ABSTRACT
BACKGROUND: Fine milling of dry lignocellulosic biomass, without prior chemical pretreatment, can produce a high percent theoretical yield of sugars during subsequent enzymatic saccharification. However, the high sugar yields, necessary for a commercial biofuels process, are costly, with the milling energy input, necessary to achieve such yields even exceeding the energy content of the biomass. In this study, we show that low moisture gaseous ammonia pretreatment of switchgrass, in advance of the milling step, significantly reduces the milling energy required to give high sugar titers. RESULTS: We have found that the increase in monomeric sugar yields upon enzymatic saccharification of ball-milled, but not chemically treated switchgrass, is more closely tied to the formation of crystallites of cellulose with a negative linear dependence on the coherent domain size than to a decrease in particle size or to an increase in surface area of the biomass. The milling energy required to reach ~80 % of theoretical yield of glucose under these conditions is intolerably high, however, approximating two times the energy content of the biomass. Two different low moisture content ammonia pretreatments, prior to milling, significantly reduce the required milling energy (four to eightfold, depending on the pretreatment). These involve either heating the biomass at 150-160 °C for 1 h at 10 wt% gaseous ammonia or incubating at room temperature for 9 days at 20 wt% gaseous ammonia, the latter mimicking potential treatment during biomass storage. We have tested this combination of pretreatment and milling on switchgrass using a variety of milling methods, but mostly using ball and attritor milling. In the case of the high-temperature gaseous ammonia treatment followed by attritor milling, the increase in the monomeric sugar yield upon saccharification shows a negative linear dependence on the second or third power of the cellulose crystalline coherent domain size, implying that the surfaces as well as the ends of the cellulose fibrils are accessible to cellulolytic enzymes. CONCLUSIONS: The combination of knife milling, low moisture gaseous ammonia pretreatment followed by attritor milling that costs only ~5 % of the energy content of the biomass for a total energy input of ~11 % of the biomass energy content, is capable of delivering high sugar titers upon enzymatic saccharification. These results show, therefore, how to better integrate a mechanochemical step into the pretreatment of switchgrass in a commercial biomass to biofuels conversion process.
ABSTRACT
An Escherichia coli catalyst with tyrosine ammonia lyase activity (TAL) has been stabilized for repeated use in batch conversions of high tyrosine solids to p-hydroxycinnamic acid (pHCA). The TAL biocatalyst was stabilized by controlling the reaction pH to 9.8 +/- 0.1 and immobilizing the cells within a calcium alginate matrix that was cross-linked with glutaraldehyde and polyethyleneimine (GA/PEI). We found a GA range where the bead-encapsulated TAL was not inactivated, and the resulting cross-linking provided the beads with the mechanical stability necessary for repeated use in consecutive batch reactions with catalyst recycle. The GA/PEI calcium alginate TAL catalyst was used in 41 1-L batch reactions where 50 g L(-1) tyrosine was converted to 39 +/- 4 g L(-1) pHCA in each batch. The practical usefulness and ease of this process was demonstrated by scaling up the TAL bead immobilization and using the immobilized TAL catalyst in four 125-L bioconversion reactions to produce over 12 kg of purified pHCA.
