Direct Sugarcane Bagasse to oil (SB2O)

Direct Sugarcane Bagasse to oil (SB2O)

Herein we present an update on the project activities carried out at UoM since April 2011. Shell is interested in the direct conversion of sugarcane bagasse into oil by culturing microbes.

We were tasked to grow yeast that is able to accumulate a relatively high proportion of oil in its body. For achieving such goal, Shell commissioned the present work to The Satake Centre for Grain Processes Engineering (SCGPE), given its R&D expertise in applying bioprocesses to produce added-value products from lignocellulosic biomass.

In the previous report, it was shown that it is possible to grow oleaginous yeast in fresh pressed bagasse. We showed images of huge oily vesicles inside the yeast in suboptimal experiments. The yeast was able to grow presumably because freshly pressed bagasse still contains soluble sugars that are usually degraded by microbes thriving in the environment.

Recently, we tested a strategy to produce a suspension rich in R. toruloides but free of undesirable particles. This approach was followed to prevent costly downstream separation processes of the bagasse-yeast mixture. It was found that it is possible to biodegrade a fraction of the bagasse and produce soluble nutrients that can be diffused through a membrane into another reactor where R. toruloides grow.

We also tested an approach to grow molds on bagasse in solid state fermentation to release soluble nutrients. This system was leached with the suspension from a submerged state system containing R. toruloides. The leachate was recovered back into the submerged state system where R. toruloides was intended to grow but did not.

In submerged estate fermentation, we found that a given combination of fungal strains was able to grow on fresh bagasse. This was assumed given the fact that a ring of biomass grew at the edge of the water surface spread on the flask by orbital shaking. An assumption was later reinforced by an optic light microscopy study.

The aforementioned ring of biomass was daily homogenised into the bagasse suspension, which was subjected to direct methanolysis for the production of FAME’S. Butyric acid methyl ester (C4:0) was presumably found in seven out of eight combinations. On the other hand, cis-8,11,14-Eicosatrienoic acid methyl ester (C20:3n3) was found in the system inoculated with T. reesei and R. toruloides. Since the samples were too diluted, it was not possible to have peaks that could be related to a FAME profile with a good degree of confidence.

Chemical treatment with sulphuric acid revealed that about 60% of the bagasse was made of cellulose and hemicellulose. This pre-treatment was very efficient to obtain sugars but also contained inhibitors such that the oleaginous yeast Rhodosporidium toruloides was not able to grow.

Biological experiments (solid state fermentation) were also carried out and we observed that our fungi (Trichoderma reesei and Aspergillus awamori) grew but consumed the sugars (maximum yield: 8 mg/g dry bagasse). The same conclusion was drawn when bagasse was supplemented with N, K, P and Mg. When bagasse was supplemented with sucrose (~3.4% w/w) to simulate freshly extracted sugarcane all the microorganisms (fungi and yeast) consumed the sugars. When bagasse was supplemented with sucrose and yeast extract our fungi consumed the sugars.

About 120 mg/g dry bagasse using commercial enzymes without autoclaving, but particles smaller than 500 microns were required to obtain these results.

The second part of the report deals with fresh sugarcanes obtained from Malaysia. Tests were carried out on unextracted as well as extracted sugarcanes (=bagasse). Solid state fermentation (SSF) was carried out to measure the cellulase activity that can be obtained. A low cellulase activity in the range 0.1 – 0.7 FPU/g dry substrate (gds) was found, and the state of the sugarcanes (unextracted or extracted sugarcanes) and the particle size (large, i.e. non sieved fibers after cutting the sugarcanes with a machete and particles smaller than 2 mm) had no influence. In contrast, when yeast extract was supplemented, a cellulase activity in the range 1-1.2 FPU/gds could be obtained indicating that sugarcanes and bagasse are deficient in nitrogen.

Using commercial enzymes on unextracted sugarcanes a sugar yield of up to 600 mg/g dry sugarcanes could be obtained compared to a yield in the range 350-450 mg/g using only water for the extraction. This suggested that a simultaneous extraction and enzymatic hydrolysis would be interesting. The use of commercial enzymes on extracted sugarcanes (bagasse) provided up to 250 mg/g additional sugars compared to nothing if the bagasse is disposed of. A better enzymatic hydrolysis was observed when the material remained wet (unextracted and extracted sugarcanes).

Alkali pre-treatment improved the enzymatic attack of cellulose and xylose by commercial enzymes giving rise to sugar yields of 700 to 1000 mg sugars/g dry bagasse.
However, our fungi could not produce cellulases on alkali pre-treated bagasse.

At the end of this report, we present a summary of the different strategies used to address the problem of hydrolysing lignocellulosic materials as well as a compendium of relevant abstracts.

Confidential.