Background and Objective: Biological flocculant or bioflocculant have been used in aquaculture industry as it can be applied easily and are environmentally friendly. Biofloc technology has been used in aquaculture using bioflocculant concept or mechanisms. This study was aimed to test and applied bioflocculant-producing bacteria isolated from biofloc for flocculation process potentially used as inoculum for rapid formation of biofloc. Materials and Methods: For the purpose of this study, isolated bacteria from the biofloc sample were identified as bioflocculant-producing bacteria using yeast peptone glucose agar as it shows highly mucoid and ropy colonies morphology. From six species of bioflocculant-producing bacteria [(Bacillus infantis (B. infantis), Bacillus cereus (B. cereus), Bacillus safensis (B. safensis), Halomonas venusta (H. venusta), Nitratireductor aquimarinus (N. aquimarinus) and Pseudoalteromonas], there was 64 consortium of bioflocculant-producing bacteria were produced using the sum of combination calculation. These 64 consortium were tested for flocculation activity using kaolin clay suspension method. One-way analysis of variance (ANOVA) with post hoc and Tukey test was used to compare the differences of flocculation activity of each bacterial consortium. Results: Out of the 64 consortium, there were 19 consortium showed more than 80% of flocculation activity. The most consortium that has more than 80% flocculation activity derived from consortiums of single and combination of two bioflocculant-producing bacteria species. Consortium of B. infantis and B. cereus found to have the highest flocculation activity followed by single species of B. infantis and N. aquimarinus which were 94.3, 92.9 and 90.6%, respectively. Conclusion: In this study, each consortium of bioflocculant-producing bacteria produced different types of extracellular polymeric substances thus affecting ability in flocculation activity test. Information on extracellular polymeric substances produced by different consortium of bioflocculant-producing bacteria will potentially to be used as inoculum for rapid formation of biofloc.
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Flocculants have been widely used in almost sectors such as in heavy industry sector, mining sector, environmental sector, beverage sector, agriculture sector and aquaculture sector1-4. There are several types of flocculants that have been used in these sectors such as inorganic flocculants, chemically synthetic flocculants and bioflocculant5. In aquaculture sector, flocculants are one of the most important elements used in waste water management. As aquaculture sector grows, issues on waste water management also arise regarding on discharge of untreated waste water into the natural water bodies. Issues of waste water management concern the researchers, aquaculturist and environmentalist thus comes with the strict regulations on the standard of waste water discharged to the natural6. To overcome the issues, aquaculture sector implements the usage of flocculants for waste water treatment. Uneaten feed, suspended particle, sludge and metabolites waste were main factors contribute to the usage of flocculants to flocculate these unwanted materials.
Back days in aquaculture sector, inorganic flocculants was widely used for waste water treatment such as calcium chloride, magnesium hydroxide, sodium carbonate and sodium hydroxide7. The aim is to purify the waste water according to the standard issues by Department of Environment before discharged to nature or to be used throughout the culture period. Despite to overcome waste water problems, flocculants have been used for another purpose in this sector. By using bioflocculant, new system has been developed to maintain water quality in culture pond, natural food supplement and disease resistance8.
A system is known as Biofloc technology (BFT) was developed by Yoram Avnimelech in 1989 with the concept of a bioreactor for single cell protein production9. It is also known by several names such as Zero Exchanged Autotrophic Heterotrophic System (ZEAH)10,11, microbial floc system12, single-cell protein production system13, suspended-growth system14 and active sludge system15.
Biofloc is the aggregation of algae, fungi, bacteria, diatom, protozoa, fecal and uneaten feed and held by a loose matrix of mucus secreted by bacteria during their growth and bound by filamentous microorganisms or electrostatic attraction. Biofloc formation is closely related to the loose matrix of mucus or extracellular polymeric substance (EPS) secreted by bacteria during their growth16. Different bacteria were producing different EPS during their growth. Certain bacteria producing EPS that able to flocculate organic and inorganic materials in the water body and forming biofloc described as bioflocculant-producing bacteria17. However, lacks of study have been done on effects of bioflocculant-producing bacteria consortium on flocculation activity. Therefore, it is crucial to isolate and identify bioflocculant-producing bacteria which then tested for flocculation activity.
MATERIALS AND METHODS
Collection of biofloc samples: Bioflocculant-producing bacteria was isolated from biofloc in Penaeus vannamei (P. vannamei) farm located at Setiu District, Terengganu, Malaysia operated by the Integrated Shrimp Aquaculture Park (iSHARP), Blue Archipelago Sdn. Bhd (Fig. 1). Biofloc was collected from three selected shrimp grow-out pond following standard operating procedures by Blue Archipelago Sdn. Bhd. Three replicates consist of 2 L of water containing biofloc were placed in Imhoff cone overnight to settle down the biofloc samples. Sediment-like biofloc was further concentrated by centrifugation for 3 min at 6000 rpm18. The concentrated biofloc sample was further used for identification of pure culture and flocculation activity. The study was conducted between October, 2015-May, 2016.
Isolation of bacteria from biofloc: Marine agar was used as cultivation medium for isolation of all bacteria colonies in the biofloc pellet19. Biofloc pellet was then streaked on marine agar for bacterial growth. All plates were placed in an incubator for 24 h at 30°C. After 24 h, grown bacterial colonies on the incubation plate were sub-cultured in zigzag line until the pure culture of a single colony of bacteria was obtained. Then series of re-plating were conducted on the isolated culture of bacteria until the pure culture of bacteria were obtained. Isolated pure culture of bacteria was maintained on marine agar slants and kept in a refrigerator at 4°C as a stock culture.
Screening of bioflocculant-producing bacteria: Screening of bioflocculant-producing bacteria was conducted using the method as described by Abd-El-Haleem et al.20. The pure isolates of bacteria were transferred into yeast peptone glucose (YPG) medium which contained 10.0 g of yeast extract powder, 20.0 g of polypeptone, 20.0 g of D(+)-glucose and 15.0 g of agar-agar powder in 1 L of filtered sea water at pH 7.0 using 1.0 M sodium hydroxide and 1.0 M hydrochloric acid and incubated at 35°C for 48 h19. Isolated strains with highly mucoid and ropy colony morphologies in the YPG medium were selected20.
Location of the Pacific Whiteleg shrimp, P. vannamei culture ponds operated by Integrated Shrimp Aquaculture Park (iSHARP), Blue Archipelago Sdn. Bhd. at Setiu District, Terengganu, Malaysia5
Enrichment medium for bioflocculant-producing bacteria cultures: Screened bacteria was grown in enrichment medium which was prepared as seeding medium by mixing 10.0 g of glucose, 0.5 g of urea, 0.2 g of MgSO4.7H2O, 5.0 g of K2HPO4, 2.0 g of peptone, 0.2 g of KH2PO4 and 0.5 g of yeast extract in 1 L of filtered sea water at pH 7.0 using 1.0 M sodium hydroxide and 1.0 M hydrochloric acid and incubated at 35°C for 48 h using incubator shaker at 120 rpm at 35°C for 3 days using method derived from Zhang et al.21 modified by Cosa et al.22. The resultant culture broth was centrifuged at 8000 rpm for 15 min and the cell-free supernatant was used for the flocculating activity.
Consortium of screened bacteria: Screening of the bioflocculant-producing bacteria showed that there were six species have been identified as bioflocculant-producing bacteria. The combination of bioflocculant-producing bacteria grown in enrichment medium was determined as follows23:
|n||=||Number of sample|
|r||=||Number of sample combination|
The consortium samples were incubated in the incubator shaker with 120 rpm at 35°C for 3 days. After 3 days, consortium samples were prepared centrifuged at 8000 rpm for 15 min and the cell-free supernatant was used for flocculating activity assay.
Flocculating activity assay: Flocculating activity was measured using a modified kaolin clay suspension method24. Kaolin clay suspension (5.0 g L1) was prepared where 5.0 g of kaolin clay powder was suspended in 1 L of deionized water. Kaolin clay suspension was adjusted to pH 7.0 using 1.0 M sodium hydroxide and 1.0 M hydrochloric acid.
For flocculating activity, 240 mL of kaolin clay suspension and 10 mL of bioflocculant solution (cell-free supernatant) were added into a 250 mL beaker. The flocculating activity assay was started with rapid mixing at 230 rpm for 2 min, followed by slow mixing for 1 min at a speed of 80 rpm using JLT4 Jar/Leaching Tester Velp Scientifica. The stirring speed was reduced to 20 rpm and stirring was continued for 30 min. Stirring apparatus was stopped and the samples in the beakers were allowed to settle for 30 min.
The optical density (OD) of the clarifying solution 3 cm below the surface was measured with Shimadzu UV Spectrophotometer UV-1800 at 550 nm. A control flocculating activity assay was prepared using the similar method where the bioflocculant solution was replaced with deionized water. The percentage of flocculating activity was calculated as follows25:
where, A and B were the absorbance at 550 nm for sample and reference, respectively.
Statistical analysis: One-way ANOVA with post hoc and Tukey test was applied to analyse the flocculation activity of each bacterial sample. Significant differences between samples were determined at 0.05 level of probability. Statistical analyses were conducted using SPSS26 version 22 computer package.
Screening of bioflocculant-producing bacteria: Bioflocculant-producing bacteria screening process indicated there were six species of bacteria showed the positive result as bioflocculant-producing bacteria using YPG medium. Bioflocculant-producing bacteria was identified as Bacillus infantis (A), B. cereus (B), B. safensis (C), Halomonas venusta (D), Nitratireductor aquimarinus (E) and Pseudoalteromonas (F)17 as they showed highly mucoid and ropy colony morphologies (Fig. 2) during their growth on YPG medium plate20.
Flocculation activity of bioflocculant-producing bacteria consortium: There were 64 of bacteria consortium consist of 63 different types of combination of bioflocculant-producing bacteria and a control (zero bacteria consortium) based on a calculation using Eq. 1. These 64 consortium of bacteria were tested for flocculation activity. The flocculation activity assay indicates the effectiveness of bioflocculant-producing bacterias EPS to floc the kaolin clay used as suspended matters in the floc jar test method. From the flocculation activity assay, it shows that overall flocculation activity was ranged between 56.1 and 94.3% (Fig. 3). The consortium of AB which was B. infantis and B. cereus showed highest flocculation activity with 94.3%. The lowest flocculation activity was observed on a consortium of BC which were B. cereus and B. safensis with 56.1%.
Flocculation activity were tested using single specie of bioflocculant-producing bacteria where B. infantis showed the highest flocculation activity followed by N. aquimarinus with 93 and 90.6% (p<0.05), respectively. The other Bacillus species that showed high flocculation activity at 87.3% was described as B. cereus. The lowest flocculation activity was observed on B. safensis with 69% which showed significantly different (p<0.05) from other consortium of bioflocculant-producing bacteria. Flocculation activity recorded on H. venusta and Pseudoalteromonas sp. showed no significant different with 80 and 79%, respectively.
There were 15 consortium of two mix bioflocculant-producing bacteria species cultured and tested for flocculation activity. From these 15 consortiums, a consortium of B. infantis and B. cereus (AB) showed highest flocculation activity which was 94.3%. There were another 4 consortium that has more than 80% flocculation activity which was consortium of B. safensis and H. venusta (CD), B. safensis and N. aquimarinus (CE), N. aquimarinus and Pseudoalteromonas (BF) and B. infantis and Pseudoalteromonas (AF) with 87.1, 83.2, 81 and 80.4%, respectively. Among all combination of two mix bioflocculant-producing bacteria species, the lowest flocculation activity was observed on the combination of B. cereus and B. safensis (BC) with 56.1%.
There were 20 consortium of three mix bioflocculant-producing bacteria species indicate that flocculation activity was ranged between 87.4 and 62.1% on consortium of B. infantis, B. cereus and B. safensis (ABC) and B. infantis, B. safensis and H. venusta (ACD), respectively. There were 6 consortium were recorded able to flocculate kaolin clay more than 80% which were consortium of B. infantis, B. safensis and N. aquimarinus ACE), B. cereus, N. aquimarinus and Pseudoalteromonas (BEF), B. cereus, H. venusta and Pseudoalteromonas (BDF), H. venusta, B. safensis and Pseudoalteromonas (DCF), B. infantis, H. venusta and N. aquimarinus (ADE) and B. infantis, B. safensis and Pseudoalteromonas (ACF) with flocculation activity 83.8, 82, 81.3, 80.8, 80.3 and 80%, respectively.
From the 15 consortium of four mix bioflocculant-producing bacteria species, it shows that flocculation activity was ranged between 87.3% for the combination of B. cereus, H. venusta, N. aquimarinus and Pseudoalteromonas (BDEF) and 63% for the combination of B. infantis, B. cereus, B. safensis and H. venusta (ABCD). There are only 4 consortium have flocculate kaolin clay more than 80% which were combination of B. cereus, H. venusta, N. aquimarinus and Pseudoalteromonas (BDEF) which was 87.3%, B. infantis, B. safensis, H. venusta and Pseudoalteromonas (ACDF) which was 85.2%, B. infantis, H. venusta, N. aquimarinus and Pseudoalteromonas (ADEF) which was 82.7% and B. cereus, B. safensis, H. venusta and Pseudoalteromonas (BCDF) which was 81.3%. The other 11 consortium were ranged between 78.9 and 63%.
|Fig. 2(a-f):|| |
Morphological characteristics of bioflocculant-producing bacteria represented (a) B. infantis (b), B. cereus (c), B. safensis (d), H. venusta (e), N. aquimarinus and (f) Pseudoalteromonas sp. and Bioflocculant-producing bacteria was identified as they showed highly mucoid and ropy colony morphologies during their growth on YPG medium plate18
There is 6 consortium derived from a consortium of five mix species of bioflocculant-producing bacteria and only one consortium have more than 80% flocculation activity which are a consortium of B. infantis, B. cereus, B. safensis, H. venusta and N. aquimarinus (ABCDE) with 87.9%. The other consortium were only able to flocculate kaolin clay between 68.2 and 59.8%.
|Fig. 3:||Flocculation activity of 63 bioflocculant-producing bacteria consortium which ranging from 56.1-94.3%|
|A control consortium sample indicates as references and represents 0% flocculation activity. Standard bars represented as standard deviation|
|Fig. 4:||Consortium of bioflocculant-producing bacteria with more than 80% of flocculation activity|
|Standard bars represented as standard deviation|
There is only one consortium of six species of bioflocculant-producing bacteria which was B. infantis, B. cereus, B. safensis, H. venusta, N. aquimarinus and Pseudoalteromonas (ABCDEF). The ability to flocculate kaolin clay of this consortium defined as one of the lowest flocculation activity recorded in this experiment which was 57.8%.
Selection of consortium with high flocculation activity as inoculum for biofloc formation: From the experiment, there were 19 consortium of bioflocculant-producing bacteria species that have more than 80% of flocculation activity. This consortium of bioflocculant-producing bacteria species very effective in flocculating kaolin clay. The highest flocculation activity was from a consortium of B. infantis and B. cereus (AB) with 94.3% the followed by single bacteria which were B. infantis (A) and N. aquimarinus (E) which were 92.9 and 90.6%, respectively (Fig. 4). Only these three sample showed flocculation activity more than 90% while other consortiums have flocculation activity between 80 and 90%.
Bioflocculant-producing bacteria was successfully screened and isolated from a biofloc sample collected from P. vannamei farm operated by iSHARP, Blue Archipelago Sdn. Bhd. From the screening processes, there were six species identified as bioflocculant-producing bacteria as they grow and showed highly mucoid, ropy colony, cream colored, convex edge, smooth, round and viscous when cultivated on YPG medium17,20.
There were three bioflocculant-producing bacteria species identified from Bacillus sp. and showed various flocculation activity of single species were ranged between 69 and 93%. There were significantly different on flocculation activity between B. infantis, B. cereus and B. safensis (p<0.05). While the other species were from genus Halomonas, Nitratireductor and Pseudoalteromonas. From the consortium of bioflocculant-producing bacteria species used in flocculation activity, it showed that flocculation activity was ranged between 56.1 and 94.3%. Both the highest and the lowest flocculation activity were from a mix of two bioflocculant- producing bacteria species which were a consortium of B. infantis and B. cereus (AB) and B. cereus and B. safensis (BC), respectively. Other than that, single bioflocculant-producing bacteria species of B. infantis (A) and N. aquimarinus have flocculation more than 90% and as good as a consortium of B. infantis and B. cereus (AB) in flocculating kaolin clay.
The concept of using combination of microbes in consortium for bioflocculant was firstly approach in 2003 where the major purpose was to increase the flocculating efficiency27. Bioflocculant produced by the single strain was improved by combine two or more microbes in consortium for bioflocculant production. Several study was done on the combination of bacteria used to improve the yield of bioflocculant and flocculating activity such as combination of Rhizobium radiobacter F2 and Bacillus shaeicus in 2004 and bioflocculant compound production by a mixed culture of Rhizobium radiobacter F2 and Bacillus shaeicus F6 in 201128,29.
These experiment was sharing the same concept to as the recent study done in 2016 using bacterial mixed culture of Halomonas sp. Okoh and Micrococcus sp. Leo which were improved flocculation activity and enhanced bioflocculant production and yield27. There were also development of 3 bacteria consortium have been studied on wastewater treatment. Acinetobacter faecalis WD2, Staphylococcus sp. DD3 and Neisseria elongate TDA4 was combined and used to remove hydrocarbon pollutant by flocculating process30.
Flocculation process usually occurs in two ways which were by adhesion of ion or by adsorption of substance on the surface of mucus or EPS3. As media cultured was centrifuged to obtain their EPS for flocculation study, flocculation activity occurs in this study was due to the process of adsorption of mineral clay on the EPS substance. During bacteria grow, EPS was produced but most bacteria producing a different type of EPS31. Even among bacteria species such as Bacillus species, they are producing a different type of EPS. B. subtilis producing polysaccharides, B. consortium producing glycoprotein, B. safensis producing functional protein and some other Bacillus species producing a variety of EPS32,33.
Despite on every bacteria producing different EPS during their growth, production of EPS was affected by the medium environment. Any environmental factors that may affect their growth, may lead to the type of EPS produced34. These may affect their ability in flocculation activity. At the certain point by culturing consortium species of bacteria may lead to the factors affecting EPS produced. Bacteria may be fighting in obtained nutrient, surviving and dominating thus producing EPS for survival and defensive purpose35. As single bacteria showed at least 69% of flocculation activity, it is assumed that consortium of bioflocculant-producing bacteria species should at least have more than 69% of flocculation activity. Even though most of the consortium of bioflocculant-producing bacteria species shows high flocculation activity, there was 17 consortium have less than 69% of flocculation activity and most of it from the consortium of more than two species of bacteria. It is maybe the properties of EPS produced was not same as they produced once during cultured as single species.
Screening and isolation of bioflocculant-producing bacteria was successfully conducted. Six species were identified as bioflocculant-producing bacteria. From the 64 consortium, it shows that 18 consortium have more than 80% flocculation activity which was from single and two mixes of bioflocculant-producing bacteria species. Mostly more than two mixes of bioflocculant-producing bacteria species have less than 80% flocculation activity. This may due to changes of properties of EPS produced in a consortium of bacteria thus affect ability in flocculation activity.
This study discovers the effect of bioflocculant-producing bacteria consortium on flocculation process that can be beneficial for sustainable production of Pacific whiteleg shrimp. This study will help the researcher to uncover the critical area of screening and isolate bioflocculant-producing bacteria that many researchers were not able to explore. Thus, a new theory on biological flocculant may be discovered and disseminated to the academicians, researchers and aquaculturists.
Authors would like to thank the Ministry of Education, Malaysia (MOE) for financial support under Fundamental Research Grant Scheme, FRGS (Vot No. 59401). Authors also would like to acknowledge iSHARP, Blue Archipelago Berhad at Setiu, Terengganu, Malaysia for aquaculture facilities of P. vannamei culture ponds. Finally, to all the hatchery officers and staffs at Institute of Tropical Aquaculture, Universiti Malaysia Terengganu for all the assistance and guidance throughout the research period.
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