WO2019050389A1

WO2019050389A1 - Bioflocculant and biosorbent derived from dead cells of marine filamentous bacterium for heavy metal and waste material removal

Info

Publication number: WO2019050389A1
Application number: PCT/MY2018/050025
Authority: WO
Inventors: Go FURUSAWA; Pui Nyuk CHIANG; Shu Chien @ Alexander CHONG; Amirul Al-Ashraf Balakrishnan ABDULLAH
Original assignee: Universiti Sains Malaysia (USM)
Current assignee: Universiti Sains Malaysia (USM)
Priority date: 2017-09-06
Filing date: 2018-05-02
Publication date: 2019-03-14
Anticipated expiration: 2020-03-06
Also published as: MY188726A

Abstract

The present invention relates to a method of producing an article suitable for use as a bioflocculant or a biosorbent for waste material and heavy metal removal. The method preferably comprises the steps of growing cells of a marine filamentous bacterium in a culture medium to achieve a log phase of bacteria growth (100); harvesting by way of separating the said cells of marine filamentous bacterium from the culture medium (200), wherein the step of harvesting includes centrifugation; and fixing the harvested cells of marine filamentous bacterium by subjecting the same to treatment with formaldehyde (300), wherein the said article comprises the fixed cells of marine filamentous bacterium.

Description

BIOFLOCCULANT AND BIOSORBENT DERIVED FROM DEAD CELLS OF MARINE FILAMENTOUS BACTERIUM FOR HEAVY METAL AND WASTE

MATERIAL REMOVAL FIELD OF THE INVENTION

The present invention relates generally to flocculation and biosorption. More particularly, the present invention relates to a method for producing an article suitable for use as a bioflocculant or a biosorbent for waste materials and heavy metals removal.

BACKGROUND OF THE INVENTION

Many environmental and health problems come from the presence of metals in surface water. Flocculants including inorganic flocculants such as aluminum sulfate and organic polymer flocculants such as polyacrylamide are widely used in the industrial water and sewage treatment, drinking water purification and fermentation industries for removal of heavy metals and other contaminants. These flocculants are commonly applied in the said industries because of their effective flocculation and low production cost. However, there are growing concerns on their toxicity and poor biodegradabiiity.

Bioflocculants which are essentially biological polymers, mainly exopolysaccharides, produced by microorganisms during their growth have attracted wide interest because of their harmlessness and excellent biodegradabiiity. The bioflocculants or biosorbents will be particularly useful for treatment of heavy metal pollution. However, in almost all cases, it is necessary for centrifugation (Huang and Liu, 2013) or filtration (Vijayaraghavan and Yun, 2008, Kinoshita et al., 2013) of absorbent materials to remove heavy metals. For example, a heavy metal biosorbent using yeast cells required centrifugation (2800 rpm, 5 min) to remove yeast cells absorbing heave metals, see Chinese Patent Application No. 101229506 A. Moreover, a long incubation time was also necessary for absorbing heavy metals as mentioned in the United States Patent Application Publication No. 2008/0009054 A1 and Chines Patent Application No. 101229506A. This means that a large physical force or action is required to remove heavy metal and is being one of limiting factors for application of previous bioflocculants or biosorbents.

Several bioflocculant-producing bacteria belonging to genera Bacillus (Shu et al., 1997, Deng et a!., 2003, Zheng et al., 2008), Corynebacterium (He and Chen, 2004), Kelbsiella (Wang et al., 2007), Paenibacillus (Li et al., 2013), Proteus (Xia et al., 2008) and Pseudomonas (Lin and Harichund, 201 1 ,Azzam and Tawfik, 2015) were found during the few past decades. Although there is a lot of studies on bioflocculant is reported, its high production cost including expensive substrates, such as sugar, phosphate and some other components, are limiting factors of its practical application. Recently, to solve this problem, various wastes including starch, corn stover (Wang et al., 2013) and rice stover (Guo et al., 2015) are used as cheaper carbon sources. Either way, the complicated purification steps containing two-stage fermentation, precipitation process required large volume of ethanol and drying process are also the cause of the high production cost.

A need therefore exists for an improved method for preparing and producing an article suitable for use as a bioflocculant or a biosorbent for waste material and heavy metal removal thereby overcoming the problems and shortcomings of the prior art. Although there are many methods for the same in the prior art, for many practical purposes, there is still considerable room for improvements. SUMMARY OF THE INVENTION

The following presents a simplified summary of the invention in order to provide a basic understanding of some aspects of the invention. This summary is not an extensive overview of the invention. Its sole purpose is to present some concepts of the invention in a simplified form as a prelude to the more detailed description that is presented later.

Accordingly, the present invention provides a method of producing an article suitable for use as a bioflocculant or a biosorbent for waste material and heavy metal removal. The method of the present invention may be characterized by the steps of growing cells of a marine filamentous bacterium in a culture medium to achieve a log phase of bacteria growth; harvesting by way of separating the said cells of marine filamentous bacterium from the culture medium, wherein the step of harvesting includes centrifugation; and fixing the harvested cells of marine filamentous bacterium by subjecting the same to treatment with formaldehyde, wherein the said article comprises the fixed cells of marine filamentous bacterium. Preferably, the said fixed cells are dead cells of marine filamentous bacterium.

Preferably, the said culture medium is a broth comprising tryptone, artificial sea salt and HEPES.

Preferably, the said cells of marine filamentous bacterium are incubated at a predefined temperature, time and rate of shaking.

Preferably, the said harvested cells of marine filamentous bacterium are treated with 0.5% formaldehyde on ice for a predefined period of time.

Preferably, the article is configured for removing kaolin, diazo dye, Congo red and metal ions including Fe³⁺ and Cu²'. In accordance with another aspect of the present invention, an article suitable for use as a bioflocculant or a biosorbent for waste material and heavy metal removal as produced by the above-identified method is provided.

It is therefore an advantage of the present invention that employs dead cells of marine filamentous bacterium in the preparation and production of bioflocculant and biosorbent.

It is therefore another advantage of the present invention that the article produced thereof can be used as a bioflocculant on kaolin and a biosorbent on a diazo dye, Congo red, and heave metals, such as Fe ⁺ and Cu²⁺. It is therefore yet another advantage of the present invention that the method can be easily implemented in a highly specific and compact, cost- effective, quick and simple manner, without the use of complicated and sophisticated steps and components.

The foregoing and other objects, features, aspects and advantages of the present invention will become better understood from a careful reading of a detailed description provided herein below with appropriate reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendant advantages thereof will be readily as the same becomes better understood by reference to the following detailed description when considered in connection with the accompanying drawings, wherein:

Figure 1 illustrates a flow diagram depicting the steps involved in a method of producing an article suitable for use as a bioflocculant or a biosorbent for heavy metal and waste material removal according to one embodiment of the present invention;

Figure 2 plots removal rate of kaolin with living cells, fixed cells and heated cells according to one embodiment of the present invention;

Figure 3 plots removal rate of Congo red using dead cells by fixation into 20 mM Ca²⁺ and 50 mM Ca²⁺ solution according to one embodiment of the present invention; and

Figures 4a and 4b respectively show the curve of Cu²⁺ and Fe³⁺ absorption using dead cells by fixation, where the concentration of each metal ion is 25, 50 75 and 100 ppm according to one embodiment of the present invention. It is noted that the drawings may not be to scale. The drawings are intended to depict only typical aspects of the invention, and therefore should not be considered as limiting the scope of the invention. In the drawings, like numberings represent like elements between the drawings.

DETAILED DESCRIPTION OF THE INVENTION

The present invention essentially relates to the use of dead cells of marine filamentous bacterium in the production of bioflocculant and biosorbent for removal of waste materials and heavy metals. Particularly, the said bioflocculant and biosorbent each is effective on kaolin, diazo dye, Congo red and heavy metals such as Cu²⁺ and Fe³⁺. The present invention advantageously describes the easy method of preparation of the dead cells as bioflocculant and biosorbent. Some marine filamentous bacteria belonging to the family Saprospiraceae show cell aggregation to capture its prey (Furusawa et al., 2003, 2015). The filamentous cell morphology and the production of extracellular polysaccharides (EPS) in these bacteria play a critical role in the cell aggregation. Besides that, the cell aggregation may be rapidly formed by adding calcium ion (Furusawa et al., 2015). This means that these bacteria are useful for bioremediation of wastewater.

It is an objective of the present invention to resolve the problems of high production cost and the complicated process for removing waste materials and heavy metals.

Accordingly, the present invention provides a method of producing an article suitable for use as a bioflocculant or a biosorbent for heavy metal and waste material removal. It is preferred that the said novel bioflocculant comprises of dead-cells of filamentous bacterium and is configured for removing kaolin, diazo dye, Congo red and heavy metals including Fe³⁺ and Cu²⁺. It is also not necessary to centrifuge for removing kaolin, Congo red and Fe³⁺ and Cu²⁺.

The marine filamentous bacterium may be isolated from a coastal area by using a method as described in Furusawa et al. (2015). The method of the present invention preferably comprises few steps as shown in Figure and begins with step 100 At this step 100, the cells of marine filamentous bacterium shall be grown or cultured in a culture medium to achieve a log phase of bacteria growth. The culture medium is preferably a broth comprising tryptone, artificial seas salt and HEPES. The strain cultured in the above broth or artificial seawater medium demonstrates bioflocculant activity. The cells of marine filamentous bacterium are incubated at a predefined temperature, time and rate of shaking.

Subsequently, the method proceeds to step 200 of harvesting by way of separating the said cells of marine filamentous bacterium from the culture medium. It is preferred that the step of harvesting includes centrifugation. Other separation processes however may be adopted to achieve the effect of the present invention.

Following the step 200, the method executes step 300 for fixing the harvested cells of marine filamentous bacterium by subjecting the same to treatment with formaldehyde. Essentially, the harvested cells of marine filamentous bacterium are treated with about 0.5% formaldehyde on ice for a predefined period of time. Preferably, the said article comprises the fixed cells of marine filamentous bacterium. More preferably, the said fixed cells are dead cells of marine filamentous bacterium. The bioflocculant activity of the dead cells prepared using 0.5% formaldehyde is almost similar to that of the living cells.

Based from the above-identified steps, the novel article including bioflocculant and biosorbent of the present invention advantageously does not involve any ethanol precipitation and lyophilisation. A compliant embodiment of the present invention will now be described by way of non-limiting examples with reference to the accompanying drawings. EXAMPLE 1

Preparation of Medium

For making a culture medium or a broth, about 2.4% of artificial sea salt (w/v), about 0.5% of tryptone (w/v) and about 10 mM HEPES (pH 7.6) were mixed and topped up to 1 L with distilled water. The culture medium was sterilized by autoclaving at about 121 °C for about 15 min. For agar plates, about 1.5% (w/v) of agar was added to the broth and the medium was autoclaved.

EXAMPLE 2

Bacteria Culture Condition

A few small bacterial colonies grown on the agar plate were inoculated to 100 ml the culture broth. The cells were incubated at about 30 °C for about 18~24 hr with 200 rpm. About 10ml of cell suspension was harvested by centrifugation at 4000 rpm for about 5 min.

EXAMPLE 3

Preparation of Dead Cells

The harvested cells were re-suspended in about 0.5 ml of 10 mM HFPES buffer (pH 7.6) with 7 mM CaCI₂ and added of formaldehyde (final concentration; 0.5%), and the cells were incubated for about 1 hr on ice. The suspension was finally centrifuged by 4000 rpm for about 5 min.

EXAMPLE 4

Res u Its and D i scu ssion

Flocculating Activity

Measurement of Flocculating Activity

Dead cells from 10 ml cell suspension were added to 10 ml of 0.2% of kaolin solution with 10 mM HEPES (pH 7.6) and 7 mM CaCI₂ in 50 ml flask. Living cells were used as positive control and the samples without CaCI₂ and heat- treated cells were used as negative controls. The flask was shacked for 1 min at 100 rpm and after stopping the shaking, immediately, 1 ml of the supernatant was carefully removed and transferred to 1 ml cuvette. The absorbance was measured using UV spectrophotometer at a wavelength of 600 nm. The flocculating activity was calculated as follows:

Flocculating activity = (A-B)/A x 100 where A and B are OD 600 (optical density at 600 nm) of the control which is 0.2% kaolin suspension, and samples, respectively.

Results of Flocculating Activity

The result of flocculating activity of dead cells was approximately 88% as well as the sample of living cells while the flocculating activity of the sample without CaCI₂ and the sample using heat-treated cells were about 5% and 0%, respectively.

Biosorption Activity

Measurement of Biosorption Activity on Congo red

Dead cells from 10 ml cell suspension were added to 10 ml of Congo red solution (15 g/ml) with 10 mM HEPES (pH 7.6) and 20 mM or 50 mM CaCI₂ in 50 ml flask. The mixture was incubated for 1 hour at 30°C with shaking (100 rpm). The aggregates were settled down at the bottom of the flask for 1 min, and then, the supernatant was carefully removed and transferred to 1 ml cuvette. The absorbance was measured using UV spectrophotometer at a wavelength of 490 nm. Biosorption activity was calculated as follows:

Biosorption activity = (A-B)/A x 100 where A and B are OD 490 (optical density at 490 nm) of the control, which is 15 Mg/ml of Congo red solution, and samples, respectively. The control is the sample without cells. Results of Biosorption Activity on Congo Red The results of biosorption activity of the sample with 20 mM and 50 m

CaCI₂ were 53.8% and 88.1 %, respectively.

Measurement of Biosorption Activity on Heavy Metals Dead cells from 30 ml cell suspension was added to 30 ml of Cu²* (25, 50,

75 and 100 ppm) or Fe³⁺ (25, 50, 75 and100 pprn) with 10 mM HEPES (pH 7.8) and 7 mM CaCI₂ in 100 ml flask. The mixture was incubated for 1 , 2, 5, 10 and 20 min at 30°C with shaking (100 rpm). The aggregates were settled down at the bottom of the flask for 1 min, and then, 15 ml of the supernatant was carefully removed and transferred to 50 mi Falcon tube. The metal ions remaining in the supernatant were measured using Atomic Absorption Spectroscopy (AAS).

Results of Biosorption Activity on Heavy Metals In the case of Cu²⁺, approximately 90% of Cu²⁺ was removed from 25 and

50 ppm samples after 0 min incubation, and >80% of Cu²⁺ was removed form 75 and 100 ppm samples after 20 min incubation. in the case of Fe³⁺, approximately 90% of Fe³⁺ was removed from 25 and 50 ppm samples after 2 min incubation, and after 20 min incubation, Fe^J+ concentration of all samples was below 1 ppm.

The terms "a" and "an," as used herein, are defined as one or more than one. The term "plurality," as used herein, is defined as two or more than two. The term "another," as used herein, is defined as at least a second or more. The terms "including" and/or "having," as used herein, are defined as comprising (i.e., open language).

While this invention has been particularly shov/n and described with reference to the exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as defined by the appended claims.

Claims

1 . A method of producing an article suitable for use as a bioflocculant or a biosorbent for waste material and heavy metal removal, characterized in that, the method comprising the steps of:

growing cells of a marine filamentous bacterium in a culture medium to achieve a log phase of bacteria growth (100);

harvesting by way of separating the said cells of marine filamentous bacterium from the culture medium (200), wherein the step of harvesting includes centrifugation; and

fixing the harvested cells of marine filamentous bacterium by subjecting the same to treatment with formaldehyde (300),

wherein the said article comprises the fixed cells of marine filamentous bacterium.

2. The method according to Claim 1 , wherein the said fixed cells are dead cells of marine filamentous bacterium.

3. The method according to Claim 1 , wherein the said culture medium is a broth comprising tryptone, artificial sea salt and HEPES.

4. The method according to Claim 1 , wherein the said cells of marine filamentous bacterium are incubated at a predefined temperature, time and rate of shaking.

5. The method according to Claim 1 , wherein the said harvested cells of marine filamentous bacterium are treated with 0.5% formaldehyde on ice for a predefined period of time.

6. The method according to Claim 1 , wherein the article is configured for removing kaolin, diazo dye, Congo red and metal ions including Fe³~ and Cu²\

7. An article suitable for use as a bioflocculant or a biosorbent for waste material and heavy metal removal as produced in Claim 1 .