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Toxicity of opal Australian paper weak black liquors towards actinobacillus succinogenes 130Z

- Halder, Dibyadeep

  • Authors: Halder, Dibyadeep
  • Date: 2022
  • Type: Text , Thesis , Masters
  • Full Text: false
  • Description: The objective of this study was to gain a better understanding of Opal Australian Paper’s (AP’s) waste streams as a potential substrate for biomanufacturing. Three low-value process streams were included in this study: Continuous Kraft Mill (CKM) weak black liquor (WBL), Neutral Sulphite Semi-Chemical (NSSC) WBL and WBL extracted from NSSC clean-pulp (CP). The primary focus of the study was to determine the chemical composition and extent of toxicity of these WBLs towards Actinobacillus succinogenes 130Z. This genetically engineered bacterium can produce a valuable platform chemical, succinic acid, using various carbohydrate sources. This project sought to increase knowledge of AP’s WBL and its suitability as a biomanufacturing substrate. Literature has been reviewed on various pre-treatment pathways for this waste stream, A. succinogenes 130Z and its effectiveness on producing succinic acid, and the analytical techniques that have been used to answer the project objectives. The effectiveness of pre-treating the NSSC and CP WBLs was measured using the following characteristics: (a) the colour content (specific absorbance) using UV-Vis spectroscopy, (b) indicative yields of several organic constituents through pyrolysis-gas chromatography-mass spectroscopy, (c) the atomic H/C and S/C ratios using an elemental analysis and (d) toxicity of the samples using a combination of semi-quantitative (plating trials) and quantitative (determination of minimum bactericidal concentration) techniques. The impact of an additional pre-treatment step of granular activated carbon for NSSC WBL was also researched here. InfraRed spectroscopy and toxicity analysis helped to conclude that CKM WBL is extremely toxic to Actinobacillus succinogenes 130Z and contained negligible amounts of residual sugar units. Characterising the NSSC and CP WBLs at the molecular level revealed the untreated WBLs are rich in lignin-derived methoxyphenols (up to 160 mg/kg) and contained low concentrations of elemental sulphur (up to approx. 1 mg/kg). Infra-red spectroscopy and gas chromatography indicated that the fermentable sugars in the WBLs were very low (< 1.2 mg/kg) even after pre-treatment. Both methoxyphenols and sulphur are known to be toxic to many bacteria and A. succinogenes 130Z could not tolerate more than 0.45 mg/ml (db, dry basis) and 0.78 mg/ml (db) of NSSC and CP WBLs respectively. Employing Amberlite® XAD7HP resins to pre-treat the NSSC and CP WBLs reduced their toxicity 2- to 5-fold, enabling the bacterium to survive in about 2.5 mg/ml (db) and 1 mg/ml (db) of NSSC and CP WBLs respectively. One possible reason for this is the significant reduction in lignin content afforded by this pre-treatment technique. These results conclude that AP’s WBLs are toxic and need to undergo pre-treatment prior to utilisation as a substrate for succinic acid production via fermentation.
  • Description: Masters by Research

Toxicity of opal Australian paper weak black liquors towards actinobacillus succinogenes 130Z

- Halder, Dibyadeep

  • Authors: Halder, Dibyadeep
  • Date: 2022
  • Type: Text , Thesis , Masters
  • Full Text: false
  • Description: The objective of this study was to gain a better understanding of Opal Australian Paper’s (AP’s) waste streams as a potential substrate for biomanufacturing. Three low-value process streams were included in this study: Continuous Kraft Mill (CKM) weak black liquor (WBL), Neutral Sulphite Semi-Chemical (NSSC) WBL and WBL extracted from NSSC clean-pulp (CP). The primary focus of the study was to determine the chemical composition and extent of toxicity of these WBLs towards Actinobacillus succinogenes 130Z. This genetically engineered bacterium can produce a valuable platform chemical, succinic acid, using various carbohydrate sources. This project sought to increase knowledge of AP’s WBL and its suitability as a biomanufacturing substrate. Literature has been reviewed on various pre-treatment pathways for this waste stream, A. succinogenes 130Z and its effectiveness on producing succinic acid, and the analytical techniques that have been used to answer the project objectives. The effectiveness of pre-treating the NSSC and CP WBLs was measured using the following characteristics: (a) the colour content (specific absorbance) using UV-Vis spectroscopy, (b) indicative yields of several organic constituents through pyrolysis-gas chromatography-mass spectroscopy, (c) the atomic H/C and S/C ratios using an elemental analysis and (d) toxicity of the samples using a combination of semi-quantitative (plating trials) and quantitative (determination of minimum bactericidal concentration) techniques. The impact of an additional pre-treatment step of granular activated carbon for NSSC WBL was also researched here. InfraRed spectroscopy and toxicity analysis helped to conclude that CKM WBL is extremely toxic to Actinobacillus succinogenes 130Z and contained negligible amounts of residual sugar units. Characterising the NSSC and CP WBLs at the molecular level revealed the untreated WBLs are rich in lignin-derived methoxyphenols (up to 160 mg/kg) and contained low concentrations of elemental sulphur (up to approx. 1 mg/kg). Infra-red spectroscopy and gas chromatography indicated that the fermentable sugars in the WBLs were very low (< 1.2 mg/kg) even after pre-treatment. Both methoxyphenols and sulphur are known to be toxic to many bacteria and A. succinogenes 130Z could not tolerate more than 0.45 mg/ml (db, dry basis) and 0.78 mg/ml (db) of NSSC and CP WBLs respectively. Employing Amberlite® XAD7HP resins to pre-treat the NSSC and CP WBLs reduced their toxicity 2- to 5-fold, enabling the bacterium to survive in about 2.5 mg/ml (db) and 1 mg/ml (db) of NSSC and CP WBLs respectively. One possible reason for this is the significant reduction in lignin content afforded by this pre-treatment technique. These results conclude that AP’s WBLs are toxic and need to undergo pre-treatment prior to utilisation as a substrate for succinic acid production via fermentation.
  • Description: Master by Research
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