Consortium of Bioflocculant-Producing Bacteria as Inoculum on Flocculation Process for Sustainable Production of Pacific Whiteleg Shrimp, Penaeus vannamei

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.

Fig. 1:	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:

(1)

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 L^–1) 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:

(2)

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.

RESULTS

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 bacteria’s 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%.

DISCUSSION

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 medium^17,20.

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 2011^28,29.

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 EPS^32,33.

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.

CONCLUSION

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.

SIGNIFICANCE STATEMENTS

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.

ACKNOWLEDGMENT

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.