Wet processing of adolescent Pennisetum purpureum for enhanced sugar release and co-product generation

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.

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

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 sugar-release 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.

Figure 1. Banagrass over 10 ft high at the Poamoho Experiment Station

Stream 1(Unjuiced/Wet)

Stream 2(Unjuiced/Dry)

Stream 3(Juiced/Wet)

Stream 4(Juiced/Dry)

Harvestedbiomass

~25% of harvest

Biomass juice Screw press

Shredder

Oven

Figure 3. Four preprocessing streams generated after harvest

Results and Discussion

PreprocessingAcid

Concentration% (v/v)

Temp°C

Time(min)

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

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.

Table 1. Optimal dilute-acid pretreatment conditions

Figure 4. Cumulative sugar release under optimal pretreatment conditions

Essential AA Non-essential AA

Arg AlaHis Asp+ASNIle Glu+Gln

Leu GlyLys ProMet SerPhe TyrThr -Val -

Table 3. Summary of amino acids in banagrass juice and fungal biomass

Table 2. Banagrass juice characteristics

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.

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).

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 sugar-release 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

References

•United States Department of Energy (USDOE) Award #: DE-FG36-08GO88037•Dr. Richard Ogoshi, Dr. Scott Turn, Ms. Saoharit Nitayavardhana

1 Miller GL. 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal Chem 31: 426- 428.2 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.

Figure 2. Banagrass from Waialua, Oahu

Devin Takara* and Samir K KhanalDepartment of Molecular Biosciences and Bioengineering