Enhanced bioremediation is a favorable approach for petroleum pollutant cleanup, which depends on the growth of oil-eating microorganisms. In this study, we show that, by using the modified T-RFLP (mT-RFLP) methodology, one of the four major microbial populations derived from oil sludge has failed to propagate in MS medium supplemented with 2% yeast extract (YE). rDNA sequence-based analysis indicated that the four populations were Donghicola sp. CT5, Bacillus sp. CT6, Alcaligenes sp. CT10, and Pseudomonas sp. ZS1. Four purified strains grow well individually in MS medium supplemented with 2% YE, suggesting that ZS1 growth is antagonized by other strains. Co-growth analysis using mT-RFLP methodology and plate inhibitory assay indicated that ZS1 exhibited antagonistic effect against CT5 and CT6. On the other hand, co-growth analysis and plate inhibition assay showed that CT10 antagonized against ZS1. To investigate the potential compounds responsible for the antagonism, supernatant of CT10 culture was subjected to GC–MS analysis. Analysis indicated that CT10 produced a number of antimicrobial compounds includ- ing cyclodipeptide c-(L-Pro-L-Phe), which was known to inhibit the growth of Pseudomonas sp. Growth test using the purified c-(L-Pro-L-Phe) from CT10 confirmed its inhibitory activity. We further showed that, using both gravimetric and GC analysis, CT10 antagonism against the oil-eating ZS1 led to the diminishing of crude oil degradation. Together, our results indicate that bioremediation can be affected by environmental antagonists. Keywords: Alcaligenes sp., Antagonism, Bioremediation, Cyclodipeptide, Oil-degrading microorganism, Pseudomonas sp. Introduction et al. 2010; Al-Mailem et al. 2017; Varjani and Upasani Petroleum leakage is a major threat to land and marine 2016). environment (Holliger et al. 1997). Physical methods Many studies have focused on the physical and chemi- involving removal of solid and liquid pollutants are cal conditions at the pollution sites that affect the perfor - tedious and expensive; and chemical methods using mance of enhanced bioremediation (Díaz-Ramírez et al. chemically synthesized surfactants can cause second- 2003; Venosa and Zhu 2003). Physical conditions such ary pollution (Kanaly and Harayama 2010; Murphy et al. as temperature and salinity affect the growth of many 2005). Enhanced bioremediation method using indig- microorganisms. Similarly, chemical conditions include enous oil-eating microorganisms and biosurfactants is mineral salts and pollutant toxic compounds also influ - believed to be a favorable method for oil spill cleanup ence the growth of various microorganisms. Hence, (Patowary et al. 2016; El-Bestawy et al. 2014; Karamalidis indigenous isolates of microorganisms are advantageous for enhanced bioremediation (Patowary et al. 2016; El- Bestawy et al. 2014). However, it remains unclear whether *Correspondence: email@example.com antagonism between microorganisms including the oil- Ocean College, Zhejiang University, Marine Science Building #379, eating ones will affect the cleanup of oil pollutants. Zhoushan Campus, 1 Zheda Road, Dinghai District, Zhoushan 316000, ZJ, China Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Liang et al. AMB Expr (2018) 8:88 Page 2 of 13 Antagonisms between microorganisms are not uncom- cultivated in glass conical flask at 30 °C in MS medium mon. Many antibiotics are discovered through the obser- supplemented with 2% YE or 1% crude oil. Cell growth vation of compounds that are nontoxic to humans but was monitored by either colorimetric (optical density at exhibit antagonistic effect against pathogenic microbes. the wavelength of 600 nm) or gravimetric methodologies The most well-known example is the discovery of penicil - (cell dry weight). All measurements were performed in lin by Fleming (Fleming 1929). Before long, Waksman has triplicate, unless otherwise stated. established the plate inhibitory method for systematic Pseudomonas sp. ZS1, Alcaligenes sp. CT10, Dong- screening for soil microbes, especially the Actinomyces hicola sp. CT5 and Bacillus sp. CT6 strains were spp. that are capable of inhibiting pathogenic microbes deposited in the China General Microbiological Cul- (Waksman and Woodruff 1940). A number of antibiotics ture Collection Center with the accession numbers of were successfully identified and characterized including CGMCC-13460, CGMCC-1.16509, CGMCC-1.16485 streptomycin and neomycin that have extensively been and CGMCC-1.16486 (respectively) and whose 16S applied to the treatment of numerous infectious diseases rDNA sequences were deposited in NCBI GenBank (Waksman and Woodruff 1940). with the accession number of KY437088, KY437091, Restriction fragment length polymorphism (or KY437089 and KY437090, respectively. RFLP) analysis of amplified rDNA allows identifica - Oligonucleotide DNA sequences 27F, 5′-AGA GTT tion of microorganisms such as mycobacterium spe-TGATCMTGG CTC AG-3′ and 1492R, 5′-TAC GGY TAC cies (Vaneechoutte et al. 1993). Terminal fluorescence CTT GTT ACG ACTT-3′ (Moreno et al. 2002) used in labeled RFLP (T-RFLP) analysis is a method for identi- PCR amplification of 16S rDNA were purchased from fication of mixed microbial populations with the help BGI (BGI, Shenzhen, China). of DNA sequencer (Liu et al. 1997). We have previously modified the T-RFLP (mT-RFLP) method by replac - Preparation of genomic DNA and PCR analysis ing DNA sequence gel with mini-PAGE gel to study To obtain microbial genomic DNA for PCR amplifica - dynamic change of microbial populations without the tion, mixed or clonal microbial cultures were pelleted need for DNA sequencer, an equipment uncommon in by centrifugation and the resulting pellet was resus- many biology laboratories (Cheng et al. 2017). By using pended in lysis solution using Genomic DNA Extraction the mT-RFLP analysis, we have isolated the rhamnolipid- kit (Axygen Scientific Inc., Tewksbury, MA, USA) and producing oil-eating Pseudomonas sp. ZS1 strains from extracted according to the manufacturer’s instruction. the mixed culture of petroleum sludge-originating The 16S rDNA fragment was PCR amplified by using microbes cultivated in MS medium supplemented with the microbial genomic DNA as template and 16S rDNA- 2% glucose (Cheng et al. 2017). specific primers 27F and 1492R (Moreno et al. 2002). The In this study, we show that the growth of Pseudomonas PCR condition was set as follows: after the initial dena- sp. ZS1 is suppressed in the mixed culture of sludge-orig- turation at 95 °C for 5 min, 30 cycles of 95 °C for 30 s, inating microbes in medium without glucose. Co-growth 55 °C for 30 s, and 72 °C for 90 s, and a final extension at and plate inhibition analyses reveal that an Alcaligenes 72 °C for 10 min. The resulting PCR fragment was sub - sp. CT10 strain exhibits antagonistic effect against Pseu - jected to sequencing analysis in BGI (BGI, Shenzhen, domonas sp. ZS1. GC–MS analysis shows that a number China) and compared with NCBI’s nucleotide sequences of antimicrobial compounds including cyclodipeptide using BLAST tools (http://www.ncbi.nlm.nih.gov). c-(L-Pro-L-Phe) present in supernatant of CT10 cul- ture. Both gravimetric and GC analyses show that CT10 Modified T‑RFLP or mT‑RFLP analysis impedes the oil-degradation by ZS1, implying that antag- To examine the dynamic change of mixed microbial onisms between environmental microorganisms can populations under various growth conditions, we modi- affect the outcome of bioremediation. fied the T-RFLP method (Liu et al. 1997) by using the mini-PAGE gel instead of sequencing gel. In brief, the 16S Materials and methods rDNA fragments were PCR amplified on genomic DNA Strains, DNA, and cultures as template derived from microbial populations using the Petroleum sludge was collected in April 2016 at Sanjiang 27F-fluorescence labeled and 1492R unlabeled primers. Ferry Terminal, Zhoushan, Zhejiang province, China. The resulting fragments were subjected to Hha I (New Microbial strains were resuspended and maintained in England Biolabs Inc., Ipswich, MA, USA) digestion and mineral salt (MS) medium (1 L contains: 0.6 g Na HPO , 8% mini-PAGE gel electrophoresis. Fluorescence signals 2 4 0.2 g KH PO , 4.0 g N aNO , 0.3 g M gSO , 0.01 g C aCl , were captured using the Gel Imaging System Tanon 5200 2 4 3 4 2 0.01 g FeSO , 1 g or 0.1% yeast extract or YE) (Zajic (Tanon Scientific Inc., Shanghai, China) with a Sybr - and Supplison 1972). For propagation, strains were Green fluorescence channel. Liang et al. AMB Expr (2018) 8:88 Page 3 of 13 Co‑growth assay film thickness of 5%-phenyl-methylpolysiloxane) (Restek Equal amount of overnight cultures was mixed and inoc- Co., Bellefonte, PA, USA) and helium (purity 99.999%) ulated to fresh medium to a concentration of 0.1 OD . was used as the carrier gas. The temperature of the injec - Cell populations at various time points during growth tion port was set to 250 °C while the sample injection was −1 were examined using mT-RFLP analysis (see above). Gel made in splitless mode with a purge flow 50 mL min image was recorded using the Gel Imaging System Tanon for 1 min. The temperature program was started with 5200 (Tanon Scientific Inc.,). an initial temperature at 50 °C and held for 2 min at −1 this temperature, then 6 °C min to 300 °C for 20 min −1 Preparation of supernatant crude extract for growth a flow rate of 1 mL min and run time 63.67 min. The inhibition analysis mass spectrometer was operated in electron ionization Growth inhibitory factors in cell-free supernatant of (EI) mode with the ion source temperature at 230 °C. Pseudomonas sp. ZS1 and Alcaligenes sp. CT10 cultures The MS quad temperature was set at 150 °C. The elec - were prepared by following the methods previously tron energy was 70 eV. Full-scan MS data were acquired reported by Zhang et at. (Zhang and Miller 1992) and in the range of 50–500 m/z to obtain the fragmentation Bharali et al. (Bharali et al. 2011), respectively. In brief, spectra of CEAC. The LabSolutions (Shimadzu Co.) was supernatant of ZS1 culture was acidified to pH 2.0 using used to determine all the peaks in raw GC chromato- HCl. The resulting precipitate was collected by centrifu - gram. Library search was done for all the peaks using the gation at 13,400g for 30 min and dissolved in bicarbonate National Institute of Standards and Technology NIST/ (pH 8.6) and extracted twice with chloroform-ethanol EPA/NIH (NIST 14 Library). All results were combined (2:1 v/v) solution. The organic phase was evaporated and into a single peak table (Table 1). the resulting paste or crude extract was used in growth inhibition assay. Likewise, supernatant of CT10 culture Purification of bioactive compounds was acidified to pH 2.0 using HCl and kept at 4 °C over - The oily yellow residue (2.7 g) was subjected to column night. The turbid supernatant was extracted twice with chromatography on a silica gel column (Qingdao Haiyang an equal volume of ethyl acetate and collected through Chemical Co., Ltd., Qingdao, China) pre-equilibrated a separating funnel. Subsequently, the organic phase was with dichloromethane and eluted with a gradient solvent evaporated and the resulting paste or crude extract was system dichloromethane-methanol (v/v, 20:1 to 0:100). used in growth inhibition test and GC–MS analysis for Seven fractions were collected and tested for antimi- bioactive compounds. crobial potential with plate inhibitory assay. Fraction 4 (1.1 g), showing inhibitory activity against Pseudomonas Plate inhibitory assay sp. ZS1, was subjected to silica gel column chromatog- To examine the growth inhibitory activities of the raphy and eluted with hexane-dichloromethane (3:1 supernatant, crude extract (see above) of supernatants v/v). Repeated chromatography led to pure compound 1 was dissolved in chloroform to a final concentration of (10 mg). −1 −1 50 mg mL . Filter discs containing 20 μL 50 mg mL supernatant crude extract were placed on top of MS agar Structure elucidation of bioactive compounds plates that were inoculated with the test strains. As con- The structure of the compound 1 was determined using trol, filter discs containing 20 μL solvent chloroform and NMR spectroscopy (Bruker DRX 500 NMR instrument, −1 20 μl 50 mg mL ampicillin in water were also placed on Bruker, Rheinstetten, Germany). CDCl (Deuterated 1 13 the same plate. Images were taken 1–3 days after incuba- chloroform) was used as solvent in H and C NMR tion at 30 °C. experiments. H NMR spectra were recorded in CDCl using tetramethylsilane (TMS) as internal standard at GC–MS analysis of compounds extracted from supernatant 500 and 400 MHz, C NMR spectra were recorded at of Alcaligenes sp. CT10 culture 125 and 100 MHz, chemical shifts are given in parts per The compounds extracted from supernatant of Alca - million and coupling constants in Hz. ligenes sp. CT10 culture (CEAC) was analyzed by gas chromatography coupled with mass spectrophotometer Determination of minimum inhibitory concentrations (GC–MS). 1 µL of CEAC was directly injected into the (MICs) injection port of gas chromatograph (Shimadzu 2010Plus To investigate the minimum inhibitory concentra- GC system, Shimadzu Co., Tokyo, Japan) coupled with tion of compound 1 or cyclodipeptide c-(L-Pro-L-Phe), a mass spectrometer system (MS) (Shimadzu QP2020 we followed the protocol described Singh-Babak et al. with quadrupole analyzer). The GC was operated on an (Singh-Babak et al. 2012). In brief, the compound and Rtx-5MS GC column (30 m × 0.25 mm, id. with 0.25 µm ciprofloxacin (Aladdin Industrial Co. Shanghai, China) Liang et al. AMB Expr (2018) 8:88 Page 4 of 13 Table 1 Compounds derived from supernatant of Alcaligenes sp. CT10 cultures a b c No. RT Compound name M.W. Formula %Pk Comment 1 5.44 Ethylbenzene 106 C H 0.86 – 8 10 2 5.63 1,4-Dimethylbenzene 106 C H 2.35 – 8 10 3 5.67 1,3-Dimethylbenzene 106 C H 0.7 – 8 10 4 6.12 Styrene 104 C H 5.86 – 8 8 5 11.87 n-Hendecane 156 C H 6.76 – 11 24 6 15.66 2(3H)-benzofuranone 134 C H O 2.79 Insecticidal 8 6 2 Fan et al. (2008) 7 16.15 Benzeneacetic acid 136 C H O 34.54 Antimicrobial 8 8 2 Zhu et al. (2011) 8 19.96 Anthranilic acid 137 C H NO 3.16 Antiendotoxic 7 7 2 Fang et al. (2005) 9 20.37 trans-2-Decenoic acid 170 C H O 9.44 – 10 18 2 10 26.16 Tributyl phosphate 266 C H O P 7.69 – 12 27 4 11 31.70 Hexahydro-3-(1-methylethyl)pyrrolo[1,2-a]pyrazine- 210 C H N O 4.9 Antimicrobial, antifungal 11 18 2 2 1,4-dione Yan et al. (2004) Rhee (2004) 12 32.06 Hexahydro-3-(1-methylethyl)pyrrolo[1,2-a]pyrazine- 210 C H N O2 6.84 Antimicrobial, antifungal 11 18 2 1,4-dione Borthwick (2012), Campbell et al. (2009) Yan et al. (2004) Rhee (2004) 13 40.12 Hexahydro-3-(phenylmethyl)pyrrolo[1,2-a]pyrazine- 244 C H N O 1.81 Antimicrobial, antifungal 14 16 2 2 1,4-dione Kumar et al. (2013) 87.52 ( Total) RT for retention time in minute %Pk for percent of peak area Comment includes bioactivity and references was subjected to twofold serial dilution from 1025 to filter paper for drying in an oven. Dried filter paper was −1 1 µg mL in 100 µL of Luria–Bertani (LB) broth (Bertani weighted prior to and after addition of cells. Crude oil 1951) using multiwell plate in duplicate. Fresh overnight mass was determined after removal of cell mass by cen- culture of ZS1 in LB was diluted to a final concentra - trifugation. Oil in supernatant was extracted using hex- tion of 5E−04 OD . The resulting culture of 100 µL ane that was evaporated prior to weighting. Samples was transferred and mixed with twofold serial dilutions at 36 days were also analyzed using GC–MS and GC of compound or ciprofloxacin. The plate was incubated analyses. at 30 °C for 24 h prior to OD measurement. The mini - mum concentration of the well without bacterial growth Gas chromatography analysis of crude oils in cultures was defined as minimum inhibitory concentration (MIC). of CT10 and ZS1 The MIC of compound 1 and Ciprofloxacin was 32 and The composition of crude oils was analyzed using the −1 2 µg mL , respectively. GC–MS methodology similar to the analysis of com- pounds in supernatant of CT10 cultures (see above). Gravimetric analysis of crude oil consumptions To analyze level-changes of individual molecules in To estimate the consumption of crude oil by Pseu- crude oils extracted from supernatant of cultures, 1 µL domonas sp. ZS1 in presence and absence of Alcaligenes of sample was directly injected into the injection port sp. CT10, cell mass and crude oil quantity (maximum of gas chromatograph (Shimadzu Co.) equipped with level was set to 100%) were determined in microbial cul- flame ionization detector (FID) and Rtx-5 column tures (i.e., ZS1, CT10, and mixture of ZS1 and CT10) in (30 m × 0.32 mm, id. with 0.25 µm film thickness) (Restek 180 rpm shake flask at 30 °C containing MS medium sup - Co., Bellefonte, PA, USA). The sample injection was plemented with 1% crude oil. Both cell mass and crude made in split mode and the split ratio was 20:1. The tem - oil mass were determined gravimetrically. In brief, cells perature of the injection port and detector temperature were pelleted from 50 mL culture by centrifugation, were set to 280 and 305 °C, respectively. The temperature resuspended in 0.5 mL MS medium, and transferred to program was started with an initial temperature at 70 °C Liang et al. AMB Expr (2018) 8:88 Page 5 of 13 −1 and held for 2 min at this temperature, then 25 °C min to 140 °C, followed by an additional increase of 3 °C −1 −1 min to 240 °C, then 10 °C min up to 300 °C, held for 15 min. The total duration of the temperature program was 59.13 min. Nitrogen was used as carrier gas, and its −1 flow rate was 30 mL min . Hydrogen gas flow rate and −1 air flow rate were 40 and 400 mL min , respectively. Level of individual compositions was estimated based on the peak area and degradation rate was based on the for- mula below: DEG% = LEVEL − LEVEL /LEVEL ctl smp ctl where DEG% is the rate of degradation, L EVEL and ctl LEVEL are compound level in control and in sample, smp respectively. Results Analysis of population dynamics in mixed culture derived from oil sludge The oil sludge-derived mixed microorganisms were sus - pended in MS medium and subsequently inoculated into the fresh MS medium supplemented with 2% yeast extract (YE) (see “Materials and methods”). The growth of the mixed culture was monitored by colorimetric methodology (OD ) (Fig. 1a). To investigate the micro- bial population dynamics, total DNA was extracted from the culture at various time points during growth and then subjected to the modified T-RFLP (mT-RFLP) analysis (see “Materials and methods”). In this analysis, each RFLP fragment would represent a unique microbial population. We found that four popula- tions, namely CT5, CT6, CT10, and ZS1, were present in the initial culture (at 0 h time point). However, 50 h after growth, only three populations CT5, CT6, and CT10 remained (Fig. 1b). Strains from the four major popula- Fig. 1 Growth of ZS1 strain is inhibited by other sludge-derived tions were isolated from the initial culture (0 h) based microbes in MS medium supplemented with 2% YE. a Growth curve on the mT-RFLP patterns. Analysis of the 16S rDNA of the sludge-derived mixed culture in MS medium with 2% YE. b Dynamic change of microbial populations in mixed culture. Image sequences indicated that the four strains were Dong- of mT-RFLP analysis. Four major microbial populations in the initial hicola sp. CT5, Bacillus sp. CT6, Alcaligenes sp. CT10, culture (at 0 h) are numbered. c Phylogenetic tree analysis based on and Pseudomonas sp. ZS1 (Fig. 1c). Of these four strains, 16S rDNA sequences. The tree is built using CLUSTALW and NJPLOT. Pseudomonas sp. ZS1 was previously isolated from the Sequence accession number of all strains is shown in parentheses oil sludge (Cheng et al. 2017). Given that all four major strains grew well individually in MS medium supple- mented with 2% YE (Additional file 1: Figure S1), this ZS1 inhibited the growth of CT5 and CT6, rather than result suggested that ZS1 growth was suppressed by one the reverse (Additional file 1: Figure S2). Plate inhibi- of the CT5, CT6, and CT10 strains. tory assay indicated that this was a result of rhamnolipid (Additional file 1: Figure S2). On the other hand, in the Antagonisms found between the four major populations co-growth analysis between ZS1 and CT10, mT-RFLP To investigate the potential antagonism between ZS1 analysis indicated that ZS1 population failed to growth at and CT5, CT6, or CT10, co-growth analysis was per- 10 h after co-growth (Fig. 2a, b). This was the first time formed (see “Materials and methods”). In the co-growth to observe that Alcaligenes sp. exhibited antagonistic analysis between ZS1 and CT5 or CT6 using mT-RFLP activity against Pseudomonas sp. To investigate whether method to monitor change of populations, we found that Liang et al. AMB Expr (2018) 8:88 Page 6 of 13 compounds were detected (Fig. 3a). Of the 13 peaks, peak 11 and 12 represented the same molecule cyclodipep- tide c-(Pro-Leu), suggesting that the two stereoisomers c-(D-Pro-L-Leu) and c-(L-Pro-L-Leu) were separated (Fig. 3b). To this end, a number of compounds that were shown to be bioactive such as insecticidal (peak 6, 2(3H)-benzofuranone) (Fan et al. 2008), antiendotoxic (peak 8, anthranilic acid) (Fang et al. 2005), antimicro- bial and antifungal (peak 7, phenylacetic acid; peak 11 and 12, hexahydro-3-(1-methylethyl) pyrrolo[1,2-a]pyra- zine-1,4-dione; peak 13, hexahydro-3-(phenylmethyl) pyrrolo[1,2-a]pyrazine-1,4-dione) (Fan et al. 2008; Zhu et al. 2011; Kumar et al. 2013; Yan et al. 2004; Rhee 2004) (Table 1). Hexahydro-3-(phenylmethyl) pyrrolo[1,2-a] pyrazine-1,4-dione (peak 13) was cyclodipeptide c-(D- Pro-L-Phe) or c-(L-Pro-L-Phe), which was isolated from Bacillus sp. N strain and showed to be inhibitory against Pseudomonas sp. at a MIC (minimal inhibitory concen- −1 tration) of 32–64 µg mL (Kumar et al. 2013), suggesting that the antagonistic effect from Alcaligenes sp. against Pseudomonas sp. was partly attributed to the cyclodi- peptides c-(D-Pro-L-Phe) and c-(L-Pro-L-Phe). To test this possibility, we undertook the purification process for the inhibitory activity against Pseudomonas sp. ZS1 (see “Materials and methods”). The purified compound Fig. 2 Alcaligenes sp. CT10 antagonizes against Pseudomonas sp. 1 13 was subsequently subjected to H and C NMR spectro- ZS1. a Growth curve of the mixed CT10 and ZS1 cultures. b Dynamic scopic analysis. change of CT10 and ZS1 populations in mixed culture. Arrow indicates the point that the population diminished. c Plate halo assay Structure determination of the compound 1 (Fig. 3c): showing that the growth of ZS1 is inhibited by supernatant extract white powder; C H N O ; ESI–MS m/z: 244 [M + H] ; 14 16 2 2 derived from CT10 cultures H NMR (CDCl , 500 MHz) δ : 7.34 (2H, dd, J = 7.5, 3 H 9 Hz), 7.26 (1H, t, J = 9 Hz), 7.23 (2H, d, J = 7.5 Hz), 5.88 (1H, S), 4.29 (1H, dd, J = 2.5, 7.5 Hz), 4.05 (1H, t, J = 7.5 Hz), 3.61 (2H, m), 3.58 (1H), 2.80 (1H, dd, J = 10.0, inhibition factors against ZS1 were secreted into the 14.5 Hz), 2.32 (1H, m), 1.99 (1H, m), 1.89 (2H, m); C medium from CT10, supernatant extract of CT10 culture NMR (CDCl ,125 MHz): 169.5, 165.1, 135.9, 129.2, was prepared (see “Materials and methods”). Plate inhibi- 129.1, 127.5, 59.1, 56.2, 45.4, 36.8, 28.3, 22.5. Based on tion assay using supernatant extract from CT10 culture the NMR spectroscopic analysis of c-(L-Pro-L-Phe) by on a disc paper indicated that it exhibited apparent inhib- Kumar et al. (Kumar et al. 2013), the compound 1 from itory effect against ZS1 (Fig. 2c). This result indicated ZS10 was identified as cyclodipeptide c-(L-Pro-L-Phe), that Alcaligenes sp. CT10 secreted the unknown factor which exhibited a potent inhibitory activity against ZS1 that antagonized against Pseudomonas sp. ZS1. −1 at a MIC of 32 µg mL against ZS1 (see “Materials and methods”). Cyclodipeptide c‑(L‑Pro‑L‑Phe) from CT10 displays inhibitory activity against ZS1 Gravimetric analysis of oil degradation by ZS1 strain To investigate the potential antagonistic factors against is disrupted by the presence of CT10 strain ZS1, supernatant extract derived from CT10 culture To investigate if efficiency of oil degradation by ZS1 was subjected to GC-MS analysis (see “Materials and strain would be affected in presence of Alcaligenes sp. methods”). The result indicated that 13 major peaks or CT10, the oil degradation experiments were performed (See figure on next page.) Fig. 3 GC-MS analysis of compounds extracted from supernatant of Alcaligenes sp. CT10 culture. a Total ion chromatograph. Peaks with matched molecules are numbered. b MS spectra of individual compounds indicated. c An inhibitory compound to ZS1 from CT10. NMR analysis indicates that the compound 1 is the cyclodipeptide c-(L-Pro-L-Phe) Liang et al. AMB Expr (2018) 8:88 Page 7 of 13 Liang et al. AMB Expr (2018) 8:88 Page 8 of 13 Fig. 4 Oil-eating activity of Pseudomonas sp. ZS1 is impeded in the presence of Alcaligenes sp. CT10. Arrow indicates the presence of floating oil on the surface of cultures. a Change of cell mass and crude oil quantity in ZS1 culture. Upper panel shows the percentage of cell mass (Cell) and crude oil mass (Oil) detected in cultures at various time points indicated. Bottom panel shows the presence (with arrow) or absence (without arrow) of floating oil in culture flask. The 50% reduction of crude oil occurs at 10 days after growth. b Change of cell mass and crude oil quantity in CT10 culture. The display is identical to a. c Change of cell mass and crude oil quantity in ZS1 and CT10 mixed culture. The display is identical to a. d Dynamic change of ZS1 and CT10 populations in mixed culture indicated in (c) in MS medium supplemented with 1% crude oil (see when CT10 strain was present in the culture (Fig. 4c). “Materials and methods”). Oil residues remained in the mT-RFLP analysis confirmed that ZS1 failed to grow in medium at various time points during growth was deter- presence of CT10 (Fig. 4d). These results indicated that mined gravimetrically by using hexane extraction and antagonisms against oil-eating microbes could abolish its weighted after evaporation (see “Materials and meth- oil degradation activity. ods”). We found that 50% of oils was degraded in ZS1 culture 10 days after growth (Fig. 4a). On the other hand, GC–MS analysis of oil degradation by ZS1 strain is impeded there was hardly any oil degradation activity detected in by the presence of CT10 strain culture of Alcaligenes sp. CT10 (Fig. 4b). However, oil Based on the GC–MS analysis, the crude oils used in degradation ability of ZS1 strains was nearly abolished this study were found to contain 23 linear aliphatic (See figure on next page.) Fig. 5 GC analysis of crude oil degradation in culture of Pseudomonas sp. ZS1 in presence or absence of Alcaligenes sp. CT10. a GC analysis of hexane extract derived from medium 36 days after shaking without bacteria. A GC spectrum of crude oil in medium is shown. b Linear and branched aliphatic hydrocarbons C H detected. Left panel shows an enlarged image of the Fig. 5a. MS spectra of the linear (upper right penal) 17 36 and branched (bottom right panel) C H are shown. c A GC spectrum of oils in ZS1 culture at 36 d after growth. An inset shows the branched 17 36 C H but not linear C H remained noticeable. d A GC spectrum of oils in CT10 culture at 36 days after growth. e A GC spectrum of oils in mixed 17 36 17 36 CT10 and ZS1 culture at 36 days after growth Liang et al. AMB Expr (2018) 8:88 Page 9 of 13 Liang et al. AMB Expr (2018) 8:88 Page 10 of 13 Table 2 Degradation of crude oil in cultures of one or both of Alcaligenes sp. CT10 and Pseudomonas sp. ZS1 a b c d e f g h i LAH (C#) RT (min) Ctl (level) CT10 (level) CT10/ZS1 (level) ZS1 (level) CT10 (%deg) CT10/ZS1 (%deg) ZS1 (%deg) 9 7.68 8570 0 0 0 100.00 100.00 100.00 10 10.34 9518 1810 393 0 80.98 95.87 100.00 11 13.09 11,662 4742 3503 0 59.34 69.96 100.00 12 15.76 14,531 9192 9351 0 36.74 35.65 100.00 13 18.29 20,599 17,001 18,330 453 17.47 11.02 97.80 14 20.68 23,975 22,193 22,858 598 7.43 4.66 97.51 15 22.94 26,059 25,468 22,826 664 2.27 12.41 97.45 16 25.07 21,935 21,923 19,366 777 0.05 11.71 96.46 17 27.09 21,009 20,503 17,364 309 2.41 17.35 98.53 18 29.02 16,658 16,654 14,154 0 0.02 15.03 100.00 19 30.85 16,659 16,722 15,411 300 − 0.38 7.49 98.20 20 32.59 16,032 15,933 13,415 592 0.62 16.32 96.31 21 34.26 16,938 17,050 13,738 547 − 0.66 18.89 96.77 22 35.85 16,626 16,535 13,159 378 0.55 20.85 97.73 23 37.38 17,060 17,005 13,042 624 0.32 23.55 96.34 24 38.85 15,283 15,178 11,839 428 0.69 22.53 97.20 25 40.27 15,747 15,757 12,688 658 − 0.06 19.43 95.82 26 41.63 14,552 14,426 11,597 447 0.87 20.31 96.93 27 42.94 13,404 13,273 12,236 532 0.98 8.71 96.03 28 44.23 20,840 11,537 10,560 694 44.64 49.33 96.67 29 45.58 10,534 10,344 10,563 723 1.80 − 0.28 93.14 30 47.08 7432 7411 7377 0 0.28 0.74 100.00 31 48.77 6251 6117 6643 0 2.14 − 6.27 100.00 Total LAH. 361,874 316,774 280,413 8724 12.5 22.5 97.6 Total oil 481,954 428,608 374,615 13,911 11.1 22.3 97.1 LAH (C#) for linear aliphatic hydrocarbons with carbon numbers RT for retention time in minute ctl (level) for levels in control CT10 (level) for levels in Alcaligenes sp. culture CT10/ZS1 (level) for levels in mixed Alcaligenes sp. and Pseudomonas sp. cultures, respectively ZS1 (level) for levels in Pseudomonas sp. culture CT10 (%deg) for oil degradation rate in Alcaligenes sp. culture CT10/ZS1 (%deg) for oil degradation rate in mixed Alcaligenes sp. and Pseudomonas sp. culture ZS1 (%deg) for oil degradation rate in Pseudomonas sp. culture hydrocarbons ranged from C9 to C31 (Additional file 1: On the other hand, oils were reduced by 12.5% com- Figure S3). Crude oils in supernatant of various cultures pared to the control levels in culture of Alcaligenes sp. 36 days after growth were hexane extracted for GC analy- CT10 36d after growth (Fig. 5d, see Table 2). However, sis (see “Materials and methods”). Oils recovered 36 days degradation rate of some hydrocarbons such as C9, after incubation in medium without bacteria was used C10, C11, and C28 was high (degradation rate > 35%). In as control for initial levels of various hydrocarbon mol- a mixed culture of ZS1 and CT10, we found that 22.5% ecules (Fig. 5a). We noted that a residue of branched ali- of total oils were degraded 36d after growth (Fig. 5e, see phatic hydrocarbon n-heptadecane present in the crude Table 2), much lower than that of 97.6% degradation in oil (Fig. 5b). Based on GC analysis, we found that 97.4% ZS1 culture, though a bit higher than that of 12.5% in of crude oils were degraded in culture of Pseudomonas CT10 culture. These results were in agreement with the sp. ZS1 36d after growth, though a trace amount of gravimetric analysis that crude-oil degradation ability of branched hydrocarbon n-heptadecane remained to be Pseudomonas sp. ZS1 strain could be inhibited in pres- detected (Fig. 5c, Table 2). ence of Alcaligenes sp. CT10. Liang et al. AMB Expr (2018) 8:88 Page 11 of 13 Discussion synthetases (Schwarzer et al. 2003) and cyclodipeptide Enhanced bioremediation is believed to be a useful synthases (Gondry et al. 2009) in microorganisms. They method for oil pollutant cleanup (Patowary et al. 2016; often serve as precursors for modification with various El-Bestawy et al. 2014). However, there are limitations tailoring enzymes that result diverse compounds with (Díaz-Ramírez et al. 2003; Venosa and Zhu 2003). Physi- numerous bioactivities such as thaxtomin A and glio- cal and chemical conditions are known to affect the toxin. Thaxtomin A is derived from hydroxylation of growth of the oil-eating microorganisms at pollutant precursor c-(L-Trp-L-Phe) (Healy et al. 2002), whereas sites. In this study, we show that biotic factors such as gliotoxin is generated through oxidation, sulfurization, antagonistic species can also influence the growth of the and methylation of precursor c-(L-Phe-L-Ser) (Gardiner oil-eating microorganisms (see Figs. 4, 5). Hence, oil deg- and Howlett 2005). Thus, CDPs have shown great poten - radation during enhanced bioremediation can be compli- tial for new drug development (Borthwick 2012). cated by not only physical and chemical factors, but also Holden et al. (1999) have proposed that CDPs inter- biological factors. fere quorum sensing signals in bacteria and hence affect We have previously screened for biosurfactant-produc- bacterial growth. However, this ideal is challenged by ing microorganisms using the mT-RFLP methodology to Campbell et al. (2009) whom have shown that none of monitor the enrichment under selective growth condi- the CDPs tested exhibit activation or inhibition of quo- tions. In this study, we show that by using this method, rum sensing signals. Hence, the mechanisms for CDPs to antagonism between microbes is readily detected (see inhibit bacterial growth remain elucidation. Figs. 1, 2). All major populations observed in mixed cul- Based on GC analysis, we find that degradation rate ture of the oil sludge-originating microorganisms are of crude oil by Pseudomonas sp. ZS1 reaches as high as found to be involved in one of the antagonistic interac- 97.6% (see Fig. 5, Table 2) in 36d. However, when Alca- tions, implying that antagonism between microbes is not ligenes sp. CT10 is present, the degradation rate reduces ignorable in environmental niches. by 4.3-fold (degradation rate of 22.5% vs. 97.6%). Degra- Biosurfactant rhamnolipid is known to inhibit bacteria dation of selected hydrocarbons such as C9, C10, C11, such as Serratia marcescens, Enterobacter aerogenes, and and C28 by CT10 is observed, suggesting a complex of Klebsiella pneumoniae (Haba et al. 2003). In this study, hydrocarbon degradation by various environmental we show that rhamnolipid produced by Pseudomonas microorganisms. sp. ZS1 inhibits the growth of Donghicola sp. CT5 and Additional file Bacillus sp. CT6 (see Additional file 1: Figure S2). It is possible that in a bacterial consortium for bioremedia- Additional file 1: Figure S1. Growth curve analysis of four strains isolated tion (Patowary et al. 2016; El-Bestawy et al. 2014), the from oil-sludge. Figure S2. Pseudomonas sp. ZS1 antagonizes against growth of oil-eating microorganisms could be inhibited Donghicola sp. CT5 and Bacillus sp. CT6. Figure S3. GC–MS analysis of by other biosurfactant-producing microbes. In fact, it has crude oil compositions used in this study. been observed that microbial populations change during bioremediation (MacNaughton et al. 1999). Hence, real- Abbreviations time monitoring the change of microbial populations GC–MS: gas chromatography coupled with mass spectrometry; mT-RFLP: during bioremediation would permits rapid interven- modified terminal-labeled restriction fragment length polymorphism; NMR: nuclear magnetic resonance. tion for improving oil-eating bacterial growth and thus increasing the efficiency of oil pollutant cleanup. Authors’ contributions In this study, we show that Alcaligenes sp. exhib- JLiang, TC, and YH carried out the biological and chemical studies; JLiu con- ceived of the study, participated in its design and coordination, and draft the its antagonistic activity against Pseudomonas sp. (see manuscript. All authors read and approved the final manuscript. Fig. 2). Based on the GC–MS analysis, a number of bio- active compounds are found to be produced by CT10 Author details Ocean College, Zhejiang University, Marine Science Building #379, Zhoushan (see Table 1). In particular, we have purified the com - Campus, 1 Zheda Road, Dinghai District, Zhoushan 316000, ZJ, China. Ocean pound 1, known as cyclodipeptide c-(L-Pro-L-Phe) that Research Center of Zhoushan, Zhejiang University, Zhoushan 316021, ZJ, shows a potent inhibitory activity against ZS1 at a MIC of China. −1 32 µg mL . Acknowledgements Cyclodipeptides (CDPs) or 2,5-diketopiperazines The authors would like to thank Mr. G. Zheng and J. Xiao for assistance in (DKPs) are the smallest cyclic peptides that widely collection of the petroleum sludge in the San-Jiang Ferry Terminal, Zhoushan Islands, Zhejiang Province, China. spread in nature as secondary functional metabolites or side products of protein metabolism in microorganisms, Competing interests plants, and animals (Borthwick 2012; Prasad 1995). CDPs The authors declare that they have no competing interests. are primarily synthesized by the non-ribosomal peptide Liang et al. AMB Expr (2018) 8:88 Page 12 of 13 Consent for publication Haba E, Pinazo A, Jauregui O, Espuny MJ, Infante MR, Manresa A (2003) Phys- All authors have read and agreed to submit to AMB Express for publication. icochemical characterization and antimicrobial properties of rhamnolip- ids produced by Pseudomonas aeruginosa 47T2 NCBIM 40044. Biotechnol Ethics approval and consent to participate Bioeng 81(3):316–322. https ://doi.org/10.1002/bit.10474 This study does not contain materials derived from human or animal. Healy FG, Krasnoff SB, Wach M, Gibson DM, Loria R (2002) Involvement of a cytochrome P450 monooxygenase in thaxtomin A biosynthesis by Funding Streptomyces acidiscabies. J Bacteriol 184(7):2019–2029 This work was partly supported by Zhoushan Municipal Science and Technol- Holden MT, Ram Chhabra S, de Nys R, Stead P, Bainton NJ, Hill PJ, Manefield M, ogy Bureau, Zhejiang Province, China (Grants numbers: 2014C51020 and Kumar N, Labatte M, England D, Rice S, Givskov M, Salmond GP, Stewart 2016C51026) and Zhejiang Dong-Jie Biological Science and Technology LLC, GS, Bycroft BW, Kjelleberg S, Williams P (1999) Quorum-sensing cross talk: Zhejiang Province, China (K18-529102-006) to JLiu. isolation and chemical characterization of cyclic dipeptides from Pseu- domonas aeruginosa and other gram-negative bacteria. 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