Transcript Wet processing of adolescent Pennisetum purpureum for enhanced

Wet processing of adolescent Pennisetum purpureum for enhanced
sugar release and co-product generation
Devin Takara* and Samir K Khanal
Department of Molecular Biosciences and Bioengineering
Sugar analyses by DNS (data not shown) suggested the optimal conditions
summarized in Table 1 for each of the four processing streams generated. The data
was confirmed by statistical analyses using an analysis of variance (ANOVA) and
Duncan’s Test. Figure 4 compares the cumulative sugar-release obtained under
optimal conditions. It was determined that Stream 3 (juiced, wet banagrass)
resulted in the highest sugar yield, and was subsequently used as a substrate for
ultrasonic investigations.
Introduction
Banagrass (Pennisetum purpureum) is a perennial grass found in the state of
Hawaii and other tropical regions around the world. Because banagrass grows
rapidly with high yields, it has been regarded as an ideal feedstock for cellulosic
biofuel production.
Despite advances in technology, the costs of pretreatment and enzymes account
for nearly 40% of the total production cost of biofuels. In an effort to reduce these
expenses, recent studies have emphasized the development of a biorefinery
concept, which mimics petroleum refineries by generating multiple co-products
from a single feedstock.
~25% of harvest
Stream 1
(Unjuiced/Wet)
Table 2. Banagrass juice characteristics
Stream 2
(Unjuiced/Dry)
Shredder
Parameters
Harvested
biomass
Stream 4
(Juiced/Dry)
Oven
Stream 3
(Juiced/Wet)
Screw press
Biomass juice
Figure 3. Four preprocessing streams generated after harvest
Figure 2. Banagrass from Waialua, Oahu
Objectives
The overall goal of this study was to evaluate the use of banagrass (4 months
old) as a feedstock for cellulosic biofuel production. It was hypothesized that the
wet processing of immature banagrass, which inherently has a high moisture
content (~90%), would create a potential for the generation of co-products, and
require less severe chemical pretreatments than conventional methods. The
following specific objectives were established to test our hypothesis:
•
•
•
•
Conduct baseline optimization studies for the dilute-acid pretreatment of four
preprocessing streams with respect to acid concentration, temperature, and
time
Compare the sugar release from the four processing streams to determine
the maximum sugar yield under optimal conditions
Characterize the juice extracted during wet processing for value-added
product generation
Conduct a preliminary investigation of incorporating ultrasonic technology
into conventional pretreatment strategies
Methodology
Banagrass was hand-harvested from the Poamoho Experiment Station (Waialua,
Oahu), and shredded. Half of the banagrass was de-watered in a screw-press,
which effectively removed about 40% of the liquid contained in the biomass, and
created Stream 3 (see Figure 3). The other half of the shredded banagrass, which
was not pressed, made up Stream 1. Conventional processing methods typically
dry the biomass at 105°C. Consequently, half of Stream 1 was dried (generating
Stream 2), and half of Stream 3 was dried (generating Stream 4).
Results and Discussion
250
Table 1. Optimal dilute-acid pretreatment conditions
Acid
Concentration
% (v/v)
Temp
°C
Stream 1 (U/W)
5.0
120
30
Stream 2 (U/D)
5.0
120
30
Stream 3 (J/W)
5.0
120
45
Stream 4 (J/D)
2.5
105
45
Preprocessing
Time
(min)
mg sugar/g biomass (dry wt.)
Figure 1. Banagrass over 10 ft high at the
Poamoho Experiment Station
The optimization of dilute-acid pretreatment was conducted with sulfuric acid at
the following concentrations: 1.0, 2.5, and 5.0% (v/v). Triplicate samples were
heated in an autoclave at varying temperatures (105°, 120°, and 135°C) and times
(30, 45, and 60 min).
Because wet processed banagrass has a short storage life, the sugar
quantification for optimization experiments was conducted by the colorimetric
dinitrosalicylic acid (DNS) method.1 Standards for two sugars of primary interest
(glucose and xylose) were used to validate this method.
Samples pretreated at optimal conditions for each preprocessing stream were
enzymatically hydrolyzed following an established protocol.2 The cumulative sugarrelease from each stream was used to determine the attainable sugar yields, and
the highest yielding stream (Stream 3) was used as a substrate for a preliminary
investigation with ultrasonication at varying treatment times (10, 20, and 30 sec).
High pressure liquid chromatography (HPLC) was used to elucidate and quantify the
individual sugars released by pretreatment at optimal conditions.
Banagrass juice, obtained after screw-pressing, was characterized using standard
protocols for chemical oxygen demand (COD) and suspended solids (SS) among
other parameters. Samples were also sent to the Oceanic Institute for amino acid
analyses.
Values
pH
6.07 ± 0.01
Total solids (%)
5.29 ± 0.01
Suspended solids (%)
1.39 ± 0.15
Volatile solids (%)
3.54 ± 0.03
Volatile suspended solids (%)
1.14 ± 0.12
Total chemical oxygen demand (g/l)
55.48 ± 3.97
Soluble chemical oxygen demand (g/l)
49.08 ± 8.39
Total Kjeldahl nitrogen (TKN) (g/l)
1.67 ± 0.00
Potassium (g/l)
9.67 ± 0.80
Table 3. Summary of amino acids in banagrass
juice and fungal biomass
Essential AA
Nonessential AA
Arg
His
Ile
Leu
Lys
Met
Phe
Thr
Val
Ala
Asp+ASN
Glu+Gln
Gly
Pro
Ser
Tyr
-
The characteristics of banagrass juice collected during wet processing are
summarized in Table 2. Rhizopus microsporus, an edible fungus, was cultivated on
the juice with no nutrient supplementation. The harvested fungal biomass and
banagrass juice were sent to the Oceanic Institute to evaluate its potential in
aquaculture applications. Essential and non-essential amino acids found in our
samples, which are important for fish feed applications, are listed in Table 3.
Data from ultrasonication studies (not shown) did not significantly enhance the
sugar-release when compared statistically at a 95% confidence level. The theory of
sonication is extremely complex, and a high variance was observed with triplicate
samples.
Conclusion
Our data suggest that the wet processing (and juicing) of banagrass promotes a
significant improvement in the cumulative sugar yield attainable from the cellulosic
biomass. Furthermore, aside from a reduction in cost (by eliminating the need for
drying), immature banagrass can be harvested more frequently, and has the
potential to generate a liquid stream that can serve as a substrate for aquaculture
feed production.
In contrast to studies conducted with a starch-based feedstock (cassava),3
ultrasonication on cellulosic material did not significantly enhance the sugarrelease of banagrass. A further understanding of the interaction of sound waves on
the chemical and morphological structure of cellulosic material maybe required for
the effective use of ultrasonication.
Acknowledgements
200
150
Xylose
Glucose
100
•United States Department of Energy (USDOE) Award #: DE-FG36-08GO88037
•Dr. Richard Ogoshi, Dr. Scott Turn, Ms. Saoharit Nitayavardhana
Arabinose
References
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1
0
Stream 1
(U/W)
Stream 2
(U/D)
Stream 3
(J/W)
Stream 4
(J/D)
Figure 4. Cumulative sugar release under optimal pretreatment
conditions
Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31: 426428.
2 Selig, M., N. Weiss, and Y. Ji. 2008. Enzymatic Saccharification of Lignocellulosic Biomass.
3 Nitayavardhana, S., Rakshit, S.K., Grewell D., van Leeuwen, J., and Khanal S.K. 2008. Ultrasound
pretreatment of cassava chip slurry to enhance sugar release for subsequent ethanol production.
Biotechnology and Bioengineering 101 (3):487-496.