Abstract

The disinfecting ability of hibiscus seed proteins against Escherichia coli, total coliforms
and faecal coliforms in water treatment was investigated. Hibiscus seeds are widely
available as a primary source of protein in tropical countries. Using varying doses of
extracts, partial inactivation of faecal coliforms and E. coli was achieved. Hibiscus
sabdariffa crude extract (SCE) achieved 67% faecal coliform inactivation whereas Hibiscus
cannabinus (kenaf) crude extract (KCE) achieved 57% using dosages of 200 mg/l.
Additionally, E. coli were observed to be more sensitive to KCE than SCE, with maximum
inactivations of 65% and 60%, respectively. Furthermore, complete bacterial regrowth was
witnessed with the extracts after 72 h storage time. However, purified sabdariffa protein
(PSP) and purified kenaf protein (PKP) showed complete (100%) bacterial inactivation post-
treatment, using a dose of 1·48 mg/l. Additionally, PSP and PKP achieved 92% and 90%
turbidity removal, respectively, from a value of 11·9 NTU. Therefore, the use of PSP and
PKP in water treatment could improve access to clean water in developing countries.
Introduction
Water treatment involves the processing of raw water to improve its quality to meet
prescribed standards for domestic, industrial and commercial uses. It is strategically
employed to prevent the ingestion of harmful contaminants (Hyung and Kim, 2009).
Traditional water treatment processes employ dis- infection processes to eliminate bacteria
and viruses in water, using chlorine-based compounds (Farré et al., 2012), ozone and
ultraviolet light, and the health benefits are well- documented and understood (Orme et al.,
1990). Alvarez and O’Brien (1982) and Pyle et al. (1992) proposed the use of some halogens
such as iodine and bromine in public water supplies, but these chemicals have not yet
received acceptance in the water industry. However, water processing in disinfection units is
not utilized fully in developing countries because of cost issues associated with the
procurement and import of disinfecting chemicals. The absence of these critical units has
increased the vulnerability of many rural communities in developing countries to waterborne
disease and death.
H. cannabinus (also known as kenaf) is a member of the Malvaceae family. It is an annual
herbaceous plant that grows under a wide range of weather and climatic conditions (Meints
and Smith, 2003). It is widespread in most of the tropics and is found in Ethiopia,
Zimbabwe, Mozambique and Uganda (Katende et al., 1999). The seed capsules are fine,
loose and hairy structures that contain five segments with a total of 20–26 seeds per capsule
(Dempsey, 1975). Kenaf is a multi-purpose and versatile crop used for energy and natural
fibre in indus- trial applications (Meera and Agamuthu, 2012). The rich fibre content of the
plant offers a perfect material for cordage, rope making and paper manufacture.
Materials and methods

Seed collection
Both kenaf and sabdariffa seed samples were obtained from a local market in Nigeria. The
seeds were harvested from mature plants and the seed kernels were manually removed from
the seedpods/capsules and then washed with tap water in the lab- oratory to remove any
surface contamination. The seed was ground into a fine powder for 2 min using a Tema
laboratory disc mill. The ground seed powders were then sieved in a set of sieves arranged
in descending order. The powders retained in 212 μm and 300 μm sieve sizes were
combined, thoroughly mixed and used to prepare suspensions.

Chemicals and reagents

Analytical-grade sodium chloride (NaCl), used in extracting crude seed and the elution of
purified proteins, and aluminum sulfate, employed in conducting control tests, were
obtained from Fisher Scientific, UK. Sodium phosphate monobasic monohydrate (Sigma-
Aldrich, Germany) and sodium phosphate dibasic (Sigma-Aldrich, UK) were used in
preparing the buffer solution while 98% hexane was used to wash the column to avoid
blockage. All suspensions were prepared using deionized water.

Preparation and extraction of coagulants

The crude seed extracts were prepared from the ground seed powders following the work
of Jones and Bridgeman (2016a): 1·0 M sodium chloride solutions were added to the
seed powder to make 2% (weight/volume (w/v)) suspensions.

Lipid extraction
The combined seed powders were de-fatted using high-grade hexane in a Soxhlet extractor.
20 g of the ground powder was extracted in the extraction thimble of the apparatus. For
efficient extraction, 2 l of solvent (high-grade hexane) was heated to 60°C. The process was
run continually for 8 h, with each complete cycle taking 2–3 min. The residue from the
extraction thimble was dried overnight at room temperature (19 ± 2°C) and the dried
residue was then ground into a fine powder using a pestle and mortar. The ground oil-free
powder was then employed in the subsequent protein purification process.

Protein purification
Protein purification was conducted according to Jones and Bridgeman (2016b): a 1 ml
HiTrap Q HP anionic ion exchange column (GE Healthcare, Sweden) was used for this
purpose.

Water Sample
River water samples were collected from Bourn Brook, adjacent to the University of
Birmingham train station, in a set of 1 l sterilised plastic containers. In total, 72
representative samples from six locations across the river (i.e. 12 1 l samples from each
location) were collected for the experiments. The collected water (72 l) was then emptied
into a single sterilised plastic bucket and the low-turbidity water was allowed to settle
naturally before any tests were conducted. The water sample was kept in a refrigerator at 4°C
and tests were conducted within 4 h of collection.
Jar test experiments
To evaluate the optimum coagulant dose for the removal of bacteria in water, jar tests were
carried out using conventional apparatus (7790-900B, Phipps and Bird, USA) in triplicate,
on six 1 l beakers (Jones and Bridgeman, 2016a).

Microbial analysis
Microbial analysis was performed using Colilert-18/Quanti- Tray (Idex Inc., UK) for the
detection of total coliforms, faecal coliforms and E. coli, following Jones and Bridgeman
(2016a). This method was selected because of its ease of operation, flexibility, accuracy and
speed.

Preparation of aluminium sulfate as a coagulant

For use as a coagulant, 2 g of aluminium sulfate was prepared by dissolving 2% w/v in 100
ml of deionised water. The suspension was mixed thoroughly using a magnetic stirrer
(Stuart Scientific, UK) for 10 min to obtain the coagulant.

Result
The inactivation of total and faecal coliforms and E. coli was investigated with extract doses
range from 50–200 mg/l. Figures 1 and 2 show the inactivation of faecal coliforms and
E. coli in raw water using SCE and KCE. At the lower dosage, the inactivation potential of
sabdariffa between the triplicate values showed a standard deviation of 0·82 while that of
kenaf was 0·52. At the higher dosage, the standard deviations between the triplicate results
were 1·2 and 1·5 for sabdariffa and kenaf, respectively.

Fig 1: Inactivation of faecal coliforms in water

treated with SCE and KCE
 Fig 2: Inactivation of E. coli in water treated with
SCE and KCE

Conclusions
A major conclusion of this work is that microbial analysis revealed some disinfecting
properties in the extracts even though they did not produce complete inactivation of the
microbial population. As anticipated, protein purification resulted in complete inactivation
of total coliforms using PSP and PKP at a reduced dosage than that of aluminium sulfate
under the same coagulation conditions. Most of the microbial population present in the raw
water showed significant sensitivity to the purified protein. Similarly, examination of the
two most significant indicator organisms faecal coliforms and
E. coli revealed excellent inactivation performance by both PSP and PKP. While partial
inactivation of total coliforms, faecal coliforms and E. coli was observed in the water
treated with crude samples, significant regrowth witnessed after 72 h storage time could be
due to the presence of residual organic matter in the extract that might have been used up
as substrate by partially inactivated organisms to aid their growth. Interestingly, however,
the purified samples showed robust performance against faecal coliforms and E. coli even
after pro- longed storage. In addition, after resuspension of the sludge, complete
inactivation of these organisms was still observed, indicating that both E. coli and faecal
coliforms are highly sensitive to the antioxidants. It is also likely that residual protein in
the treated water could disinfect secondary microbes in the treated water during the 72 h
storage period.