Elsevier

Process Biochemistry

Volume 110, November 2021, Pages 26-36
Process Biochemistry

Combine strategy of treated activated charcoal and cell surface protein curli induction for enhanced performance in Escherichia coli immobilization

https://doi.org/10.1016/j.procbio.2021.06.012Get rights and content

Highlights

  • NaOH treatment increased surface area, pore volume and macropore size.

  • NaOH-treated activated charcoal increased cell adsorption by >120 %.

  • Production of curli enhanced the adsorption of E. coli by 50 %.

  • NaOH treatment and curli induction together increased overall cell adsorption by >160 %.

Abstract

Immobilization of Escherichia coli (E. coli) on commercial activated charcoal was enhanced by mild chemical treatment coupled with curli production from E. coli. The chemical used to treat the activated charcoal were sodium hydroxide, hydrochloric acid, ammonium hydroxide, and acetic acid while nickel (II) chloride was used to promote the production of curli. Characteristics of the activated charcoal before and after chemical treatments were analyzed including its surface properties, pore size, and crystalline structure. The immobilization of E. coli was enhanced greatly after sodium hydroxide treatment which gave rise to more than 120 % cell immobilized compared to the untreated activated charcoal which was mainly attributed to the larger size of macropore, surface area, and pore volume. Curli were produced by the induction of nickel (II) chloride and further enhanced the cell immobilization by at least 50 %. Overall, the combine strategy enhanced cell immobilization by more than 160 %. The resulting biocatalyst from the enhanced cell immobilization managed to be reused up to 10 cycles for the enzyme cyclodextrin glucanotransferase expression while retaining up to 60 % of the enzyme’s initial activity.

Introduction

Activated carbon (AC) is an interesting and versatile material in a way that it can be used for a wide range of applications such as for storage of gasses [1], removal of dyes and metal ions [2], and even for medical purposes [3]. However, AC is used most commonly as an adsorbent for the removal of varying pollutants from various matrices due to their naturally high adsorption capability. The high adsorption capability of AC is mainly contributed by the complex and heterogeneous structure obtained through its activation process. Carbonaceous material can be activated either physically or chemically [4]. The activation process will cause an abundance of porous structures, high surface area, and varying chemical structures on the surface to be formed [5]. These characteristics, however, may depend on the raw material used for producing the AC.

Modification of AC can be done to modify or enhance its existence characteristic. The most common modification done to AC is physical and chemical modification. Physical modification usually involves the use of extremely high heat and this type of modification solely increases the pore volume and surface area of the AC [6]. Chemical modifications usually are divided into acidic and basic treatment. Acidic treatment may change the character of AC by introducing the acidic surface functional group to the AC [7]. The changes will increase the uptake of inorganic species such as metal ions. Basic treatment will introduce basic functional groups to the surface of AC and this helps adsorb negatively-charged species, usually of the organic species such as phenol [8]. Both of these treatments may give varying results on the surface area and pore size of AC. Another less common modification of AC is the biological modification in which involves the process of bioadsorption. This bioadsorption process involves the attachment of bacteria to AC via the immobilization process [6]. Bioadsorption or the immobilization of bacteria on AC has been done in previous studies and mostly to remove chemical species from an aqueous solution such as lead [9], nitrite [10], and phenol [11]. The porous structure of activated charcoal can provide a protective environment for the bacteria from fluid shear forces. On top of that, the presence of varieties of functional groups on the surfaces of activated charcoal will aid in bacterial attachment.

Immobilization is a general process that involves the process of attachment or entrapment. Other than bacteria, the immobilization process can also be applied to varieties of biocatalysts including enzymes, and even to animal and plant cells [12]. Immobilization of bacteria came about as an alternative for enzyme immobilization. With the immobilization of bacteria, which often involves enzymes produced on the surface of the bacteria or expressed extracellularly, the long and expensive procedure for enzyme separation and purification can be eliminated. Moreover, this process also enables the procurement of products and the recovery of the catalyst with ease. Thus far there have been four established methods for bacteria immobilization: covalent bonding or cross-linking, encapsulation, entrapment, and adsorption [13]. Covalent bonding or cross-linking involves the formation of covalent bonds between the support material and cell in the presence of a binding (cross-linking) agent. Encapsulation and entrapment are both an irreversible immobilization where the bacteria are confined in the support material, usually in a form of a capsule for encapsulation and support material made such as agar and alginate for entrapment. Adsorption is the simplest method of reversible immobilization and was the first example of cell immobilization. This type of immobilization involves physical interaction between the bacteria and the surface of the support material which may include the interaction between forces such as van der Waals forces, electrostatic and hydrophobic interaction as well as hydrogen bonds [14]. These immobilization methods have their very own advantages and disadvantages and their selection should be based on individual needs and criteria.

Apart from the removal of chemicals from aqueous solution, immobilizing bacteria to AC, which essentially is the creation of a whole-cell biocatalyst, could offer great potential in other applications such as protein expression. Protein expression from an immobilized cell has been studied previously [15], but none has been done using AC. AC can be utilized as a candidate for such a task because AC, apart from its unique adsorptive capability, can be obtained in abundance at a low cost, is structurally and thermally stable as well as having resistance towards microbial degradation [16]. Moreover, AC is an inert material that will not impair the growth of bacteria and preserves their physiological activity [12]. It should be noted that the term modification to AC, in this paper represents treatment applied to as-received commercial activated charcoal after activation. Modification of the bacteria’s surface involves the manipulation of curli. Curli are a type of fimbriae that are thin and aggregative produced by many Enterobacteriaceae including Escherichia coli (E. coli) [17] and Salmonella typhimurium [18]. Curli constitutes the major protein component of the extracellular matrix and they possess multiple functions including cell adhesion and biofilm formation, host cell invasion, immune system activation, and protection from environmental stresses. The curli expression may be affected by several environmental signals and the presence of certain chemicals including temperature, osmolarity, and salt. However, curli are mostly expressed during the stationary phase and at a low temperature of below 30 °C, although there are a few exceptions of strains that can grow curli at 37 °C [19]. Enhancing the production of curli may increase the attachment of bacteria to the AC. The main objective of our research is to produce a whole-cell biocatalyst for enzyme expression. A whole-cell biocatalyst is the utilization of a whole cell, which usually involves the immobilization of the cell to certain matrices, in order to produce product through a biotransformation process [20]. To the best of the author’s knowledge, combining the treatment of AC and manipulation of curli to enhance E. coli immobilization to AC has not been done yet. Therefore, this study focuses on the modification of AC using chemical treatments to improve its physiochemical properties for enhanced E. coli immobilization. In addition to that, the surface of E. coli was manipulated by producing curli using nickel additive during the immobilization process to further enhance the cell attachment. The combination of the former and later methods proved to increase E. coli immobilization significantly, leading to better reusability in enzyme expression and cell viability.

Section snippets

Bacterial strain and media

Bacteria strain of E. coli BL21 (DE3) harboring the enzyme cyclodextrin glucanotransferase (CGTase) (EC 2.4.1.19) was obtained from Genetic Engineering Lab, Universiti Teknologi Malaysia [21]. The medium used to grow the bacteria was 2xYT (16 g/L tryptone, 10 g/L yeast extract, and 5 g/L NaCl). Agar plate was prepared using the Luria-Bertani agar plate (10 g/L tryptone, 10 g/L NaCl, 5 g/L yeast extract, and 15 g/L agar).

Activated carbon and its characterization

The activated carbon used in this study is a commercial untreated activated

Elemental analysis

The UTAC was subjected to 10 M of NaOH, HCl, ammonium hydroxide, and acetic acid. Table 1 summarizes the elemental analysis and ash content of the UTAC before and after the chemical treatments. All the chemical treatments have decreased the oxygen content in the AC with N-AC showing the lowest oxygen content of 13.3 %, followed by Aa-AC, H-AC, and Am-AC with 14.9 %, 15.0 %, and 15.2 %, respectively. The removal of oxygen content from an activated carbon could happen when an elevated temperature

Conclusion

The enhancement of the immobilization process can be attributed to the treatment of AC causing larger surface area and pore volume, retaining relatively high ash content, increasing the difference in surface charges between E. coli and support material, and an increase in hydrophobicity. All these factors provide favorable conditions for the adherence of E. coli while modification of the bacteria surface by promoting the production of curli further enhanced the immobilization process.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the Ministry of Education Malaysia (KPM) and Universiti Teknologi Malaysia (UTM) through the Collaborative Research Grant (grant number Q.J130000.2409.04G82).

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