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Development of the flow through bioreactor of 3D-structured bacteria for biodegradation of aromatic chloroderivatives from contaminated water

Periodic Reporting for period 1 - cryo-bacteria-reactor (Development of the flow through bioreactor of 3D-structured bacteria for biodegradation of aromatic chloroderivatives from contaminated water)

Reporting period: 2016-09-15 to 2018-09-14

Phenol derivatives are poisonous and carcinogenic to both humans animals and aquatic life and are often found in wastewater discharged from pharmaceutical treatment factories and oil refineries. Chlorophenols in particular are highly resistant to biodegradation in the environment and considered a serious pollutant. The currently used industrial methods for phenol derivatives remediation are not efficient, multi-step, complex and costly, and generates additional waste which in turn should be utilised. Bioremediation processes utilising bacteria represents an alternative to existing chemical methods. Immobilisation of bacteria on a substrate reveals many benefit over free bacteria systems, such as higher biomass content, high metabolic activity, resistance to toxic chemicals, allowing continuous process operating and avoiding the biomass- liquid separation requirements. The immobilised bacteria can be reused several times opening opportunities for developing cost-effective processes for wastewater treatment. In this project we apply a novel technology for developing a novel flow through 3D structured bacterial cells systems for bioremediation. The main objective of the project was to develop an environmentally friendly one-step immobilisation of bacteria to obtain bioreactor with a high density of immobilised cells; low diffusion restriction of contaminant to the cells and exploit the system as a flow through bioreactor for degradation of phenols and chlorophenols.
During the project a novel method of direct cross-linking of the bacteria cells into a 3D-structured macroporous, highly permeable system was developed and their efficiency for purification of contaminated water from chlorinated phenols and polyphenols as well as non-chlorinated phenol derivatives was analysed.
Four bacteria strains (non-pathogenic) known to be able to degrade phenol derivatives were selected such as (Pseudomonas mendocina, Rhodococcus korensis, Acinetobacter radioresistence and Athrobacter chlorophenolicus). They were directly cross-linked into a highly water permeable 3D-structured macroporous material using novel cross-linking polymers. This entailed the production of robust 3D microporous, non-toxic, material (cryogels) containing mostly (90%) immobilised phenol-degrading bacteria. The chemical composition and concentration of the polymers used as cross-linkers for bacterial cells were optimised to obtain strong cryogel material with retained metabolic activity of the cells. Synthesised new polymers were comprehensively characterised by the use of modern advanced methods such as infrared microscopy, proton nuclear magnetic resonance, measurement of the surface charge, molecular weight distribution and thermostability. Toxicity of polymers to different bacteria strains as well as the effect of freezing on the viability of cross-linked cells was assessed using spectrophotometric MTT assay and laser scanning confocal microscopy. Overall, six polymers and their combinations were utilised to produce spongy like materials for bioremediation purposes using four bacterial strains. For the first time we are able to report reproducible procedures of synthesis of new cross-linking agents as well as a detailed reproducible protocol of bioreactor preparation. Optimal combination of novel cross-linking polymers (PEI-al and PVA-al) for the preparation mechanically stable and elastic macroporous 3D bioreactors based on bacteria were selected for remediation study. The viability of the cells before and after spongy material preparation, as well as after remediation cycle were examined and visualised using different types of microscopy.
Obtained macroporous 3D bioreactors were used toward bioremediation of phenol, cresol, nitrophenol, chlorophenols and dichlorophenols. Mixtures of phenolic compounds in river and underground water and waste waters or in model buffered solutions at pH 6, 7.2 and 8, respectively. Three generations of 3D bioreactors were prepared and tested in a dynamic and static mode, respectively. Based on a series of experiments the most effective bacterial strains were Acinetobacter radioresistens and Arthrobacter chlorophenolecus, which were selected and used for a more comprehensive study of bioremediation of chlorophenol derivatives (2-chlorophenol, 4-chlorophenol and 2.4-Dichlorophenol) and tested in dynamic mode and static mode. It was shown that the bioremediation took place significantly slower in static mode compared to dynamic mode. The bioremediation process in an open system took place 3-4 times faster compared to a model system (sterile conditions), which is a positive sign, pointing to the successful application if we need to transfer the technology to pilot scale and then to industrial scale. Bioremediation efficiency of 3D bioreactors was measured using phenol, methylphenols or chlorophenol spiked river water or waste water (municipal wastewater from a wastewater treatment in Hailsham, UK and Kosice, Slovakia). We demonstrated that prepared 3D bioreactors could be used for bioremediation of a complex mixture (phenol and chlorophenols) of contaminants resulting in low energy cost purification of water. Overall 130 experimental set ups in triplicate were performed to optimise bioremediation parameters and select optimal composition for detailed investigation. A low content of polymer in the structure makes disposal easier after its use and this is therefore a greener technology. The bioremediation system can be successfully applied for phenol concentration up to 300 ppm.
Advantages of the developed technology is the absence of the separation step of bacteria from water after the bioremediation step, a possibility to reuse material a number of times, without the need of additional use of chemicals, bacteria are non-pathogenic and therefore the water can be safely discharged to environment without additional purification. The material can be reused at least 10 cycles without decline of the bioremediation efficiency. There is no need for the presence of additional source of carbon in water to proceed the bioremediation process.
During the project we developed a novel approach for making bioreactors and the treatment of contaminated water to solve sophisticated problems of Environmental remediation. We have demonstrated a novel “green” approach that is capable of degrading various phenol derivatives in an environmentally friendly way without the use of expensive and toxic chemicals. This in turn will significantly reduce the carbon dioxide foot print of waste water treatment processes and therefore will have enormous social and ecological impact.
The optimised method of production of material with a high number of live immobilised bacteria, low polymer content and controlled porosity is a promising means for development of novel materials based on live/or enzymatically active bacterial cells for various environmental, biotechnological, and medical applications. This will open opportunities for the development of production of known valuable products in a cheaper and easier manner as well as in an environmentally friendly way.