Compositional Analyses For Summative Mass Closure

For a feedstock to support microbial growth and bioconversion, it must contain certain essential nutrients which can roughly be divided into: carbon source, energy source, nitrogen source, minerals and vitamins (Nielsen et al., 2012). The summative mass closure gives an indication of the chemical composition of a potential feedstock and this determines the types of bio-conversion processes to which the biomass can be applied.

The moisture contents of the brans were the first parameter determined as these provided the oven dry weight (ODW) upon which the results of other analyses were estimated. It was determined with a halogen moisture analyser which has several advantages over traditional methods including that it employs higher operating temperatures to dry samples and thus has shorter drying times than the traditional oven method. It also uses smaller amounts of sample while retaining its accuracy (while attempting to selesct the ideal program on the analyser, the results obtained from the barley rap program were compared with a traditional oven method and the differences were found to be less than 5 %). The moisture content of the white bran was found to be higher at about 11.6% than the red bran at 6.3%. This disparity was not surprising because the methods of preparing the bran are not standardised and so could indicate a less vigorous water extraction at the sieving stage, or indicate that it was not dried for as long as the red bran was. As this value was over the 10% benchmark for biomass samples (Sluiter et al., 2010), the white bran was oven dried till the MC was brought to 6.8%.

This prevents mould spoilage and also ensures that moisture does not interfere with subsequent compositional analyses as, for instance, high moisture contents could interfere with dilute acid hydrolysis experiments and prevent complete digestion. The bran MC figures

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were found to be similar to that reported by Corredor et al (2007) who reported 8% MC for bran from a dry decortication process.

Sample preparation was conducted to ensure the biomass was in the ideal state for compositional analyses investigations. This involved sieving the bran into fractions that were neither too fine nor too coarse. The recommended size for biomass analysis is -20/+80 which refers to the fraction that passes through the 20 mesh sieve but is retained by the smaller 80 mesh sieve (Sluiter et al, 2010). About 83.8% of the white bran and 87.1% of the red bran fell into this range. However, it was observed that the -80 fraction formed a considerable proportion of the brans, about 12-15%, and discarding it could use an overall change in composition as certain anatomical fractions segregate disproportionally into the fines fraction (Sluiter et al., 2010). On the other hand, the amount of the +20 fraction was insignificant in both brans so that was also retained. In subsequent experiments, the brans were thus not sieved after milling.

Ash content represents the mineral/inorganic matter of the biomass removed after all the vaporisable, organic matter has been removed by combustion (Tiwari and Singh, 2012). The white and red brans had ash contents of approximately 1.8% and 1.5%, respectively. These figures are similar to a reported figure of 1.82% (dry weight basis) for sorghum grains (FAO, 1995). For comparison, these values are less than the ash content of herbaceous feedstock such as barley straw, alfalfa stems and canary grass reported to be 4.5%, 7.1% and 8.0%

respectively (Amarasekara, 2013).

The carbohydrate content, including both storage and structural carbohydrates, was also determined as this was vital to give an indication of the carbon source for consideration for potential fermentative processes. The total content was determined by acid hydrolysis and it was observed that WB and RB had almost equal amounts of about 69.03 % and 70.48%

respectively (Table 3.4); this is only slightly lower than 73 % reported for the whole grain (Fuleky, 2009). Glucose content was the highest at about 58 – 60 % and trace amounts of xylose, arabinose, mannose; cellobiose and maltose were not detected indicating that the digestion of cellulose and starch had proceeded to completion. These observed high carbohydrate values was a very significant finding as they indicate an abundance of potential carbon source in sorghum brans for bioconversion processes. It also indicates that the processing method is not very efficient resulting in high residual starch content in the bran.

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Lignin is an amorphous polyphenolic compound formed from the polymerisation of three phenylpropanoid monomers namely coniferyl, sinapyl and p-coumaryl alcohols. Lignin has been shown to exist in cereal grains using the acetyl bromide (and other methods), even after excluding the possibility of interference by low molecular weight phenolic compounds (Bunzel et al., 2004). The lignin contents of the brans were determined by the acetyl bromide method which is based on the solubilisation of lignin and the determination of absorbance values at 280 nm. This method is simple and quick to perform, and reportedly provides higher lignin recovery (Moreira-Vilar et al., 2014). It was estimated that the lignin contents of WB and RB were 6.7 % and 10.5 % respectively. These Figures are higher than those reported for whole grains because lignin (along with cellulose and hemicellulose) is a structural material found in the pericarp, testa and aleurone tissues (see Figure 1.9) and mainly retained in the bran (Arendt and Zannini, 2013). They are however in congruence with the sum total of the insoluble dietary fibre and alkali-extracted dietary fibre for rye bran as reported by Bunzel et al. (2004).

In addition to carbon sources, microorganisms require other nutrients for growth and bioconversion processes. Protein is the second major component of sorghum (FAO, 1995), with the protein content indicating the presence of constituent amino acids which fermenting organisms require in order to proliferate. The red bran had a higher protein content at 16.9 % than white bran at 15.6 % protein, which is higher than the amount found in whole sorghum grains where a range of 8.6 % – 18.2 % has been reported (Virupaksha and Sastry, 1968).

The sorghum grain contains up to 80 % of its protein in the endosperm (Figure 1.9) (Taylor and Schüssler, 1986), where they exist as protein bodies composed mainly of prolamin and in the protein matrix located in the peripheral and inner endosperm comprising mainly glutelin (FAO, 1995). However, the germ comprises albumins and globulins which form about 16 % of grain Nitrogen (Taylor and Schüssler, 1986) while 3% is located in the pericarp. The high bran protein content could be because the bran contains all of the pericarp content, in addition to parts of the endosperm and germ. This high protein content also indicates that brans may have a good potential for nutritional applications.

The crude lipid content of WB and RB were 2.3 % and 3.7 %, respectively which is quite similar to reported values of 2 % to 3 % for decorticated sorghum bran (FAO, 1995) and to that of whole sorghum reported to be 3.0 % (Corredor et al., 2007) and 3.3 % by Jambunathan and Subramanian (1988). The similarity with whole grain values could be ascribed to the fact that the germ fraction which contains most of the oils has been ground

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and mixed in the slurry from which the bran was extracted. The oils are thus uniformly dispersed by the milling process.

The summative mass closure of the sorghum brans were achieved with white bran being elucidated up to 102.9 % and RB to 109.4 %. The values surpassing the 100 % mark could be due to several reasons, such as an over estimation of the protein content due to the method used, and the conversion factor of 6.25 which is not specific for sorghum bran, or simply due to experimental error.

3.4.2 Other Compositional Analyses

Other parameters were determined to give a clearer picture of the composition of the sorghum brans. Thus, even though the total carbohydrate content had been determined, starch was specifically evaluated. From Table 4.9 it was observed that both brans had a high starch content with 49.7 % and 53.0 % of the WB and RB respectively being composed of starch left after the slurry was filtered (see Section 2.2.1). This was a surprisingly high value considering that whole sorghum grains contain 56 % - 76 % starch (Jambunathan and Subramanian, 1988) and the sieving process was aimed at ensuring starch removal. However, high values have been previously reported with 16.4 - 33.2 %, reported for wheat bran, 51.0

% for barley bran and 52.3 % for oats bran respectively (Clegg, 1956; Englyst et al., 1983;

Bhatty, 1993). These high amounts observed in this work could be ascribed to the poor grinding ability of the machine used, and to inadequate squeezing and rinsing during the sieving process. It is however important to note that as the major component of sorghum, the variability of this method means sorghum bran residual starch content will vary within batches. These starch values contribute 82 % and 87 % of total glucose obtained from carbohydrate hydrolysis respectively with structural carbohydrate components being much lower at 5.7 % - 7.5 % and 5.2 % - 5.4 % for cellulose and hemicellulose respectively (Table 3.10). The cellulose and hemicellulose contents found in the present study were considerably lower than the 18 % and 11 % respectively reported by Corredor et al. (2007) but this could be due to the higher starch content observed in this work, and the fact that their process didn’t involve milling the whole grain, but abrasive decortication which selectively removes the bran only. The discovery of such high amounts of starch was another very significant finding, as this indicated that starch degradation would be a suitable way to generate glucose-rich hydrolysates for subsequent fermentations.

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Similarly, the reducing sugars were investigated by the DNS method as there was very little information available concerning this component of sorghum bran and its potential contribution to the bioconversion is worth noting. The values of 0.85 % and 1.16 % observed for WB and RB respectively (Table 3.11) were found to be higher than an average of 0.12 % reported by Jambunathan and Subramanian (1988) for whole grains. This might be due to the liberation of sugars from the starch by microbial action during the steeping/soaking process of bran preparation.