Microbially Derived Energy
Programme plan 2008-2009
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
GHG from soil cores in lab
2:1
2:2
Modified pot experimental
set up
2:3
Establismnemt of Nit and
Den genes techniques
2:4
GHG main experiment
2:5
DP
3
Literature hygiene and
plant pathogens
3:1
Protocol indicator
organisms
3:2
Protocol plant pathogens
3:3
Collection of biofertilizers
and screening
3:4
DP
GFP tagging experiment
3:5
Integrated evaluation
4:1
4
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. & Svensson 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 and 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 142:461-467.
35