The development and optimisation of a lab-scale process for biological treatment of lignin-rich wastewater using biofilms formed by Nuerospora Discreta

Lignin is a complex biopolymer found in lignocellulosic materials used as raw material in pulp and paper making. Lignin is processed as a by-product of low value and is discarded in the wastewater. This wastewater is highly polluting due to dissolved lignin degradation products, which give it an intense colour and high chemical oxygen demand (COD), causing harm to aquatic life, plants, and animals. Removal or degradation of lignin has been shown to improve water quality in industrial wastewater, however, the complex structure of lignin makes it difficult to be degraded. Advancements in wastewater treatment methods, such as the conventional physiochemical and thermochemical methods employed, have a detrimental impact on the environment due to the production of hazardous by-products and high energy requirements. Biological treatment of lignin using fungi has the potential to overcome many of these roadblocks and lead to a successful process.

This thesis aims to develop a single-step biological process for treating wastewater from the kraft process used in paper-making. Neurospora discreta, an ascomycete fungus, has been reported to degrade lignin effectively in lignocellulosic biomass, as it possesses the ligninolytic enzymatic machinery required for lignin degradation. It also has a unique ability to form robust biofilms at the air and liquid interface. In this research, N. discreta was evaluated for its ability to treat lignin-rich wastewater for the first time.

The process optimisation was initially developed on synthetic wastewater using alkali lignin, followed by studies using wastewater provided by a pulp and paper-producing company. Firstly, the Taguchi statistical design of experiments, was used to identify the critical process levers for enhancing lignin degradation. Secondly, the addition of naturally formed lignin degradation intermediates in the fungal-treated wastewater spent media was evaluated as a strategy to increase lignin and COD removal. Finally, the biofilms were tested in a continuous repeated batch process, where actively metabolising mature biofilms were transferred to fresh wastewater in repeated cycles. The process was then scaled-up eightfold to tray reactors.

This research has developed a fungal biofilm-based sustainable, eco-friendly and scalable alternative for lignin and COD removal in industrial wastewater. The fungal biofilm treatment proved to be efficient in removing 67.8% of standard kraft lignin in synthetic wastewater. The process efficiencies, while treating real wastewater from pulp and paper mill, were improved significantly by using lignin degradation intermediates as additives. The lignin and COD removal efficiencies of 70% were noted in cultures fed with lignin degradation intermediates compared to 57% and 50% respectively, in unfed culture. Enzyme activity for polyphenol oxidase (PPO), versatile peroxidase (VPO) and laccase were also seen where VPO was reported for the first time in a Neurospora species. The repeated-batch treatment process was evaluated and resulted in an efficient scalable process.