University-Based Research and
Development Project
Performed by The
With Support Through
The
and the
Summary
Scientists and engineers at
our four state universities are working to improve
- Promote existing biomass research at
- Rely on existing
In response to the request
for partnership, the four comprehensive universities of The State of
Mississippi:
Depolymerization of Lignocellulose by Fungal Cells and
Immobilized Enzymes
Development of a Bioadsorbent for the Biodiesel
Industry
The
High-Value Lignin Co-Products through Pretreatment and
Microbial Conditioning
The
(1) Biomass Waste to Energy via Energy-Enhanced
Biomass
(2) Pine Sawdust Conversion to Sugars for Fermentation
to Fuel Grade Ethanol Using a Twin Screw Extruder
These projects integrated
existing university research with commercial partners to bring technology
closer to commercialization. Project objectives focused on U.S. DOE-identified
research needs and barriers to biorefinery development as stated in the
Multi-Year Technical Plan. The report addresses biomass investigated, enabling
technologies developed and obstacles overcome, products developed, commercial
partnerships and collaborators.
At the beginning of these
projects, we anticipated findings that would contribute to increased activities
for growing, harvesting, and processing lignocellulose and oil crops. The aim
was to enable production of fuel and chemicals to meet local and state needs,
thereby creating commercial opportunities. A look at the “before and after
picture” helps to put our progress in perspective. Prior to the project, we had
awareness and some experience with various methods: biomass pretreatment,
enzymatic hydrolysis, compositional analysis, enzyme production and separation,
acid hydrolysis, waste water treatment, and solid fuel formulation. Upon
completion of these projects, we have mastered and demonstrated these
capabilities. We have obtained key pieces of information to support commercial
activities such as solid fuels production, pretreatments leading from sawdust
and grasses to ethanol, and production, separation and use of enzymes to
further support cellulosic ethanol production. Some of these results offer
almost immediate commercial opportunity. Others take us significant steps
toward pilot and demonstration-scale testing. We have enhanced our physical
capital, such as high performance chromatography tools for the lab, and our
human capital through expertise such as enzyme production and lignin isolation.
Thus we have taken
substantial steps toward our envisioned goals of, “providing commercial opportunities
– ethanol production, transportation and solid fuel blending, engineered wood
products, and biodiesel production….and scientific and technological advances
leading to in-state know-how and formal intellectual property rights will
generate additional commercial opportunities regionally and nationally.”
High-Value Lignin Co-Products through
Pretreatment and Microbial Conditioning
The
Contract
Number; MTA- SBI – 1003
PI:
Clint Williford, Department of
Chemical Engineering, drwill@olemiss.edu
(Web page http://www.olemiss.edu/depts/chemical_eng/cww.html)
Co-PI: Charles Burandt,
Executive Summary
Lignin, the second most
abundant biopolymer, comprises 15-25% of most biomass. It provides structural
integrity and protects cellulose from decomposition. While beneficial to
plants, this chemical recalcitrance impedes conversion of lignocellulose to
ethanol and other chemicals. To produce
cellulosic ethanol, capital-intensive pretreatments remove the lignin, but
yield a low value product consigned to combustion. DOE has identified these
problems as key technical and economic obstacles to commercialization. The
following figure shows one version of the U.S. DOE’s “Sugar Platform” process
flow diagram. Biomass is pretreated to disrupt the structure and make the
cellulose accessible. Enzymes perform saccharification, converting the
cellulose polymer into sugars. Yeasts then ferment the sugars to ethanol. This
diagram includes conditioning, for example with fungi, to facilitate
pretreatment, and yield amore useable lignin co-product.
In our prior work, we applied
a bacterial consortium from wastewater sludge to lignin and observed depolymerization.
The following plot shows an HPLC response curve, which shifts up and down
indicating initial polymerization; but, then depolymerization.
With Ym - Polymerization
followed by subsequent depolymerization
We also
applied the consortium to lignin mounted for visualization with an atomic force
microscope. The top set of images show initial lignin ‘droplets,” which are
reduced in size and number (bottom image) after incubation with the bacteria.
Our project at the
We applied two pretreatments,
ammonia fiber expansion and extrusion/expansion, and conditioned four grasses
with a bacterial consortium and a fungus. We determined the impact on
subsequent enzymatic hydrolysis to fermentable sugars- the feed for ethanol
production. Lignin was isolated so as to resemble as closely as possible its
native state, and assessed for its degree of degradation during pretreatment.
The lignin analysis to date is an important step to better understanding their
potential for adhesives and polymeric binders. While changes to sugar
production were modest in this evaluation, we did produce enzymes, using the
low-cost grass feedstock. Since enzymes are a significant component in most
cellulosic ethanol processes, our findings are relevant to improving process
economics.
Major Project Tasks:
Task 1 – Obtain and
derive lignin and lignocellulose from corn stover using advanced pretreatments.
A.
Feedstock
Development:
We have grown, obtained, and
prepqred switchgrass, miscanthus, sudan-sorghum, and corn stover. These have
been shredded and dried in 5 gal bucket quantities. In addition, a high sugar
rye grass has been investigated as a winter crop.
Due to the ready availability
of existing infrastructure for handling grass crops in our area and local
expertise in their production, only grass species and their cultivars were used
in our studies.
Growth, harvesting, and
preparation observations were made for the following:
- Sorghum bicolor ssp. Fodder type “sweet” sorghum.
- Panicum virgatum Switchgrass
- Miscanthus floridulus “Giant Miscanthus”
- Lolium perenne “High Sugar Grass.”
Panicum virgatum Switchgrass
Material of an old accession of switchgrass was collected at the
Whitten Plant Materials
Resource Center Lafayette Co.
Switchgrass established well
and out-competed weeds with its vigorous and dense growth. It required limited
water and l fertilizer applications.
Winter
switchgrass
Processing
switchgrass
B.
Pretreatment
We implemented a
collaborative research with
In AFEX process, biomass is
treated with liquid ammonia in a closed heated reactor, the pressure is
released rapidly exploding the structure of the biomass and thus reducing its
recalcitrance. The ammonia can be recovered and used. Depending on the biomass,
the process conditions like moisture, temperature, ammonia to biomass ratio and
treatment time can be optimized. The process conditions, though not optimized,
are present in the table.
Task 2 – Apply lignin-depolymerizing bacteria to corn
stover. Determine solubilization, compositional changes and lignin
depolymerization. Cornstover was
treated with the preconditioned waste water sludge consortium for 20 days and
the effects of treatment are assessed in terms of susceptibility to enzyme
hydrolysis which requires the samples to be analyzed for their composition.
Composition analysis is a two step procedure of hydrolyzing the biomass samples
to estimate the total cellulose content.
The first step is 1hour of concentrated acid hydrolysis, where polymeric cellulose chains are broken to smaller chains, followed by the dilute acid hydrolysis where the smaller chains are broken to monomeric sugars. The hydrolyzed sugars solution is neutralized with calcium carbonate to analyze the solution for sugars using HPLC technique (Column: Aminex HPX-87P, mobile phase: water detector: refractive index detector).
Composition
Analysis
Composition
analysis was conducted to check any changes in composition occurred during the
process of conditioning. The procedure followed was a NREL protocol LAP 8.
Changes in glucan % were observed, though not large after conditioning, while
there was a significant decrease in xylan % after 10 day conditioning. Increase
in glucan percentages allows a decrease in biomass loading during enzyme
hydrolysis to yield the same glucose production.
Control
(left) and conditioned sample (right)
Task 3 – Evaluate lignin co-product as an adhesive, additive or polymer. We performed an
initial extraction (Soxhlet) and determined the extractives content in each
sample, implemented an extraction and analysis protocol through the Department
of Wood and Paper Science at NC State University. Ethanol/benzene,
Dichloromethane and hot water extraction has been performed on unpretreated and
enzymatically hydrolyzed samples. Klason lignin and acid soluble lignin content
was determined. NMR analysis was performed to investigate changes o functional
groups.
Klason lignin content
|
Sample Name |
Klason lignin(%) |
Acid soluble(%) |
Total Lignin Content(%) |
|
Cornstover |
18.23 |
2.17 |
20.42 |
|
Miscanthus |
16.27 |
1.88 |
18.15 |
|
Sorghum |
13.27 |
1.93 |
15.14 |
|
Switchgrass |
15.26 |
1.65 |
16.19 |
1H-NMR of Acid pretreated enzyme hydrolyzed sample
Task 4 – Perform functional assay of pretreated biomass with and without microbial conditioning. As per Task 2, we are using enzymatic hydrolysis as an assay tool which follows NREL LAP 09. The following figure shows results of enzyme hydrolysis of AFEX pretreated cornstover, switch grass, giant miscanthus and sorghum. The results are provided by MBI International.
Task 5 – Screen additional microbial agents for depolymerization and disengagement of lignin from lignocellulose. We have obtained and applied P. chrysosporium fungus to our four feedstocks. The method mimicked solid state (field) conditioning based on U.S.DOE research (Keller, et al., 2003). This was an attempt to investigate the effect of a known microbe on four different biomass substrates. Changes were observed in enzymatic digestibility and viscosity of cornstover treated with Phanerochaete chrysosporium (Keller, et al., 2003). Enzyme activity tests are a required part of analysis to determine the microbial activity.
Microbial culture from digestive tract of beetle.
____
Keller, F. A., Hamilton, J.
E., and Q. A., Quang (2003). Microbial Pretreatment of Biomass: Potential for Reducing Severity of Thermochemical
Biomass Pretreatment, Applied Biochemistry and Biotechnolog, Volumes
105, Number 1 – 3.
Ezyme Activity
Significant Observations/Findings
1. Consortium bacteria has effects of
decolorizing biomass
2. Phanerochete chrysosporium produces cellulases relatively higher
with switch grass
3. Exposure to moisture
increases the xylose yields
Conclusions
1. Consortium bacteria can be used in the
bioremediation of both aqueous and solid effluents
2. Switch grass has the highest potential as a
substrate for enzyme production
3. Exposing the biomass to
moisture for prolonged periods can increase the xylose yields.
Professional Activities and Products
Presentations at technical/professional conferences
“High-Value Lignin Co-Products through Pretreatment
and Microbial Conditioning” by Swetha
Mahalaxmi, Naresh K budhavaram, Clint Williford, James Rawlins at 29th
SIM Annual Meeting 2007.
“Microbial
Conditioning and Pretreatment of Grasses for Ethanol and Lignin Co-Products” by Swetha Mahalaxmi, Ashwini Thakre, Clint Williford, Colin R Jackson,
Charles Burandt at AIChE Annual Meeting 2007.
“Phanerochaete chrysosporium conditioning of
grasses: Enzyme acitivity and Hydrolysis to Sugars”
by Swetha Mahalaxmi, Colin R Jackson,
Clint Williford, at 30th SIM Annual Meeting 2008.
In addition, presentations
were made at the Bio-products conference in
Web site or other Internet sites that reflect the
results of this project
Project summaries were
prepared for the MTA website; and
Networks or collaborations fostered
We have established a close
collaboration with one of our UM Biology faculty whose specialization concerns
biomass degraders in the soil. Meetings on renewable fuels have been attended
in
Technologies/Techniques
Methods mastered: Lignin
isolation, NMR characterization, enzymatic hydrolysis, and compositional
analysis, HPLC analysis of sugars, electrophoresis, and application of ammonia
fiber expansion pretreatment
Journal Publication Abstracts
1. Determination
of lignin by size exclusion chromatography using multi angle laser light
scattering by Aarti V. Gidh;
Stephen R. Decker; Todd B. Vinzant; Michael E. Himmel; Clint Williford (pp.
102-110). Published 17 April 2006 in J Chromatography, 1114(1):
102-10.
2. Fungal-Induced
Redistribution of Kraft lignin Molecular Weight by Multi-Angle laser Light
Scattering by Aarti
V. Gidh; Stephen R. Decker; Todd B. Vinzant; Michael E. Himmel; Clint
Williford, published in Chemical Engineering Communications,
publisher Taylor and Francis Ltd, Volume 193, Number 12, 2006,
pp. 1546-1561(16).
3. Detailed Analysis of Modifications in Lignin After Treatment With
Cultures Screened for Lignin Depolymerizing Agents by Aarti Gidh, Dinesh
Talreja, Todd B. Vinzant, Clint Williford, and Alfred Mikell, in Applied Biochemistry and Biotechnology, Spring 2006, Volume 131,
Issue 1-3, pps. 829-843, (ISSN:0273-2289).
4. Characterization of lignin using multi-angle
laser light scattering and atomic force microscopy by A.V.
Gidh , S.R.
Decker, C.H.
See, M.E.
Himmel , and C.W.
Williford, in
Analytica Chimica Acta., 555(2),
p. 250
Internships
National Renewable
Energy Laboratory
Novozymes
MBI international
1. Determination of lignin by size exclusion chromatography using multi angle
laser light scattering by
Aarti V. Gidh; Stephen R. Decker; Todd B. Vinzant; Michael E. Himmel; Clint
Williford (pp. 102-110). Published 17 April 2006 in J Chromatography,
1114(1): 102-10.
A method was developed using
high-performance size exclusion liquid chromatography (HPSEC) with multi-angle
laser light scattering (MALLS), quasi-elastic light scattering (QELS),
interferometric refractometry (RI) and UV detection to characterize and monitor
lignin. The combination proved very
effective at tracking changes in molecular conformation of lignin
molecules over time; i.e. changes in molecular weight distribution, radius of gyration, and hydrodynamic radius. Until
this study, UV detection (280nm) had been the primary lignin determination
method for chromatography. Three different HPLC columns were used to study the
effects of pH, flow conditions, and
mobile phase compositions (dimethyl sulphoxide, water, 0.1M NaOH, and lithium
bromide) on the chromatography of lignin. Since light scattering accuracy is
highly dependent on solute concentration, both the UV and RI detectors were
calibrated for use as concentration detectors. Shodex Asahipak GS-320 HQ column
with 0.1M NaOH (pH 12.0) run at 0.5ml/min was found to give the highest
separation and most consistent recovery. The study also revealed that the lignin aggregated at pH below 8.5. This
aggregation was detected only by MALLS and was not observed on UV or RI detectors.
It is very important to take this loss in apparent concentration due to
aggregation into consideration before collecting reliable depolymerization
data.
Keywords: Lignin; Light scattering; Method development; HPLC; Aggregation
2.
Fungal-Induced Redistribution of Kraft lignin Molecular Weight by
Multi-Angle laser Light Scattering by Aarti
V. Gidh; Stephen R. Decker; Todd B. Vinzant; Michael E. Himmel; Clint
Williford, published in Chemical Engineering Communications,
publisher Taylor and Francis Ltd, Volume 193, Number 12, 2006,
pp. 1546-1561(16)
Culture broths from Phanerochaete
chrysosporium and Trametes cingulata , combined with co-factors such as
hydrogen peroxide, dithiothreitol, copper, iron, and manganese ions were
examined for the ability to modify lignin structure. High-performance size
exclusion chromatography (HP-SEC) coupled to multi-angle laser light scattering
(MALLS) detection was used to determine the effect of several white rot fungi,
pH values, enzymes, and co-factors on the molecular weight distribution of
treated kraft lignin. The analytical procedure tracked changes in molecular
weight distribution, radius of gyration, and hydrodynamic radius. Results
showed changes in the molecular weight distribution of lignin components when
treated with combinations of factors. The induced cultures showed more lignin
depolymerization for the specific lignin samples in which they were initially
grown. The distribution in the radius of gyration became narrower with time,
indicating that molecular conformation changed to a more uniform molecular
shape. H 2 O 2 and DTT showed the most significant changes in lignin molecular
weight distribution.
Keywords: Aggregation; Depolymerization; Fungi; Light-scattering; Lignin; Ligninase
3. Detailed Analysis of Modifications in Lignin After Treatment With Cultures Screened for Lignin Depolymerizing Agents by Aarti Gidh, Dinesh Talreja, Todd B. Vinzant, Clint Williford, and Alfred Mikell, in Applied Biochemistry and Biotechnology, Spring 2006, Volume 131, Issue 1-3, pps. 829-843, (ISSN:0273-2289)
Termites, beetles, and other arthropods can digest living and decaying wood plus other lignocellulosic plant litter. Microbial sources like other wood-eating insect guts and wastewater treatment sludge were screened for lignin depolymerization. Near infrared spectroscopy and atomic force microscopy (AFM) along with high-performance liquid chromatography (HPLC), were used to track changes in functional groups, size, shape, and molecular weight of lignin molecules during incubations. Odontotaenius disjunctus (Betsy beetle) guts dissected whole or separately as midgut, foregut, and hindgut, consumed corn stover but did not show lignin depolymerization. The sludge-treated lignin did show some reduction in molecular weight on the HPLC, particle size (350–650 nm initially to 135–220 nm by day 30) and particles per field on AFM. pH and the presence of nutrients had a substantial effect on the extent of depolymerization. Cultures in lignin and nutrients showed higher growth than cultures with lignin only. Colony characteristics within the beetle gut and the sludge were also evaluated.
Keywords: Lignin; beetles; NIR; HPLC;
AFM; depolymerization
4. Characterization of lignin using multi-angle
laser light scattering and atomic force microscopy by A.V.
Gidh , S.R.
Decker, C.H.
See, M.E.
Himmel , and C.W.
Williford, in
Analytica Chimica Acta., 555(2),
p. 250
Small differences in the
isolation techniques of lignin can result in significant changes in its
molecular structure and configuration. Light scattering (evaluated at 18
different angles in a plane), Atomic Force Microscopy (AFM) and Near Infrared
Spectroscopy (NIR) proved very effective for evaluating the characteristics of
lignin. Zimm plots were generated using Zimm, Debye and
Internships
Internship by A.V. Gidh,
National Renewable Energy Laboratories, September ‘02 to March ’03, Contributed to annual report Lignin Depolymerase from Trametes
cingulata, May 16, 2001 – August 31, 2002 for Subcontract
XCO-1-31048-01
·
Method
development for lignin analysis on HPLC-SEC coupled with Ultraviolet (UV),
Multi angle laser light scattering (MALLS) and Refractive index (RI) detectors.
Tracking changes in lignin’s molecular weight distribution, radius of gyration,
hydrodynamic radius and concentration caused due to depolymerization.
·
Batch
characterization studies on lignin using Zimm, Debye and
·
Growth incubation
studies with fungal isolates (e.g. Phanerochaete
chrysosporium and Trametes
cingulata) and enzymes (laccase) to track lignin depolymerization.
Internship by A.V. Gidh, Novozymes, Summer 2004 May ‘04 to August ’04, contributed to report: The Effect of Different Fermentation Parameters
on SSF for Fuel Ethanol,” SSF (Simultaneous Saccharification and Fermentations)
of corn starch with various enzymes to improve enzyme efficiency, yeast
metabolism and bioethanol yield. (This internship was conducted independently
and supported by Novozymes. However, it contributed to an understanding of
screening techniques that were adapted for our research.)
Internship by S. Mahalaxmi, MBI International, Summer 2006: Four grass feedstocks were pretreated by ammonia fiber expansion (AFEX). Ms. Mahalaxmi learned analytical techniques using HPLC. Samples were evaluated for composition and subsequently for yields of sugars after enzymatic hydrolysis.
Research
Collaboration visit by