Genetic engineering to enhance microalgal-based produced water treatment with emphasis on CRISPR/Cas9: A review

Abstract

In recent years, the increased demand for and regional variability of available water resources, along with sustainable water supply planning, have driven interest in the reuse of produced water. Reusing produced water can provide important economic, social, and environmental benefits, particularly in water-scarce regions. Therefore, efficient wastewater treatment is a crucial step prior to reuse to meet the requirements for use within the oil and gas industry or by external users. Bioremediation using microalgae has received increased interest as a method for produced water treatment for removing not only major contaminants such as nitrogen and phosphorus, but also heavy metals and hydrocarbons. Some research publications reported nearly 100% removal of total hydrocarbons, total nitrogen, ammonium nitrogen, and iron when using microalgae to treat produced water. Enhancing microalgal removal efficiency as well as growth rate, in the presence of such relevant contaminants is of great interest to many industries to further optimize the process. One novel approach to further enhancing algal capabilities and phytoremediation of wastewater is genetic modification. A comprehensive description of using genetically engineered microalgae for wastewater bioremediation is discussed in this review. This article also reviews random and targeted mutations as a method to alter microalgal traits to produce strains capable of tolerating various stressors related to wastewater. Other methods of genetic engineering are discussed, with sympathy for CRISPR/Cas9 technology. This is accompanied by the opportunities, as well as the challenges of using genetically engineered microalgae for this purpose.

Keywords: CRISPR/cas9; bioremediation; genetic engineering; microalgae; produced wastewater.

FIGURE 1

Simplified workflow for ZFN, TALEN and CRISPR/Cas9 mediated microalgae genome editing. (A) ZFN, (B) TALEN and (C) CRISPR/Cas9. The 3 tools have 4 steps (1) using bioinformatic tools to identify the targeted sequence and design accordingly, (2) construct the delivery method by choosing the vector, (3) select the delivery method to insert the plasmid into the microalgae cell, (4) subculture the microalgae on a selective medium and determine the efficiency of gene editing.

FIGURE 2

Genetic engineering tools-induced genome editing in Microalgae. The double-stranded breaks (DSBs) introduced at the target site by CRISPR/Cas or TALEN or ZFN complexes stimulates the endogenous DNA repair machineries, non-homologous end joining (NHEJ) in the absence of the donor template or the homology-directed repair (HDR) in presence of the donor template. The NHEJ is generally associated with the introduction of insertions and/or deletions (indels) of varying lengths at the DSB site, often leading to the disruption of the reading frame of the target gene. The HDR pathway results in a precise insertion or deletion at the DSB site by homologous recombination. The preferred tool is the CRISPR/Cas9 which is highlighted in red as it is very accurate, easy, and fast.