Plastic leachates negatively affect Prochlorococcus growth

Leachates of common plastic items (HDPE shopping bags and PVC matting) were made using a base of sterile artificial seawater. Exponential phase cultures of Prochlorococcus were inoculated into media containing a range of leachate dilutions (generated by volume/volume dilutions in AMP1 media of 50, 25, 12.5, 6.25 and 3.125% for HDPE; 10, 2, 1, 0.5 and 0.25% for PVC) alongside equivalent controls (AMP1 media with no leachate addition). The leachate dilutions used for growth and photophysiology monitoring were chosen following preliminary tests that indicated PVC leachate had a considerably greater effect than HDPE at equivalent concentrations.

Growth was impaired for both Prochlorococcus MIT9312 and NATL2A across the full range of HDPE and PVC leachate dilutions tested, as measured by flow cytometric counts of chlorophyll fluorescent cells (Fig. 1). MIT9312 responded more quickly to both HDPE and PVC leachate exposure than NATL2A. In MIT9312, significant (p < 0.01) population reductions were observed at 48 h for all tested HDPE and PVC leachate dilutions compared to the controls, whereas in NATL2A only the two most concentrated HDPE and PVC leachates were significantly different from controls at 48 h (p-values in Supplementary Data 1). After 72 h of exposure, the population density of both strains was reduced for all levels of HDPE and PVC leachate tested (Fig. 1a–d). For each plastic, higher concentrations of leachate resulted in greater reductions in population density, indicating that leachate exposure affected cell populations in a dose-dependent manner (Fig. 1a–d).

Fig. 1 Population growth of Prochlorococcus MIT9312 and NATL2A in the presence of diluted HDPE and PVC leachates compared to 0% leachate control (AMP1 media). Growth curves (a–d) and growth rates (e, f) for each treatment are shown. Data points are mean values of triplicate biologically independent samples with error bars representing the standard deviation (error bars not visible where values were smaller than symbols). Lower case letters at the 72 h timepoints in a–d signify which treatments were found to be significantly different (p < 0.01) in population density at 72 h post exposure; source data and exact p-values for all timepoints are provided in Supplementary Data 1 Full size image

Population growth rates also declined in a dose-dependent manner across the three days of the experiment (Fig. 1e, f). Both strains of Prochlorococcus showed an overall negative growth rate across the experiment when exposed to HDPE leachate dilutions of 12.5 to 50%. PVC leachate exposure resulted in a markedly greater effect on MIT9312 growth compared to NATL2A. All tested PVC leachate dilutions resulted in negative growth rates for MIT9312, whereas NATL2A was able to maintain a positive growth rate for 0.25 to 1% PVC dilutions.

Plastic leachates impair photosynthesis in Prochlorococcus

In addition to effects on population growth, leachate exposure had a strong, dose-dependent effect on photochemical efficiency of photosystem II (PSII) in both Prochlorococcus MIT9312 and NATL2A (Fig. 2a–d). As with population growth, differences were observed with respect to dose sensitivity and timing of response of the two strains. The effective quantum yield of PSII (Φ PSII ) was significantly (p < 0.01) reduced on HDPE leachate exposure in MIT9312 across all tested dilutions and all but the lowest concentration (3.125%) in NATL2A (Fig. 2a, b, Supplementary Table 2). Both strains showed significant reductions in Φ PSII at 24 h for HDPE leachate dilutions of 12.5 to 50%. Strain-specific differences, however, were apparent at 48 h: Φ PSII was affected in MIT9312 at all tested dilutions and had declined to undetectable levels for 12.5% HDPE leachate whereas Φ PSII reductions were more moderate in NATL2A. Exposure to 10% PVC leachate (the highest tested concentration) resulted in a very rapid decline in effective quantum yield of PSII in both strains. Measurements of Φ PSII 3 h post exposure showed that there was no detectable PSII activity in either strain in this treatment by this time. Intermediate and low PVC concentrations again resulted in more rapid and pronounced declines in Φ PSII for MIT9312 than NATL2A and treatment with 0.25% PVC leachate did not significantly affect NATL2A Φ PSII (Fig. 2c, d, p-values in Supplementary Table 2).

Fig. 2 Photosynthetic capacity of Prochlorococcus MIT9312 and NATL2A in the presence of diluted HDPE and PVC leachates compared to 0% leachate control (AMP1 media). Measurements of effective quantum yield of PSII (Φ PSII ) (a–d) and oxygen production rates (e–h) for Prochlorococcus MIT9312 and NATL2A control and leachate exposed populations after 3, 24 and 48 h of exposure. Data points are from triplicate independent biological samples (except for oxygen production rate measurements for NATL2A 3 h control in HDPE experiment where n = 2). In e–h, a subset of leachate dilutions were tested and once negative oxygen production rates were recorded (indicating respiration rates were likely higher than oxygen production rates) no more measurements were made. Asterisks are used to signify treatments for which measurements were significantly different (p < 0.01) to the control at the examined time point; source data and exact p-values for all timepoints are provided in Supplementary Data 2 Full size image

Clear declines in oxygen production rates were also observed following HDPE and PVC leachate exposure. For both strains exposed to HDPE leachates oxygen production rates started declining from 24 h (Fig. 2e, f, Supplementary Table 2). A slight increase in the oxygen production rate was observed in MIT9312 after 3 h exposure to 6.25% HDPE leachate, which may imply an initial effort to obtain energy when subjected to stress. Exposure to both 2 and 10% PVC leachate had a significant negative effect on oxygen production rates within 3 h in both strains (Fig. 2g, h, Supplementary Table 2). By 24 h, oxygen production had halted for MIT9312 cultures exposed to all tested PVC concentrations, whereas NATL2A continued to produce oxygen at similar levels to the control for 0.5% PVC until 48 h.

Leachate exposure results in global transcriptional changes

Global transcriptomic analyses (RNA-Seq) were conducted to determine the early-stage transcriptional response to leachate exposure using dilutions that impaired population growth in both strains following 24–48 h exposure (50% HDPE; 2% PVC compared to no leachate controls). In MIT9312 exposure to leachates resulted in significant (p < 0.01) differential transcription of a large number of coding genes: 589 for PVC and 403 for HDPE, whereas a substantially lower proportion were impacted in NATL2A: 66 for PVC and 136 for HDPE (Supplementary Data 3 and 4). Highly responsive genes (log 2 fold changes greater than ±1) were mapped onto Clusters of Orthologous Groups (COGs) and gene ontology (GO) categories to identify processes that were transcriptionally affected by the leachate exposure treatments (Supplementary Table 1).

Examination of highly transcriptionally responsive genes showed a number of processes were strongly influenced by leachate exposure in MIT9312 (Fig. 3, Supplementary Table 1). Exposure to both leachates led to increased transcription of common stress response genes, such as groEL, dnaK, clpP and ftsH. Both leachate treatments also resulted in strong positive transcriptional responses in a large number of hli (high-light inducible) family genes. In Prochlorococcus MED4, hli genes have been shown to be transcriptionally responsive to specific stress conditions, suggesting they have a role in oxidative and other stresses linked to photosynthesis22. An uncharacterized, Prochlorococcus-specific transcriptional regulator (PMT9312_RS06030) was also very strongly upregulated under exposure to both leachates and may have a role in coordinating the cellular response to plastic leachate-induced stress.

Fig. 3 Schematic model of Prochlorococcus MIT9312 showing key genes and functions with significant responses to short-term HDPE and PVC plastic leachate exposure based on transcriptomic data. The sets of highly responsive genes in both MIT9312 and NATL2A organized by functional categories are listed in Supplementary Table 1 and complete lists of differentially transcribed genes for MIT9312 and NATL2A are found in Supplementary Data 3 and 4, respectively Full size image

In MIT9312, genes associated with primary production were among the most highly affected following leachate exposure. Transcription was strongly reduced for a number of MIT9312 carbon fixation associated genes on exposure to both leachates, compared to the control (Fig. 3, Supplementary Table 1). Among the set showing the highest fold change was a cluster of five genes (PMT9312_RS02820–40) encoding carbon dioxide concentrating mechanism protein CcmK, both RUBISCO subunits and carboxysome shell proteins. Genes involved in photosynthesis and light-harvesting also showed clear transcriptional level changes under both leachate exposures, fitting with the observed photophysiological responses. PVC leachate exposure resulted in substantial reduction in transcription of almost all genes encoding photosystem I subunits (psaA,B,C,D,E,F,J,K,L), most photosystem II (psbB,H,J,M,O,T,Z) subunits, cytochrome b 6 -f complex subunits (petA,C,G), and photosynthetic pigment biosynthesis (Supplementary Data 3). By contrast, the MIT9312 transcriptional impact of HDPE leachate on photosynthesis-related genes was less pronounced, with transcription significantly reduced for pcbD encoding Chl a/b binding light-harvesting protein and photosystem II associated genes (psbB,J,K,T,O and PMT9312_RS06075 encoding PsbF-like protein), whereas two psbA genes showed a significant increase in transcription (Supplementary Data 3). In response to both PVC and HDPE plastic leachate treatments, genes implicated in photosystem repair (Psb28, reported to be involved in PSII repair mechanisms in cyanobacteria23 and PsbN, involved in repair from photoinhibition in higher plants24) were substantially upregulated.

The MIT9312 leachate transcriptional response also involved altered transcription of a number of transport and cell wall/membrane associated genes (Fig. 3, Supplementary Data 3). In both PVC and HDPE experiments, genes encoding putative multidrug efflux ABC transporter (PMT9312_RS04330) and tetracycline resistance major facilitator superfamily efflux pump showed significantly higher transcription in leachate exposed cultures relative to the control. In both instances, fold changes were higher for PVC leachate exposure than HDPE. In contrast to the efflux-associated genes, a number of other transporters showed differentially reduced transcription under leachate exposure conditions. HDPE leachate exposure resulted in reduced transcription of an iron ABC transporter substrate-binding protein PMT9312_RS06520, metal ABC transporter permease PMT9312_RS03095 and porin PMT9312_RS06535 relative to the no leachate control. PVC leachate exposure resulted in a relative reduction in transcription of a different set of genes which may also be associated with metal transport: a putative manganese ABC transporter PMT9312_RS03085-90, cation:proton antiporter PMT9312_RS08725 and anion transporter PMT9312_RS01090. A number of genes within the cell wall/membrane biogenesis functional category also showed strong reductions in transcription under leachate exposure conditions which may also affect movement of substances into and out of the cell.

In NATL2A, far fewer genes were significantly differentially transcribed. Of the genes that did show a strong transcriptional response to plastic leachate exposure, many are annotated as hypothetical proteins and do not have functions ascribed (Supplementary Data 4). Within the functionally annotated set of strongly differentially transcribed genes were a number of putative regulators that showed transcriptional increases in leachate exposed cultures (Supplementary Table 1). DNA-binding response regulator PMN2A_RS05195 increased under HDPE exposure, whereas GntR family transcriptional regulator PMN2A_RS09350 increased under PVC exposure, indicating that regulation of the leachate exposure response may be mediated by different genes in each strain (Supplementary Table 1). Other significant transcriptional changes observed in NATL2A include reduced transcription of genes involved with nitrogen metabolism under HDPE leachate exposure with substantial decreases observed for genes encoding a ferredoxin-nitrite reductase, nitrite transporter, urea ABC transporter substrate-binding protein (PMN2A_RS09785, RS09790, RS08475) as well as the global nitrogen regulator NtcA (RS01460). PVC leachate exposure impacted transcription of genes associate with cell wall/membrane biogenesis and transport, such as sugar transferase (PMN2A_RS07615) and a porin (PMN2A_RS02250).

Leachates are a mixture of organic and inorganic substances

Exploratory analyses were performed on the HDPE and PVC leachates to investigate their composition and attempt to determine possible contributors to the observed growth and transcriptomic impacts. Untargeted Liquid Chromatography–Mass Spectrometry (LC–MS) analysis of the organics in the leachates indicated that the plastic leachates contained a complex mixture of organic components, whilst not identifying specific compounds. The HDPE and PVC leachates contained 5877 and 10658 components, respectively, that were absent or observed at substantially lower levels in the AMP1 media. Of these, 5799 were observed in the leachates from both plastics, with PVC leaching a number of additional components. Many components also appeared to be more highly abundant in the PVC leachate compared to either the HDPE leachate or the basal AMP1 media (Supplementary Fig. 1).

Although the broad chemical screening applied did not allow identification of individual chemical species, library searches of the mzCloud mass spectral database and Compound Discoverer Extractables and Leachables mass list were performed. The most highly enriched leachate components had no matches in either database. Database matches (tentative identifications) were obtained for a relatively small number of strongly enriched (log 2 fold > 2) components in the PVC and HDPE leachate (168 and 53, respectively, Supplementary Data 5).

Leachate elemental composition analyses conducted using Inductively Coupled Plasma–Optical Emission Spectrometry and Mass Spectrometry (ICP–OES and ICP-MS) identified elements enriched in leachates relative to basal medium. Zinc (Zn) was observed at higher concentrations in HDPE leachate than AMP1 basal medium (~31X). HDPE leachate also contained manganese and nickel at concentrations above the method detection limit (MDL), whereas levels in the control were below this level, indicating that these are also enriched to some degree in these leachates.

In the PVC leachate Zn was highly enriched (~564X) relative to basal media, and strontium levels were also enriched (~7X higher than basal media) (Supplementary Table 2). Copper was also measured at levels above the method detection limit (MDL) in the PVC leachate sample but was below this limit in the control suggesting some degree of enrichment.