Clemson University Clemson University TigerPrints TigerPrints All Dissertations Dissertations 5-2021 Pretreatment of Cellulosic Biomass Through Bioleaching to Pretreatment of Cellulosic Biomass Through Bioleaching to Reduce Inorganic Ingredients Reduce Inorganic Ingredients Ning Zhang Clemson University, [email protected]Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Recommended Citation Zhang, Ning, "Pretreatment of Cellulosic Biomass Through Bioleaching to Reduce Inorganic Ingredients" (2021). All Dissertations. 2766. https://tigerprints.clemson.edu/all_dissertations/2766 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected].
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Clemson University Clemson University
TigerPrints TigerPrints
All Dissertations Dissertations
5-2021
Pretreatment of Cellulosic Biomass Through Bioleaching to Pretreatment of Cellulosic Biomass Through Bioleaching to
Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations
Recommended Citation Recommended Citation Zhang, Ning, "Pretreatment of Cellulosic Biomass Through Bioleaching to Reduce Inorganic Ingredients" (2021). All Dissertations. 2766. https://tigerprints.clemson.edu/all_dissertations/2766
This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected].
PRETREATMENT OF CELLULOSIC BIOMASS THROUGH BIOLEACHING TO REDUCE INORGANIC INGREDIENTS
A Dissertation Presented to
the Graduate School of Clemson University
In Partial Fulfillment of the Requirements for the Degree
Doctor of Philosophy Biosystems Engineering
by Ning Zhang
December 2020
Accepted by:
Terry Walker, Committee Chair Yi Zheng
Caye Drapcho Julia Kerrigan
ii
ABSTRACT
Lignocellulosic biomass is one of the most abundant natural resources available,
though has not been fully exploited as a feedstock for fuel and chemicals.
Thermochemical conversions of this biomass face a range of issues as a result of the
inorganic elements present in the biomass, thus necessitating a pretreatment to remove
such elements. Bioleaching is one promising method to achieve this objective, by
utilizing microbial activities to extract and remove the inorganic components from the
biomass feedstock.
In this research three microbial species including two fungi (Fusarium oxysporum
and Aspergillus niger) and one bacterium (Burkholderia fungorum) were selected to
pretreat four lignocellulosic feedstocks – switchgrass, corn stover, wheat straw and
sorghum. Results demonstrated that among the three microbes, A. niger was the most
efficient in removing most elements by 80% after 48 hours, and sorghum was relatively
more amenable to bioleaching. With A. niger, the bioleaching with a water to feedstock
ratio (v/w) of 25 for 6 h was sufficient to leach K (85%), Cl (90%), Mg (60%), and P
(70%) from sorghum. Bioleaching was shown as more efficient than water leaching.
Studies on bioleaching mechanism indicated that the acidification resulted from organic
acids produced by A. niger during bioleaching might have contributed to the higher
leaching efficiency. Following that, the bioleaching process with A. niger was scaled up
to be carried out in custom built bioreactors. Three operating parameters were
investigated for their effects on leaching efficiency – fungal mass added to each reactor,
leaching time, and glucose concentration. Response surface methodology (RSM) was
iii
used for the experiment design and model regression. Results showed that after the
bioreactor leaching process, the residual ash percentage of the sorghum biomass was
significantly lower (3.63±0.19%, mean ± standard deviation) compared with the ash
content (4.72±0.13%) after water leaching (p<0.00001). The RSM model provided
directions for improving our bioleaching process.
iv
DEDICATION
I would like to dedicate this work to my parents Mr. X. Zhang and Ms. Liying
Gao for their support and encouragement throughout my exceptionally long period of
school life.
.
v
ACKNOWLEDGMENTS
I would like to thank my advisor Dr. Terry Walker for his support and guidance
during my study at Clemson University. And I would also like to thank my committee
member, Dr. Yi Zheng for his patience and support during my PhD study. Meanwhile, I
owe many thanks to my committee member, Dr. Caye Drapcho for her encouragement
and help. And I would like to thank Dr. Julia Kerrigan for being in my committee and for
providing the fungal culture used in my research. I also thank Dr. William Bridges for
helping with data analysis.
I would like to thank Rodney Merck and Rodney Morgan for their help with the
bioreactor fabrications for my research. I want to thank David Lipscomb for his help in
my research. I would also like to thank my former fellow lab members Karthik
Gopalakrishnan, Xiang Li and Rui Xiao for their help. In addition, I want to thank Dr.
David Freedman, other faculty and staff members of the Department of Environmental
Engineering and Earth Science at Clemson, for their support. At last I want to thank my
mother, Liying Gao for her selfless support during my long period of school life. Without
her sharing my burden of taking care of the family while being a student, I cannot make it
here.
vi
TABLE OF CONTENTS
Page
TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ............................................................................................... v LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ........................................................................................................ ix CHAPTER I. LITERATURE REVIEW .............................................................................. 1 1.1 Introduciton ........................................................................................ 1 1.2 Lignocellulosic biomass ..................................................................... 2 1.2.1 Cellulose ................................................................................... 3 1.2.2 Hemicellulose ........................................................................... 3 1.2.3 Lignin ........................................................................................ 4 1.3 Pretreatment ....................................................................................... 4 1.3.1 Biochemical conversion pretreatments ..................................... 4 1.3.2 Thermochemical conversion pretreatments .............................. 5 1.4 Bioleaching ........................................................................................ 7 1.5 Conclusion ......................................................................................... 8 II. ASSESSMENT OF BIOLEACHING MICROORGANISMS .................... 10 2.1 Introduction ...................................................................................... 10 2.2 Materials and Methods ..................................................................... 13 2.2.1 Biomass feedstocks ................................................................. 13 2.2.2 Microbial culture preparation ................................................. 13 2.2.3 Bioleaching setup .................................................................... 15 2.2.4 Analytical methods ................................................................. 15 2.2.5 Data Analysis .......................................................................... 17 2.3 Results and Discussion .................................................................... 17 2.3.1 Biomass feedstock composition .............................................. 17 2.3.2 Microbial growth characteristics ............................................. 18
vii
2.3.3 Bioleaching with different microorganisms ............................ 19 2.4 Conclusion ....................................................................................... 21 III. STUDY OF FACTORS THAT AFFECT BIOLEACHING PROCESS ..... 22 3.1 Introduction ...................................................................................... 22 3.2 Materials & Methods ....................................................................... 22 3.2.1 Effect of leaching time ............................................................ 23 3.2.2 Effect of water loading ............................................................ 23 3.2.3 Study of bioleaching mechanisms .......................................... 24 3.3 Results & Discussion ....................................................................... 24 3.3.1 Effect of time on bioleaching .................................................. 24 3.3.2 Effect of water loading on bioleaching ................................... 25 3.3.3 Study of bioleaching mechanisms .......................................... 26 3.4 Conclusion ....................................................................................... 28 IV. THE BIOLEACHING PROCESS SCALING UP ....................................... 30 4.1 Introduction ...................................................................................... 30 4.2 Materials & Methods ....................................................................... 32 4.2.1 Lignocellulosic biomass.......................................................... 32 4.2.2 Aspergillus niger ..................................................................... 32 4.2.3 Bioleaching reactors................................................................ 33 4.2.4 Response surface methodology (RSM) .................................. 35 4.2.5 Analytical methods ................................................................. 36 4.3 Results & Discussion ....................................................................... 37 4.4 Conclusion ....................................................................................... 40 V. CONCLUSIONS AND RECOMMENDATIONS ...................................... 41 5.1 Conclusions ...................................................................................... 41 5.2 Recommendations ............................................................................ 43 TABLES ........................................................................................................................ 45 FIGURES ....................................................................................................................... 50 APPENDICES ............................................................................................................... 66 Appendix A .................................................................................................. 67 Appendix B .................................................................................................. ?? Appendix C .................................................................................................. ?? REFERENCES .............................................................................................................. 79
viii
LIST OF TABLES
Table Page 2.1 Detection limits for the ICP method ............................................................ 45 2.2 Chemical compositions of biomass feedstocks............................................ 45 2.3 Elemental compositions of biomass feedstocks ........................................... 46
2.4 Major chemical compositions (dry basis) of biomass feedstocks after 48 h of leaching with A. niger and water ........................................................... 46
4.1 Actual variable values and the coded values used in RSM ......................... 47 4.2 Box-Behnken design for the RSM study ..................................................... 47 4.3 Actual results and predicted responses based on the RSM model ............... 48 4.4 Analysis of variance (ANOVA) for the quadratic model ............................ 48 4.5 The ANOVA table for lack of fit of the model ............................................ 48 4.6 Estimates of parameter coefficients and the evaluation of significance ...... 49 S2.1 ICP-OES analysis of selected elements in leached biomass feedstocks ..........
S3.1 ICP-OES analysis of selected elements in leached sorghum in time factor study ....................................................................................................... 71
S3.2 ICP-OES analysis of selected elements in leached sorghum in water ratio
factor study............................................................................................. 74 S3.3 ICP-OES analysis of selected elements in leached sorghum in the study of
leaching mechanisms ............................................................................. 76 S3.4 Major compositions (dry basis) of biomass feedstocks after 48 h of leaching
with A. niger and water .......................................................................... 77 S4.1 Residual ash percentage data ....................................................................... 78
ix
LIST OF FIGURES
Figure Page 2.1 Example of bioleaching setup ...................................................................... 59 2.2 Microbial growth curves .............................................................................. 59 2.3 Effect of bioleaching on different biomass feedstocks in 48h ..................... 60
3.1 Effect of leaching time on the removal of (A) = chloride, (B) = potassium and (C) = magnesium ................................................................................... 61
3.2 Effect of water loading on leaching efficiency of sorghum. ........................ 62
3.3 Bioleaching using different fractions of fungal culture compared with water
leaching. ................................................................................................. 63 4.1 Schematic of single reactor used for bioleaching ........................................ 64 4.2 Schematic of four bioleaching reactors set up ............................................. 65 4.3 Photograph of all four reactors set up for bioleaching ................................. 65 4.4 Comparison of ash content (based on dry weight) of sorghum straws after
bioleaching or water leaching and the untreated sorghum straws. ........ 66 4.5 Surface plot of the predicted response changing with variables X1 and X2 ..... ................................................................................................................ 66 4.6 Contour plot of the predicted response changing with variables X1 and X2 .... ................................................................................................................ 67 4.7 Surface plot of the predicted response changing with variables X1 and X3 ..... ................................................................................................................ 68 4.8 Contour plot of the predicted response changing with variables X1 and X3 .... ................................................................................................................ 69 4.9 Surface plot of the predicted response changing with variables X2 and X3 ..... ................................................................................................................ 70
x
List of Figures (Continued) Figure Page 4.10 Contour plot of the predicted response changing with variables X2 and X3 .... ................................................................................................................ 71 S4.1 Plot of Y (residual ash percentage) versus X1 (fungal loading). ...................... ................................................................................................................ 72 S4.2 Plot of residuals versus X1 (fungal loading) ................................................ 72 S4.3 Plot of Y (residual ash percentage) versus X2 (leaching time) .................... 73 S4.4 Plot of residuals versus X2 (leaching time) .................................................. 73 S4.5 Plot of Y (residual ash percentage) versus X3 (glucose concentration) ....... 74 S4.6 Plot of residuals versus X3 (glucose concentration) .................................... 74
1
CHAPTER I
LITERATURE REVIEW
1.1 Introduction
The history of mankind using biomass may date back to one million years ago,
with evidence of burned wood ash discovered from the Acheulien strata in a South
African cave (Berna, Goldberg et al. 2012). Since ancient times a major part of human
life has been closely entangled with biomass – fruit collection, crop cultivation, firewood
gathering, and beverage production from excess food. As the global society entered the
industrial age, the focus of human activities gradually shifted towards obtaining and
securing fossil fuels. However, the non-renewable nature of the fossil energy resources,
and the concomitant environmental issues have driven people to explore alternative
sources of fuels and raw materials that are eco-friendly. Also the current mainstream
practice of “take-make-dispose” or linear economy model has been based on the
presumption of abundant and inexpensive natural resources, whereas showing a variety of
disadvantages (Hassan, Williams et al. 2018). Thus, it is the right time to transition to an
economy with more emphasis on renewability – a circular economy that focuses on re-
use, recovery and recycle of resources (MacArthur 2013). To accomplish this goal,
biorefineries, instead of petroleum-based refineries should become the main engine
driving a sustainable economy. For that purpose, the vastly abundant and under-used
lignocellulosic biomass could be used in various conversion processes for fuel and
material production.
2
Much attention has focused on lignocellulosic biomass biochemical conversion
into ethanol as a second-generation biofuel (Sims, Mabee et al. 2010). Due to the
structure and composition of the biomass, pretreatment is a necessity that would increase
the accessibility of its cellulose component to hydrolytic enzymes. Various types of
pretreatment technologies are being developed, which can be categorized into physical,
chemical and biological treatments, as well as any combination from these single types
(Kumar, A. K., Sharma 2017).
Thermo-chemical conversion (e.g., combustion, gasification, and pyrolysis) is
another application for lignocellulosic biomass, in which the biomass is directly
converted into fuels and other chemicals under heat, pressure and other physical
conditions. For such conversion processes, pretreatments are required due to the presence
of certain inorganic ingredients - more commonly known as ash (Davidsson, Korsgren et
al. 2002). To remove the inorganics from the biomass, a pretreatment called leaching has
been used, which could be carried out through rinsing of biomass by water (water
leaching), acidic or alkali reagents (chemical leaching), or microorganisms (bioleaching)
(Zhang, Wang et al. 2019a). This chapter reviews the pretreatment of the lignocellulosic
biomass, and the application of bioleaching.
1.2 Lignocellulosic biomass
In addition to inorganic ingredients, lignocellulosic biomass is mainly composed
of three biopolymers – cellulose, hemicellulose and lignin. Relative compositions of the
three vary considerably depending on the type, species and source of the biomass
(Agbor, Cicek et al. 2011).
3
1.2.1 Cellulose
As the most abundant biomolecule on earth, cellulose is the main target for the
biological conversion of lignocellulosic biomass (Coffey, Bell et al. 1995). Being present
in the cell walls of most plants, cellulose provides structural support and protection to the
host species. Cellulose is a homogenous linear polymer consisting of only glucose
molecules connected by β-1,4 glycosidic linkages, with the disaccharide cellobiose as the
repeating unit. The number of glucose subunits in cellulose molecules ranges between
7000 – 15000. Multiple chains of polymerized glucose are bundled up by inter-chain
cross bridges – hydrogen bonds and Van der Waals forces to form microfibrils, which in
turn bond with other microfibrils, hemicellulose and lignin molecules to form
macrofibrils and other higher order structures (Brethauer, Studer 2015).
1.2.2 Hemicellulose
Hemicellulose molecules are either linear or branched heteropolymers that are
composed of pentoses (xylose and arabinose), hexoses (glucose, mannose, and galactose),
and the acidified form of sugars, such as glucuronic acid. Compared with cellulose,
hemicellulose has a shorter chain with 500 – 3000 subunits (Gibson 2012). Unlike the
homogenous cellulose, the composition of hemicellulose varies with different biomass
types. For instance, hemicellulose molecules in straw and grass biomass mainly consist of
xylan, while glucomannan is the major form in soft wood (Agbor, Cicek et al. 2011).
Compared with cellulose and lignin, hemicellulose presents the least difficulty
regarding the removal of it during the biomass pretreatment. However, some of its
degradation products after the pretreatment – furfurals and hydroxymethyl furfurals are
4
known to inhibit the following fermentation process. Therefore, the removal of
hemicellulose has to be carefully balanced with avoiding formation of these inhibitors
(Palmqvist, Hahn-Hägerdal 2000a, b).
1.2.3 Lignin
Lignin is the second most abundant biomolecule on earth, after the cellulose. As a
highly branched heteropolymer, lignin has an undefined structure (amorphous) and a
largely varying composition of three phenyl propane monomer subunits – p-coumaryl,
coniferyl and sinapyl alcohol. Lignin can be viewed as the “glue” that binds the cellulose
and hemicellulose together, which confers mechanical strength on the plant. And the
recalcitrance of the lignocellulosic biomass can also be attributed to the presence of
lignin. While being resistant to enzymatic hydrolysis, the degradation products of lignin
are also inhibitors of saccharification and fermentation processes (Brethauer, Studer
2015, Mood, Golfeshan et al. 2013).
1.3 Pretreatment
Common destinations of lignocellulosic biomass are: 1) biochemical conversion
into ethanol and other biochemicals; 2) thermo-chemical conversion into products such as
bio-oil, bio-gas and bio-char. Different pathways would require specific pretreatment that
facilitates biomass conversion.
1.3.1 Pretreatments for biochemical conversion
Successful biochemical conversion requires that the biomass be pretreated such
that its cellulose and hemicellulose components are more accessible to hydrolytic
enzymes that break down these polymers to their constituent monomers. Thereby the
5
fermentation can proceed with the hydrolysate as ready-to-use substrates. Pretreatments
include the following types: 1) physical, such as mechanical extrusion, milling,
microwave, ultrasound, etc.; 2) chemical, such as the use of dilute acids, alkaline
reagents, organosolv, ionic liquid, and ozonolysis, etc.; 3) biological, by using
microorganisms such as white rot fungi, soft rot fungi and brown rot fungi;
4) physiochemical, such as steam explosion that has real applications in the industry,
ammonia fiber explosion, CO2 explosion and liquid hot water (Kumar, A. K., Sharma
2017). In addition, any combination of the above types may also be considered so as to
incorporate the advantages of different pretreatments (Agbor, Cicek et al. 2011).
1.3.2 Pretreatments for thermo-chemical conversion
Thermo-chemical conversion refers to the thermal decomposition of
lignocellulosic biomass at elevated temperature and pressure inside certain special
purpose equipment. Different conversion processes include pyrolysis, gasification, direct
combustion, etc. Several products are generated by the conversion, such as the bio-oil,
bio-gas, syngas, and tar, that could either be directly burned for heat / energy generation
or upgraded into higher quality transportation fuels. Solid products generated are also
known as bio-char and can be used as fertilizers. Besides, the biomass can be blended
with coal in the co-firing process in power plants (Baxter, Miles et al. 1998).
The current thermo-chemical conversion processes are facing a range of issues
that are caused by inorganic compounds present in the lignocellulosic biomass. Alkali
metals (Na, K), alkali earth metals (Ca, Mg), Cl, S and Si are among those inorganic
elements that have been shown to lead to slagging, fouling, agglomeration, corrosion, etc.
6
These issues would reduce the heat transfer rate of the conversion equipment, increase
the maintenance cost of the process due to frequent shutting down and cleaning, and
eventually damage the equipment (Carrillo, Staggenborg et al. 2014, Dayton, Jenkins et
al. 1999).
To mitigate the above problems, pretreatments are required before thermo-
chemical conversion of lignocellulosic biomass to remove its inorganic ingredients. One
simple pretreatment is rinsing the biomass with water, also known as water leaching. In
other cases, the biomass would be left in the field for several days so that natural
precipitation would perform water leaching before collection. Therefore, water leaching
is a simple and inexpensive method of reducing the inorganic compounds in the biomass.
However, water leaching can only remove those elements that are water soluble, such as
Na, K, Cl, etc., while it cannot achieve the same degree of removal with elements that are
insoluble in water, such as Mg, Ca and Si. Another disadvantage of water leaching is that
it requires a high water to biomass ratio (water volume / biomass dry weight), thus
preventing large scale application in the industry (Liu, Bi 2011a, Zhang, Wang et al.
2019b).
To remove insoluble elements that are bound to the organic backbones of the
biomass, dilute acids including sulfuric acid, hydrochloric acid, acetic acid, or nitric acid
can be added in the pretreatment, which could remove almost all inorganic constituents
from the biomass. However, the added acids would bring additional cost to the process,
while the residual acid in the leachate would cause environmental concerns, and the
7
disposal would further increase the cost of the whole process (Liu, Bi 2011a, Davidsson,
Korsgren et al. 2002).
1.4 Bioleaching
Due to disadvantages of water / acid leaching, great challenges must be overcome
to attain objectives of removing water insoluble elements and improving the economic
sustainability of the leaching process. For such purposes, bioleaching might be the
solution, where microorganisms are used for their physiological activities that might
achieve the elemental removal under environmentally mild conditions.
Bioleaching, also known as biomining or biohydrometallurgy, has been widely
applied by the mining industry to recover metals from low grade ores. Large size mining
heaps have been established by copper mines around the world, where acidophilic
bacteria gradually solubilize copper, iron, and zinc from mining ores. Meanwhile, such
bioleaching is also carried out in production scale reactors (Acevedo 2000, Sasaki,
Nakamuta et al. 2009, Yang, T., Xu, Wen, and Yang 2009a).
In addition to application by the industry, bioleaching has been extensively
studied by researchers. Priha et al. used both pure and mixed acidophilic bacteria
(Acidithiobacillus sp. and indigenous strains related to Burkholderia fungorum) to extract
phosphorus from low grade fluorapatite, obtaining up to 97% phosphorus yield after 3
weeks, where the biogenic acids from the leaching bacteria were suggested to be the
major leaching agent (Priha, Sarlin et al. 2014). Wu et al. used a reactor to solubilize
phosphorus, fluorine and other elements from granite grains through B. fungorum
8
leaching, which achieved high recovery for all elements after 35 days (Wu, L., Jacobson,
and Hausner 2008a). Mulligan et al studied fungus such as Aspergillus niger for leaching
low grade ores. Different culturing medium compositions were tested and after 14 days of
leaching, 68% Cu, 46% Zn and 34% Ni were recovered (Mulligan, Kamali 2003).
Bosshard et al. also used A. niger to extract heavy metals from municipal solid waste
(MSW) incineration fly ash and achieved optimal metal removals with 81% Cd, 66% Zn,
57% Cu, and 52% Pb after 24 h (Bosshard, Bachofen et al. 1996). Both studies indicated
that organic acids produced by A. niger played a critical role in the leaching process.
While previous bioleaching studies have been mostly focused on bio-mining, bioleaching
could also provide an alternative solution to water leaching alone in achieving more
efficient element removal from lignocellulosic biomass. Bansal et al. used the fungus
Fusarium oxysporum to extract Si nanoparticles from rice husks and up to 96% Si was
solubilized after 24 h of co-incubation with the fungus (Bansal, Ahmad et al. 2006). This
research indicates that bioleaching would be a promising technology for lignocellulosic
biomass pretreatment before thermo-chemical conversion processes.
1.5 Conclusion
Lignocellulosic biomass is one of the most abundant resources that is inexpensive
yet has not been fully exploited. To incorporate biomass into the new circular shaped
bioeconomy, major technological hurdles need to be overcome, which include: 1)
appropriate pretreatments that would facilitate the enzymatic hydrolysis of the biomass so
the following steps in the biochemical conversion could proceed with efficiency, and 2)
proper leaching pretreatments that could significantly lower the content of inorganic
9
compounds in the biomass without considerable tradeoff in economics. Bioleaching is a
promising technology, though more research is necessary to reveal its capability in
lignocellulosic biomass pretreatment. Our hypothesis is that bioleaching could be used as
an eco-friendly and efficient pretreatment method for lignocellulosic biomass, which
would significantly reduce the inorganic ingredients in the biomass, thus improve the
quality of the biomass as the feedstock for thermochemical conversion processes. With
this research, we aim to accomplish these following objectives in the ensuing chapters: 1)
study and compare a number of selected microorganisms on their capabilities to leach
inorganic elements from a range of biomass feedstocks (Chapter II); 2) select the best
performing microbe and one type of lignocellulosic biomass that is more amenable to
bioleaching than others (Chapter II); 3) focus on the selected combination of bioleaching
microbe and feedstock biomass, investigate certain factors that might influence the
bioleaching process, and conduct a preliminary study on bioleaching mechanisms
(Chapter III); 4) scale up the bioleaching process, and evaluate the effect of different
variables on bioleaching on the new platform (Chapter IV).
10
CHAPTER II
ASSESSMENT OF BIOLEACHING MICROORGANISMS
2.1 INTRODUCTION
Lignocellulosic biomass has been widely studied as a feedstock for next
generation biofuel production. It has three major components – cellulose, hemicellulose
and lignin, and can be converted into biofuels (e.g., syngas and bio-oil) through
thermochemical pathways such as direction combustion, gasification and pyrolysis (Liu,
Bi 2011b). However, the presence of certain inorganic elements such as alkali metals (K
and Na), alkali earth metals (Mg and Ca), Si, Cl, and S in the biomass, especially
herbaceous biomass would cause issues during the thermal conversion processes, which
include fouling, slagging, agglomeration, and corrosion. For example during the fluidized
bed combustion, K and/or Na content can cause low melting point of the ash, and the
partially molten ash can result in sintering and crystallization in the fluidized bed reactor
(Thy, Jenkins et al. 2010, Steenari, Lundberg et al. 2009). Eventually the issues due to
inorganic elements can reduce the heat transfer rate of the biomass conversion
equipment, even damaging the reactors, thus increase the maintenance cost of thermal
conversion reactors (Turn, Kinoshita et al. 1998, Vamvuka, Zografos et al. 2008a, Pîşă,
Rădulescu et al. 2009, Yu, Thy et al. 2014). Furthermore, elements such as Cl, N and S
could be emitted during the conversion process in the form of acidic vapors leading to
reactor corrosion, even air pollution. In addition, alkali metals might also lead to high
viscosity of bio-oil after fast pyrolysis (Liu, Bi 2011b). Therefore, a pretreatment step is
11
necessary to remove those inorganic elements before the subsequent thermal chemical
conversion of the lignocellulosic biomass.
Rinsing with water (water leaching) is a simple and common pretreatment to
improve the biomass quality. Water leaching can effectively remove most of the water-
soluble elements such as K, Na and Cl (Jenkins, Bakker et al. 1996, Deng, Zhang et al.
2010). However, it is not effective with water-insoluble elements such as Mg and Ca
(Davidsson, Korsgren et al. 2002). Besides, water leaching requires a large amount of
water, which severely limits its application at industrial scales. Meanwhile, bioleaching
could be an alternative pretreatment method and it has been extensively studied and used
by the mining industry (bio-mining). With bioleaching, researchers utilized the microbial
activities to recover metals from low-grade ore or retrieve hazardous elements from solid
waste materials (Hocheng, Su et al. 2014, Mishra, Kim et al. 2008a, Priha, Sarlin et al.
2014, Yang, T., Xu, Wen, and Yang 2009b). Today in the mining industry, bacteria
mediated bio-mining has been a well-established process, such as being used for copper
recovery from low grade ores or bio-oxidation of refractory gold ores. The bio-mining
could be carried out in either heaps of ground ores, or specially designed, mechanically
agitated reactors (Acevedo 2000). So far a variety of microbes have been assessed for
their leaching capabilities on a wide range of raw materials. By using pure and mixed
cultures of acidophilic bacteria (e.g., Acidithiobacillus sp. and/or locally collected
bacterial strains related to Burkholderia fungorum), Priha et al. recovered phosphorus
from low grade fluorapatite, and obtained up to 97% yield after 21 days (Priha, Sarlin et
al. 2014). Wu et al. used B. fungorum to extract phosphorus, fluorine and several metal
12
elements from granite grains in a batch reactor, with a high releasing rate for all elements
after 35 days (Wu, L., Jacobson, and Hausner 2008b). Mulligan et al. studied Aspergillus
niger for leaching low grade ores, in which 68% Cu, 46% Zn and 34% Ni were removed
after 14 days of leaching (Mulligan, Kamali 2003). Bosshard et al. also used A. niger to
extract heavy metals from municipal solid waste (MSW) incineration fly ash and
recovered 81% Cd, 66% Zn, 57% Cu, and 52% Pb after 24 h of leaching (Bosshard,
Bachofen et al. 1996). Both studies with A. niger indicated that biogenic organic acids
played a crucial role in the leaching process. While most bioleaching studies have
focused on application in bio-mining, bioleaching is also expected to remove inorganic
elements from lignocellulosic biomass. In one research, Fusarium oxysporum was used to
extract Si in the form of nanoparticles from rice husks and up to 96% Si was recovered
co-incubation with the fungus for 24 hours (Bansal, Ahmad et al. 2006), suggesting that
bioleaching could be a promising technology for lignocellulosic biomass pretreatment
before thermochemical conversion processes.
In this chapter, three microorganisms were evaluated for their bioleaching
capabilities to remove inorganic elements from four biomass including switchgrass, corn
stover, sorghum, and wheat straw. The objective is to compare the leaching efficiency of
different micorbes on different biomass, and select one species to focus on in following
a All numbers were normalized based on the dry weight of feedstock biomass prior to leaching and are shown as mean± standard deviations (n=3). Each sample’s measurement was normalized first, then the mean and standard deviation were calculated.
47
Table 4.1. Actual variable values and the coded values used in RSM.
Variable Unit Symbol Coded levels -1 0 1
Fungal mass g/L X1 0.4 1.2 2 Leaching time d X2 1 3 5
Y Actual - Actual residual ash percentage based on dry weight (%), Y Predicted - Residual ash percentage based on dry weight (%) predicted by the RSM model, X1- Fungal mass added to each reactor (g dry weight), X2- Leaching time (day), X3- Glucose concentration (g/L).
Table 4.4. Analysis of variance (ANOVA) for the quadratic model.
Source DF* Sum of Squares Mean Square F Ratio Model 6 0.47 0.08 11.22 Error 8 0.06 0.01 Prob > F Total 14 0.53 0.0016
*Degree of freedom
Table 4.5. The ANOVA table for lack of fit of the model.
Source DF* Sum of Squares Mean Square F Ratio Lack of Fit 6 0.02 0.003 0.21 Pure Error 2 0.03 0.017 Prob > F Total Error 8 0.05 0.94
*Degree of freedom
49
Table 4.6. Estimates of parameter coefficients and the evaluation of significance.
Figure 2.1. Example of bioleaching setup (left: water leaching, right: bioleaching with A. niger culture)
Figure 2.2. Microbial growth curves. FO=F. oxysporum, AN=A. niger and BF=B. fungorum. FO and AN were based on biomass dry weight. BF was based on OD600. Error bars are standard deviations (n=3)
51
Figure 2.3. Effect of bioleaching on different biomass feedstocks in 48 h. FO=F. oxysporum, AN=A. niger
and BF=B. fungorum. Error bars are standard deviations (n=2)
52
Figure 3.1. Effect of leaching time on the removal of (A) = chloride, (B) = potassium and (C) =
magnesium. Error bars are standard deviations (n=2).
53
Figure 3.2. Effect of water loading on leaching efficiency of sorghum. Error bars are standard deviations
(n=3).
54
Figure 3.3. Bioleaching using different fractions of fungal culture compared with water leaching. Error
bars are standard deviations (n=3)
55
Figure 4.1. Schematic of single reactor used for bioleaching. 1- air, 2a- upper cap venting port, 2b- lower body venting port, 3- sprinkler, 4- rotameter, 5- nylon mesh membrane and plastic mesh plate, 6- liquid surface, 7- fungal pellets, 8- magnetic stirrer, 9- peristaltic pump.
56
Figure 4.2. Schematic of four bioleaching reactors set up.
Figure 4.3. Photograph of all four reactors set up for bioleaching.
57
Figure 4.4. Comparison of ash content (based on dry weight) of sorghum straws after bioleaching (n=15) and water leaching (n=12). Initial ash content of sorghum straws is 6.03±0.75%
Figure 4.5. Surface plot of the predicted response changing with variables X1 and X2. Y- Ash percentage based on biomass dry weight (%), X1- Fungal mass added, X2- Leaching time.
0
1
2
3
4
5
6
Bioleaching Water Leaching
Ash
%
58
Figure 4.6. Contour plot of predicted response changing with variables X1 and X2. X1:Fungal biomass, X2:Leaching time.
59
Figure 4.7. Surface plot of the predicted response changing with variables X1 and X3. Y- Ash percentage based on biomass dry weight (%), X1- Fungal mass added, X3- Glucose concentration.
60
Figure 4.8. Contour plot of predicted response changing with variables X1 and X3. X1:Fungal biomass, X3:Glucose concentration.
61
Figure 4.9. Surface plot of the predicted response changing with variables X2 and X3. Y- Ash percentage based on biomass dry weight (%), X2- Leaching time, X3- Glucose concentration.
62
Figure 4.10. Contour plot of predicted response changing with variables X2 and X3. X2:Leaching time, X3:Glucose concentration.
63
Figure S4.1 Plot of Y (residual ash percentage) versus X1 (fungal loading).
Figure S4.2 Plot of residuals versus X1 (fungal loading).
64
Figure S4.3 Plot of Y (residual ash percentage) versus X2 (leaching time).
Figure S4.4 Plot of residuals versus X2 (leaching time).
65
Figure S4.5 Plot of Y (residual ash percentage) versus X3 (glucose concentration).
Figure S4.6 Plot of residuals versus X3 (glucose concentration).
66
APPENDICES
67
Appendix A Raw data for Chapter II
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