Enhancing the removal of chemical oxygen demand and inorganic nitrogen from saline fish processing wastewater by bioaugmentation
Creators: name: HOANG, Thi Hong Anh
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 4104
subject: Environmental management
classificationCode: 410404
subject: Environmental management

description: The increasing global population has contributed to a significant increase in the demand for fish products, reaching 94.6 million tonnes in 2018. This has resulted in a significant increase in wastewater generation from fish production systems. As more than 90% of fish products are marine, seawater is widely used in the processing; this seawater ultimately becomes saline wastewater. Therefore, wastewater generated by fish processing facilities is characterised by high salt concentration, similar to or greater than that of seawater. However, fish processing wastewaters also contain a high level of pollutants such as nitrogenous (e.g. nitrate, nitrite, ammonia) and carbonaceous compounds (e.g. colloids including proteins, lipids, and other organic compounds found in fish bodies) which can harm fish and other inhabitants of the aquatic ecosystem if released directly into oceans and rivers. Adequate treatment of the wastewater is therefore crucial to a sustainable fish industry. However, the treatment of these wastewaters is complicated by many factors including high salinity and high nitrogen content. Hence, there is an urgent demand for cost-efficient and environmental-friendly technologies that can improve the efficacy of the current treatment processes without the demand for significant additional infrastructure. 

Several approaches have been previously studied to remove pollutants from processing wastewater including physico-chemical processes: for example, combining coagulation and flocculation processes using FeCl3, a coagulant/flocculant agent (e.g. Tanfloc SH) or copolymers. Physical processes have involved sedimentation and dissolved air flotation treatments. However, both are expensive treatments, largely due to issues with the high salt concentration. In addition, both physical and chemical treatments have limitations as they tend to produce secondary products which have to be removed. The biological approach - based on the use of the natural microbial community present in the wastewater, represents an effective and environmental-friendly process to remove pollutants from aqueous ecosystems. Several potential biological candidates, capable of heterotrophic nitrification and aerobic denitrification, including Alcaligenes faecalis and Acinetobacter calcoaceticus have been investigated as candidates for the basis of a commercial process for the conversion of ammonium and nitrates to nitrogen gas. However, these non-halophilic bacteria are unsuitable for saline fish wastewater treatment because of cell disintegration due to the osmotic pressure difference across the cellular membrane that causes the loss of cellular water. Due to high salinity, effective biological treatment of these wastewaters requires the application of halophilic microorganisms which can tolerate high salt concentrations. The overarching aim of this study was to develop a cost-effective bioremediation for the removal of total nitrogen (TN) and chemical oxygen demand (COD) from saline fish processing wastewaters based on the application of halophilic bacteria. 

With the aim of improving saline fish processing treatment by applying halotolerant bacteria which can survive in high salinity and also release proteases to assist in the remediation of the wastewater, the first research chapter involved firstly the isolation of halophilic bacteria from seawater and subsequent assessment of their ability to produce protease activity, and secondly, assessment of their ability to enhance the treatment of fish processing wastewater, in terms of COD and TN removal in the final effluent. Five of the 7 isolated organisms were identified as moderate halophilic bacteria, which exhibit optimum salinity for growth between 5% to 15% (w/v). Through 16s rRNA gene sequencing followed by BLAST searches, five moderate halophilic bacteria isolates were identified as Marinihabdus sp., Vibrio sp., Alteromonas sp., Alteromonas macleodii, and Marinobacter hydrocarbonclasticus. All five exhibited the capacity to produce extracellular protease activity (9.93 - 14.54 U mL-1). However, the bioaugmentation of two strains, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus into non-sterile saline fish processing wastewater resulted in significantly higher removal efficiencies for COD, TN, nitrate, nitrite, and ammonium. The greatest removal of COD was observed in wastewater amended with Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus after 9 days (92%), with COD reduced from the initial concentration of 1128 ± 16.0 mg L-1 to 92.7 ± 6.9 and 94.7 ± 2.0 mg L-1, respectively. The greatest TN removal (85%) was observed when Marinobacter hydrocarbonoclasticus was introduced to the wastewater, with the reduction from an initial value, 360 ± 15.46 mg L-1 on day one to 53.3 ± 2.88 mg L-1 on day 9; bioaugmentation with Marinirhabdus sp. reduced the TN to 66.67 ± 7.63 mg L-1 (an 80% reduction). The values obtained in the treatment met the second class (Level B) requirement of national municipal wastewater discharge standard of EU (COD &lt; 120 mg L-1 and TN &lt; 70 mg L-1). With their degradative capabilities, these two isolates represent potential candidates for bioaugmentation to improve the effluent treatment of saline fish wastewater. 

Since the cost associated with the use of multiple single cultures which are ultimately combined for use as bioaugmentation agents may limit the commercial success of the application, in the second research chapter, the two halophilic isolates Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus, which were found capable of efficient nutrient removal from fish wastewater with salinity above 3%, were selected to assess the application of co-culture technology with the aim to determine whether growth and protease activity in co-culture resulted in greater biomass production and protease yields compared to monoculture. Further, the range of environmental conditions (pH, temperature, and salinity) in which the proteases from the co-culture remained active compared to protease activities from the monocultures was assessed. The mixed culture reached higher densities (OD600 = 1.392), 1.6 - 2.0-fold greater than those attained by the pure cultures, Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus (OD600 = 0.700 and OD600 = 0.877, respectively). Enhanced protease production was also observed in the co-culture (38.6 Units OD-1), 1.8 - 2.4-fold higher than that of monocultures (21.6 and 16.0 Units OD-1, for Marinihabdus sp. and Marinobacter hydrocarbonoclasticus, respectively). Additionally, the temperature, pH, and salinity range of protease activity (defined as &gt;75% of the maximum value) from the co-culture was greater than that observed in protease activities from the monoculture. For proteases from the co-culture, the range was 25 - 60oC, pH 4 - 12, and 10 - 30% salinity; in monocultures, the range of protease activity was 30 - 45oC, pH 4 - 8 and 5 - 15% salinity for Marinihabdus sp., and 45 - 60oC, pH 8 - 12, and 20 - 30% salinity for Marinobacter hydrocarbonoclasticus. Overall, the results confirmed firstly that both growth and protease activity in co-culture resulted in increased biomass and protease production compared to monoculture, and secondly, a broader range of environmental conditions under which protease activity remained effective was observed for enzymes produced from the co-culture. Both these outcomes further enhance the commercial viability of this environmentally friendly approach to enhance the treatment of fish processing wastewater. 

The work conducted in the third research chapter firstly aimed to investigate whether the augmentation of fish wastewater with either Marinirhabdus sp., Marinobacter hydrocarbonoclasticus, or a consortium of the two halophilic bacteria, could successfully enhance COD- and total nitrogen- removal in fish processing wastewater, and secondly to assess the survival of the two organisms in non-sterile fish wastewater as well as the impact of treatment on the bacterial community at the end of the process. The addition of monoculture was again effective in enhancing the removal of COD; however, the addition of the co-culture significantly improved TN removal after 9 days of incubation. Bioaugmentation with either Marinirhabdus sp., or Marinobacter hydrocarbonoclasticus showed 91% and 92% of COD removal (with COD reduced from 1275 ± 15 mg L-1 to 108 ± 7.0 and 101 ± 8.7 mg L-1, respectively), while the addition of the co-culture resulted in a lower reduction (88%), from 1275 ± 15 mg L-1 to 287 ± 7.9 mg L-1. However, greatest TN removal was observed in wastewater amended with the co-culture (88% removal), with a reduction from the initial value of 275 ± 6.5 mg L-1 to 34 ± 7.2 mg L-1, higher than Marinobacter hydrocarbonoclasticus (84% removal) and Marinihahabdus sp. bioaugmentation (79% removal). Since the success of any bioaugmentation must be sustained, the survival of the two organisms in non-sterile fish wastewater at the end of the process was examined using 16S rDNA amplicon based sequencing of DNA extracted following the end of 9-day incubation from all fish wastewater treatments. The results not only indicated the fate of Marinihabdus sp. and Marinobacter hydrocarbonclasticus but also highlighted the impacts in terms of changes in bacterial community at both the class and genus levels in the wastewater during treatment. Marinihabdus sp. was not detected in either mono or co-culture augmented treatments. In contrast, Marinobacter hydrocarbonclasticus reached a relative abundance level of 36% when augmented alone and approximately 20% in co-culture amended treatments. The addition of these selected strains significantly affected the bacterial community in non-sterilised fish wastewater. For example, although Marinihabdus sp. was not detected at the end of the process even when it has been added, the introduction of the bacteria into wastewater resulted in significant changes in the bacterial species with known degradative capabilities such as Psychrobacter proteolyticus, Shewanella decolorationis, and Vibrio sp. This may, in part explain the enhanced removal of COD and TN in inoculated treatments. However, for any commercial remediation process, an important consideration is that the bioaugmented organism must maintain activity and growth throughout the incubation and be effective in meeting the goals of the process to achieve successful commercial bioaugmentation. In this respect, the presence of Marinobacter hydrocarbonclasticus was a dominant organism at the end of the treatment implying that re-augmentation was not required for continuous wastewater treatment to enhance commercial potential. 

Overall, this study has demonstrated that the addition of halophilic bacteria Marinirhabdus sp. and Marinobacter hydrocarbonoclasticus can enhance the removal of COD and TN in non-sterile saline fish wastewater. Growth and protease production in co-culture was found to be an effective way of preparing the bioaugmentation agents resulting in enhanced biomass and protease production, and a more robust protease activity in terms of efficacy over a range of environmental conditions. Only Marinobacter hydrocarbonoclasticus was found to survive and remained a dominant member of the bacterial community at the end of treatment. The ability of Marinobacter hydrocarbonclasticus to enhance the treatment and dominate the bacterial community suggests the commercial potential of this organism for bioaugmentation of aquaculture wastewater without the need for further bioremediation.