1. Introduction
Rice is the second most widely grown cereal, and it is eaten by the majority of the world’s population [
1]. Rice is a staple food for Malaysians, and it is estimated that Malaysians consume nearly 3 million tons of rice per year [
2]; the figure is expected to increase due to the continual rise in population. As a conservative estimate, washing every 1 kg of rice grains with at least 1 L of water would work out to 3 billion L of washed rice wastewater produced yearly. Rice is usually washed to remove dust and dirt before cooking and the water after the rice is washed (hereafter referred to as washed rice water or WRW) is often discarded into the environment. However, rice washing can also remove a significant amount of water-soluble nutrients from the rice. Many studies, as reviewed by Juliano [
3], have shown that rice washing can reduce Ca, P, Mg, K, protein, crude fat, crude fiber, thiamine, riboflavin, and niacin by up to 26%, 47%, 70%, 41%, 7%, 65%, 30%, 59%, 26%, and 60%, respectively, through leaching from the rice. These leached nutrients could be used as a liquid plant fertilizer and soil amendments.
Rather than being merely discarded, unused, into the environment, WRW ought to be reused as part of water governance. By the 2050, the global freshwater demand is expected to increase by 55% [
4]. This increase is largely caused by the detrimental climate change and continuous increase in world population, driving WWAP [
5] to advocate for more effective water governance so that wastewater, rather than just being discarded into the environment, is instead reused. Furthermore, the AQUASTAT database of the FAO of the United Nations estimates that more than half of the global freshwater withdrawals are simply discarded as wastewater into the environment [
5]. Only 11% of the global freshwater withdrawal corresponds to municipal water demand, of which only 3% is consumed, while the remaining 8% is simply discarded as wastewater. Therefore, as part of water governance, WRW, like any other wastewater, ought to be reused. The practice of reusing WRW can potentially lead to considerable savings in water, as well as fertilizer use.
WRW is often claimed to be a beneficial plant fertilizer and soil amendment due to the leached rice nutrients in the WRW, but claims of WRW’s benefits are without strong scientific evidence. Nabayi et al. [
2] reported that scientifically rigorous studies to determine the benefits of reusing WRW for agriculture are severely lacking. Nabayi et al. [
2] found only 41 papers on WRW studies, only 10% of them were published in citation index journals. Out of these citation index journals, only about 3% were on microbes. The nutrient contents of WRW (ethanol, P, N, and S) are reported to increase with fermentation for 6 days [
6]. Similarly, Nurhasanah et al. [
7] reported that WRW was a better medium than the popular nutrient-rich Luria-Bertani (LB) broth to promote the growth of
Pseudomonas fluorescens, a plant growth-promoting rhizobacteria (PGPR). Several studies also reported WRW to support the growth of beneficial bacteria
Rhizobium,
Azospirillum,
Azotobacter,
Pseudomonas fluorescence,
Lactobacillus, and
Bacillus, as well as beneficial fungi
Trichoderma,
Penicillium, and
Saccharomyces [
6,
8,
9].
This study aimed to determine the effect of fermentation periods on the macro- and micronutrient contents of WRW and whether (and to what degree) fermentation of WRW promotes the growth of beneficial soil bacteria, particularly N-fixing and P- and K-solubilizing bacteria, as well as catalase- and IAA-producing bacteria. The identification and the biochemical characterization of the bacteria in the WRW is necessary to determine if WRW could potentially increase soil health by introducing beneficial soil bacteria [
2]. To our knowledge, this could be the first study to report how the fermentation affects both the micro- and micronutrient content of the WRW. Sairi et al. [
9] only morphologically identified the microorganisms in the WRW, but this study identified the bacteria in the WRW using gene sequencing. Therefore, the objectives of the study were (1) to determine the potential of fermented WRW to fix N, solubilize P and K, and produce catalase and IAA; (2) to isolate, identify, and test the bacteria present in the fermented WRW for catalase and IAA potential; and (3) to determine the effect of fermentation periods on nutrient content of WRW.
2. Materials and Methods
The rice grain used in the study is ‘Rambutan’ (Padiberas Nasional Berhad, Malaysia) because of its popularity and availability in Malaysia. For washing the rice, distilled water was used with a mixer (Bossman Kaden matte BK-100S, Tokyo, Japan) at 80 rpm (0.357 g Force) for 90 s to ensure uniformity and repeatability in washing. After washing the rice, the mixture was sieved (500-micron sizes) to separate the rice grains from the WRW, after which the WRW was allowed to ferment for 0 (unfermented), 3, 6, and 9 days. These fermentation periods are labeled as F0, F3, F6, and F9, respectively. The fermentation was carried out indoors, under room temperature, and without additives in the Soil Physics Laboratory, Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia. After the rice was washed at the required water to rice ratio, the resultant WRW was kept in a series of plastic vials for predetermined periods of 3, 6, and 9 days to allow for fermentation, while the freshly prepared WRW was used as the 0-day fermented WRW. The WRW culture was aerated daily for 20–30 min by opening the vials caps throughout the fermentation period.
2.1. Chemical Analyses
Both the rice grains and the WRW at different fermentation periods were subjected to chemical analyses in addition to the pH and EC measurements. C, N, and S were analyzed by CNS analyzer (LECO Corp., St. Joseph, MI, USA). P, K, Ca, Mg, Cu, Zn, and B were analyzed using AAS (Perkin Elmer, PinAAcle, 900T, Waltham, MA, USA), after dry-ashing in the case of the rice grains [
10]. Ammonium and nitrate were determined by the Kjeldahl procedure [
11]. pH and EC were measured using a pH and EC lab meter (Metrohm, 827, Riverview, FL, USA) [
12].
2.2. Culture Media Preparation and Bacterial Population
Tryptic soy agar (TSA) was used to determine the bacterial population in the various fermented WRW samples, following the method of Tan et al. [
13]. After the inoculation of the WRW culture, plates were incubated for 24 h at 33 °C. The bacterial population was counted from each fermented WRW type (in triplicates). Each plate that has a range of 30 to 300 colonies was selected and counted as colony-forming units (CFU) per mL of sample [
14].
2.3. N Fixation, Phosphate, and Potassium Solubilization Ability of the WRW Culture
The N fixation was determined qualitatively by growing the WRW on Nfb medium (N-free solid malate medium) following Döbereiner and Day [
15]. The quantitative assessment of the N fixing bacteria were determined using the Acetylene Reduction Assay (ARA) method to quantify the N fixation rate of the WRW culture [
16,
17].
The qualitative phosphate solubilizing ability was determined by observing a halo zone around the colony (10 μL inoculum, 1 × 10
8 CFU mL
−1) after 24 h of incubation [
18]. The phosphate solubilizing index (PSI) of the fermented WRW culture was determined as outlined in Sitepu et al. [
19]. The vanadomolybdophosphoric acid method was used to quantify the amount of soluble phosphate in the culture supernatants of NBRIP (National Botanical Research Institute’s Phosphate) broth [
20,
21]. A standard curve was prepared using a stock solution containing a mixture of KH
2PO
4 and 5 mL of concentrated H
2SO
4, making up to 1 L using distilled water. Then, 10 μL of each WRW culture suspension was inoculated into each NBRIP broth; 100 mL of NBRIP culture medium in a flask without inoculum served as the control. The flasks were continuously incubated for 12 days (i12) at room temperature under constant agitation at 100 rpm, as outlined in Tan et al. [
13]. The pH of each culture medium was also checked accordingly at 6 and 12 days (i6 and i12). The assessment of the solubilized phosphate of the culture was performed at two different times (after i6 and i12 of incubation). Then, 25 mL of each culture medium was transferred to 50 mL tubes and centrifuged at 8000×
g for 20 min at each assessment time. Next, 2.5 mL of the supernatant was transferred into a 50 mL beaker, followed by the addition of 20 mL of distilled water. Afterwards, 2.5 mL of Barton’s reagent [
22] was added quickly for mixing action, which was allowed for color development for 10 min. The absorbance was determined calorimetrically using a spectrophotometer at 430 nm.
Aleksandrov agar medium was used to determine the qualitative potassium solubilization [
23] of the WRW culture. The quantitative assessment of the potassium solubilization rate was determined based on the ability of the bacteria to release K from the supplemented muscovite mica in the media. For the procedure, 1 mL of an overnight culture of fermented WRW was inoculated into 100 mL of Aleksandrov broth. The quantity of K released in the broth was measured at three different days after incubation (i5, i10, and i15) from three flasks’ replicates as outlined in Tan et al. [
13]. Centrifuging the incubated broth cultures at 10,000 rpm for 10 min was carried out to separate the supernatant from the muscovite mica and bacterial cells. Then, 1 mL of the supernatant was transferred into a 50 mL volumetric flask and the volume was gradually increased to 50 mL using distilled water and mixed thoroughly. The available K content in the supernatant was measured by Atomic Absorption Spectrometer (AAS) (Perkin Elmer, PinAAcle, 900T, Waltham, MA, USA).
2.4. Production of Indole Acetic Acid (IAA)
The ability of the fermented WRW and the bacterial isolates to produce IAA were determined following Gordon and Weber [
24]. The isolates and WRW culture were grown in Tryptic Soy Broth (TSB) in a conical flask for 3 days in a shaker incubator at 30 °C. Next, 1 mL of a fully grown of either WRW culture or an isolate was transferred into a fresh 100 mL of TSB containing 5 mL of tryptophan (2 μm sterilized Whatman No. 2 filter paper). The mixture was incubated for an additional 24 h in a shaker incubator. Afterwards, 1.5 mL of the mixture was pipetted into Eppendorf tube and centrifuged at 8000 rpm for 15 min. Then, 1 mL of the supernatant was transferred into a new tube and 2 mL of Salkowski’s reagent (150 mL of concentrated H
2SO
4, 250 mL of distilled H
2O, 7.5 mL of 0.5 M FeCl
3·6H
2O) [
25] was added and mixed vigorously and incubated for 25 min. Quantitatively, the IAA was ascertained using a spectrophotometer at 535 nm. A standard was prepared using pure IAA at 0, 5, 10, 20, 30, 40, 50, and 100 ppm. The quantity of the IAA produced was estimated from the standard curve.
2.5. Catalase Test
The isolates and the fermented WRW culture were tested for their ability to produce catalase enzyme following Khalifa et al. [
26]. Hydrogen peroxide (5%) was added drop by drop to an aliquot of an incubated cultures from either WRW or isolate culture (after 24 h of incubation) after spreading on a clean glass slide. Positive results were indicated when gas bubbles evolved within a few seconds.
2.6. Bacterial Isolations
Following the bacterial growth and population count, a direct spreading method was used to isolate the different bacteria from different samples (fermentation period) based on shape, color, and size [
27].
2.7. Bacterial Identification Using 16S rRNA Gene Sequence
Bacterial inoculum from overnight streaked culture plate was re-cultured overnight in nutrient broth. Genomic DNA was isolated from the bacterial culture (WRW) by using the Genomic DNA Mini Kit (Favorgen) (Pingtung Agricultural Biotechnology Park, Pingtung, Taiwan). Thereafter, the DNA was stored at −20 °C for further analyses. The 16S rRNA gene was amplified using universal forward 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and reverse 1492R (5′-GGTTACCTTGTTACGACTT-3′) primers (Apical Science Sdn. Bhd., Seri Kembangan, Selangor, Malaysia). Then, 30 μL reaction mixture was prepared each containing 2 μL of DNA template, 15 μL of Master mix (containing 10X PCR Reaction Buffer, dNTPs mix, Taq polymerase, MgCl2, and ultra-pure water), 10 μL of Nuclease free water, and 1.5 μL each of forward and reverse primers. PCR reactions, were carried out using a thermal cycler (MJ Mini Personal Thermal Cycler, Bio-Rad, Hercules, CA, USA) with the following cycles: denaturation for 4 min at 95 °C, 45 s at 95 °C, 45 s at 58 °C for annealing, 1 min at 72 °C for initial extension, and final extension for 10 min at 72 °C. The amplified 16S rRNA gene was purified with a Gel/PCR DNA Fragments Extraction Kit (Favorgen) and outsourced for sequencing (Apical Scientific Sdn. Bhd., Selangor, Malaysia). The sequenced data were aligned and analyzed to identify the bacterium and its closest neighbors using BLAST (NCBI, Bethesda, Rockville, MD, USA). The sequences obtained for selected bacterial isolates were manually analyzed using Sequence Scanner Software v1.0 by Applied Biosystems® (Forster City, CA, USA). The interpretation of the sequences was performed by comparing them with information in the BLAST database online (NCBI, Bethesda, MD, USA).
2.8. Sequence Submission and Phylogenetic Analyses
The partial 16S rRNA gene sequences of the identified strains in this study were deposited in GenBank database (
http://www.ncbi.nlm.nih.gov/GenBank/index.html) accessed on 15 December 2020, as reported by Nabayi et al. [
28]. The sequence was further used to construct a phylogenetic tree using the Maximum Likelihood method. All the 16S rRNA gene sequences were aligned using ClustalW2 with the most closely related bacteria sequences obtained from the NCBI database using the MEGA software version 7.
2.9. Data Analysis
All data obtained were subjected to analysis of variance using the general linear model (GLM). The treatment means were separated by the Honest Significant Difference (HSD) test at 5% with Minitab (version 19) software package (Minitab, LLC, College Town, PA, USA). All treatments were in triplicate unless otherwise stated.
5. Conclusions and Recommendations
Fermented WRW after 3 days had a greater bacterial population of 2.12 × 108 which decreased with a longer fermentation period. The greater bacterial population led to more IAA, N fixation, and P and K solubilization, which increased by 13.2–85.5%, 9.4–83.3%, 22.4–84.1%, and 21.4–83.6%, respectively, compared to other fermentation periods. Similarly, the study showed an increase in nutrients with an increase in fermentation, indicating the presence of plant beneficial microorganisms such as N-fixing and P- and K-solubilizing bacteria in WRW. The isolation and identification showed the presence of Bacillus subtilis and Enterobacter spp., among others, that are N-fixing and P- and K-solubilizing microorganisms, and biocontrol agents. Fermenting WRW for 3 days produced greater diversity of beneficial microbes. This study showed that WRW, rather than being discarded, can be reused, as the nutrient and microbial analyses showed the presence of nutrients and beneficial bacterial strains that could promote plant growth and yield and soil fertility. Therefore, it is recommended that WRW should be fermented for 3 days before its use as plant fertilizer for more beneficial microorganisms and plant nutrients.