Journal of Oil Palm Research DOI: https://doi.org/10.21894/jopr.2021.0008 A COMPARATIVE STUDY OF BACTERIAL COMMUNITIES DETERMINED BY CULTURE- DEPENDENT AND-INDEPENDENT APPROACHES IN OIL PALM PLANTED ON TROPICAL PEATLAND AYOB, ZAHIDAH* and KUSAI, NOR AZIZAH* ABSTRACT A combination of deoxyribonucleic acid (DNA)-based method and sequencing technologies have initiated a new era of soil microbial ecology to examine the patterns of bacterial communities in tropical peatland. The aims of the study are to verify and compare the bacterial communities in a 12-year-old oil palm plantation on peat of Sibu, Sarawak, Malaysia using culture-dependent and culture-independent approaches. The bacterial diversity identified from both approaches were amplified using 16S ribosomal deoxyribonucleic acid (rDNA) (341/907) primer, sequenced and analysed. This resulted in recovering a total of 227 bacterial isolates belonging to four major phyla accumulated from 22 genera. Meanwhile, about 216 denaturing gradient gel electrophoresis (DGGE) bands were excised, which corresponded to 195 different bacterial species from 20 different phyla by culture-independent method. Although both approaches detected a total of four predominant bacterial phyla (Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes), in general, different taxonomic sequences were targeted by each method. In comparison to culture-dependent, polymerase chain reaction (PCR)-DGGE method identified a higher rate of bacterial diversity and richness and also detected non-culturable bacteria. Thus, this suggests that culture-independent method was showed to be more efficient on the bacterial diversity identification that will lead towards unravelling the hidden bacterial species associated with agricultural practices carried out in Southeast Asia peatland. Keywords: tropical peatland, oil palm plantation, 16S rDNA, culture-dependent, polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Received: 28 September 2020; Accepted: 4 January 2021; Published online: 26 February 2021. INTRODUCTION fertility of tropical peatland (Schneider et al., 2015; Nurulita et al., 2016). Previous studies have reported Prokaryotes are the most abundant group of soil that many factors, including land-use change such as microorganisms, which include bacteria and peat swamp forest conversion to oil palm plantation, archaeaARTICLE and play fundamental ecological roles in plantIN species, and PRESS soil properties drive changes in the decomposition, biogeochemical cycling and the pH, temperature, water content, organic carbon maintenance of soil structure, soil quality, and content, nutrient content and soil texture, which influence the diversity of soil microbial communities (Kerfahi et al., 2014; Schneider et al., 2015; Wu * Malaysian Palm Oil Board, et al., 2015; Nurulita et al., 2016). Furthermore, 6 Persiaran Institusi, Bandar Baru Bangi, 43000 Kajang, Selangor, Malaysia. understanding the diversity of soil microbial E-mail: [email protected] communities is vital for knowing their function in 1 JOURNAL OF OIL PALM RESEARCH ecosystems and the impact of oil palm management on DGGE gels, and enables the detection of diverse practices and plant communities (Kerfahi et al., 2014). members of soil microbial organisms, including Thus, the identification of bacterial communities in unculturable microbes that are unable to proliferate deep peat cultivated with oil palm using culture- in culture media (Watanabe et al., 2004; Green dependent and culture-independent approaches et al., 2010; Chaudhary et al., 2019). Nevertheless, would postulate the impact of these communities the culture-dependent method is still essential on environmental changes in the tropical peatland. for isolating and defining the taxonomic and The 16S ribosomal deoxyribonucleic acid metabolites of pure strains, and also understanding (16S rDNA) gene is used as a universal molecular how their functions influence bacterial diversity marker in microbial diversity studies because patterns in tropical peatland (González-Rocha et al., it has highly conserved regions that display 2017; Chaudhary et al., 2019). considerable sequence variations, even within Very few studies have reported on the closely related taxa (Singh et al., 2011). The diversity diversity of bacterial communities in Southeast of bacterial communities from peat ecosystems can Asia peatland. Previously, analysis of the bacterial be determined using various approaches, most community composition of tropical peatland commonly culture-dependent methods such as was based separately on culturable or culture- spread-plate (Roslan et al., 2015; Kusai and Ayob, independent methods and compared different 2020) and culture-independent methods, namely ecosystems (Maidin et al., 2016; Nurulita et al., 2016; denaturing gradient gel electrophoresis (DGGE) Tripathi et al., 2016; Too et al., 2018; Dhandapani et (Jackson et al., 2009; Arai et al., 2014; Maidin et al., al., 2019; Kusai and Ayob, 2020; Liu et al., 2020). No 2016; Nurulita et al., 2016), terminal restriction data is available and this study intends to highlight fragment length polymorphism (T-RFLP) the comparison between the bacterial community (Situmorang et al., 2016), 454-pyrosequencing composition of culture-dependent and culture- (Kanokratana et al., 2011; Liu et al., 2020), and next- independent approaches of bacterial communities generation sequencing (Tripathi et al., 2016; Too et al., from a 12-year-old oil palm plantation on tropical 2018). Among these approaches, DGGE is one of the peatland. Therefore, in this article, we investigate most well-established and is used to explain microbial the application of these approaches to verify diversity studies by comparing the composition, and compare bacterial communities in isolation richness and structure of the microbial communities on growth media, followed by 16S rRNA-based in soil, other environments (Ellis et al., 2003; Singh identification of isolates, whereas the culture- et al., 2012; Piterina and Pembroke, 2013; Nagendran independent method involved extracting DNA et al., 2014; Teo and Wong, 2014; Maidin et al., 2016; directly from the soil for polymerase chain reaction Nurulita et al., 2016) and food samples (Chen (PCR) amplification of the 16S rRNA gene followed et al., 2008; Jianzhong et al., 2009; Miguel et al., 2010; by DGGE analysis and sequencing. Kesmen and Kacmaz, 2011). This novel method was introduced into microbial ecology in 1993 by Muyzer et al. MATERIALS AND METHODS Traditionally, culture-based methods provide limited information on microbial communities Peat Sampling because the majority of bacteria are difficult to culture in a laboratory since they require specific growth Peat was sampled in an oil palm (Elaeis media and, as a result, a large proportion of microbial guineensis Jacq.) plantation located in the vicinity of communities remain unexplored. Previous studies Sibu, Sarawak, Malaysia. Muriate of potash (MOP) –1 –1 have reported that culture-dependent methods (0.75 kg palm ), urea [CO(NH2)2] (0.25 kg palm ) present copiotrophs and high guanine-cytosine and rock phosphate (RP) (0.2 kg palm–1) have (GC) gram-positive bacteria because these bacterial been supplemented to the 12-year-old oil palms. groups are capable of growing on selective media The studied site was classified as undecomposed (Smit et al., 2001; Edenborn and Sexstone, 2007), sapric organic materials based on Malaysian whereas culture-independent methods may detect Unified Classification of Organic Soils (MUCOS) bacteria that are difficult to culture, such as those by Paramananthan (2016). This type of soil consists from the phyla Acidobacteria and Verrucomicrobia of sapric material of 1/3 fibre content and no wood (LipsonARTICLE and Schmidt, 2004; Edenborn and Sexstone, upIN to 100 cm depth PRESS (Veloo et al., 2015). The average 2007). Advancements in culture-independent annual rainfall over the area was estimated at methods have facilitated microbial diversity studies 2624.38 mm yr–1 during year 2013. Sampling was by comparing the composition and richness of the carried out during dry season with less than 100 mm bacterial communities in soil (Qaisrani et al., 2019). precipitation on August 2013. Peat samples were In addition, culture-independent methods such as collected at 10 sampling points (N1-N10) with depth DGGE has the capacity to process multiple samples between 0-30 cm, using peat auger. The sampling rapidly by analysing bands that migrate separately points were at least 10 m apart from each other with 2 A COMPARATIVE STUDY OF BACTERIAL COMMUNITIES DETERMINED BY CULTURE-DEPENDENT AND-INDEPENDENT APPROACHES IN OIL PALM PLANTED ON TROPICAL PEATLAND the central point at 2°09’ 47.065” N, 111°55’ 22.387” E Bhd, Puchong, Selangor, Malaysia). The following (Kusai et al., 2018). Peat samples were chilled in the thermal cycling scheme was used for amplification ice box during transportation from the field to the of 16S rDNA: initial hot start incubation at 94°C for laboratory and stored at 4°C until further analysis. 2 min, 35 cycles of denaturation at 94°C for 30 sec, The inorganic elements including macronutrients, annealing for 30 sec at 52°C, extension at 72°C for micronutrient and heavy metal contents were 30 sec, followed by a final extension period at 72°C analysed using Perkin Elmer ELAN DRC-e ICP- for 2 min. Deionised water was used as template for Mass Spectrometry (Perkin Elmer Sdn Bhd, negative control. The PCR products were analysed Petaling