Our results show that differences in environmental conditions between inland and coastal sinkholes, caused mainly by the inflow of seawater in the latter, influence the microbial community structure of their sediments. Furthermore, the microbial community structure also varied within the sinkholes and according to the sediment zone sampled, suggesting that a connection between the atmosphere in the outermost location of the sediments and sunlight creates an environment distinct from that found in deeper caves. Together with the different environmental factors that were measured (in situ physicochemical composition of water and sediment) these characteristics could drive niche-specific microbial community structures associated with the sediment zones. Additionally, beta-diversity analysis showed separate clustering of the sediment microbial communities from the coastal and inland sinkholes, and of the WM zone from cavern and cave zones at both sinkholes. Microbial community structure associated with karst environments have shown to be significantly influenced by environmental factors as seen in the Bahamian blue holes19, a coastal sinkhole13, a Floridan anchialine sinkhole20, and sediments from Chinese karst caves21.
Microbial communities from karst sediments can be limited by nutrients such as carbon, phosphorus, and nitrogen21, therefore, influencing their structure. Differences in the microbial community composition associated with multiple environmental factors (moisture, type of niche, nitrogen) were also reported in karst cave sediments from China21. Previous studies had shown there was no effect on the alpha diversity of water column assemblages in the Yucatan groundwater associated with the type of sinkhole (inland or coastal)6. However, this observation may be limited due to the low sample number used in the study6. For other karst sinkholes, the microbial community dynamics differ between the water column and the sediments6,22,23,24.
The karst caves and sinkholes of the underground river in Yucatan are characterized by low phosphorus concentrations and high levels of nitrate, mostly related to Anthropocentric activities (urban developments, farms and agriculture)3. The inland sinkhole at Noh Mozón showed the highest concentration of nitrate detected in the study and, not surprisingly, the area is surrounded by agricultural fields. The presence of organic matter in the sinkholes from the Yucatan peninsula are highly dependent on the connection between the cave systems, on the levels of exposure to light, and on their morphology3. High concentrations of organic carbon (661 ± 132 μM) and methane (6466 ± 659 nM) have been reported in the top layer of the water masses in coastal sinkholes before13. In this study, the highest concentration of organic carbon was observed in the sediments from the coastal sinkhole, likely originating from the surrounding vegetation and from seawater intrusion. We hypothesize that the differences in nutrients found at these two types of sinkholes influence the structure of their microbial communities.
Other environmental factors such as pH and dissolved oxygen (DO), may also contribute significantly to the composition and structure of microbial communities, as seen in freshwater lake sediments22. Davis and Garey20 reported distinct microbial communities with unique functions for each water layer from an anchialine sinkhole from the Florida karst aquifer and suggested that this occurred as a result of the influence of the hydrochemistry, including differences in the concentration carbon and other nutrients from the environment20. Analyses of the sinkhole caves from the Yucatan underwater river support observations that physical and chemical parameters create distinct ecological niches which host unique microbes, as a high abundance of exclusive (not shared) ASVs were observed in the three sediment zones at both locations.
The taxonomic diversity from the coastal and inland sinkholes included Chloroflexi, Crenarchaeota, Desulfobacterota, Proteobacteria, Nitrospirota, Bacteroidota, and Firmicutes as the most abundant phyla in the sediment samples, however, there were differences in the relative abundance associated with the type of sinkhole and sediment zone. Some of these phyla (Chloroflexi, Proteobacteria, and Bacteroidetes) have been reported in sediments from freshwater karst sinkholes from Lake Huron25 in water and sediments from other sinkholes in the Yucatan peninsula6, and in the karst caves bacteriome from southwest China21. A study that included coastal marine sediments from two sites in the Yucatan peninsula, showed high abundances of Spirochaeta, Desulfococcus, Clostridium, Psychrobacter26, four genera that were abundant in the coastal sinkhole. However, Desulfococcus, Synechococcus were also abundant in the inland sinkhole. Of the most abundant genera reported for sediments from different marine environments in the Yucatan coast23, Acinetobacter, Desulfotignum, Desulfovibrio, Pseudomonas, Sedimenticola, and Sulfurimonas were also present in the coastal sinkhole while only Pseudomonas and Sedimenticola were also present in the inland sinkhole23. The high number of families shared between the coastal sinkhole and marine sediments from the Yucatan coast, together with the salinity levels registered at the bottom layer of the water column in the coastal sinkhole, suggest an interconnection between these two environments which shapes the microbial communities present in the sediments of caverns and caves of this sinkhole. The genus Nitrospira was abundant in the WM from the coastal sinkhole and in all sediment zones from the inland sinkhole. This genus has been reported as one of the most abundant in the surface of speleothems from El Zapote coastal sinkhole2, and is considered a complete ammonia oxidizer (comammox), meaning it converts ammonia to nitrate through nitrite. A negative correlation between abundance of this genus and salinity has been reported before, which could explain the low concentration of Nitrospira in the cavern and cave from the coastal sinkhole, where the highest salinity was observed27. Connectivity between coastal sinkholes and the ocean, as well as the terrestrial input of soil organic matter (OM) has been reported for the underground karst aquifer in the Yucatan peninsula13. As in other sediments, degradation of OM is carried out by several MFGs including acetogenic bacteria, methanogens, and sulfate reducers13,20,28. When these MFGs were analyzed in coastal and inland sinkholes, differences in their relative abundances were clearly marked by the type of sinkhole and by the sediment zone analyzed, supporting the hypothesis that environmental differences drive microbial community distributions in these niches. The high abundance of sulfate-reducing bacteria (SRB) in the three sediment zones from the coastal sinkhole suggests that sulfate reduction is a predominant function. SRB degrade organic matter using sulfate with sulfide as waste or end-product19,30, originating hydrogen sulfide (H2S)29, which could explain the low concentration of sulfate the hydrogen sulfide (H2S) cloud observed and previously reported in the WM zone of El Zapote coastal sinkhole30. In this study, high levels of sulfate (SO−4) were measured in the water samples from the cavern and cave zones from El Zapote sinkhole, which could be associated with sulfate-rich deposits, such as gypsum beds, which have been reported in other sinkholes from the Yucatan peninsula (up to 2400 mg/L of sulfate concentration)3. However, we do not disregard other possible sources of sulfate, associated with seawater intrusion or as a product of sulfide or sulfur oxidation29,31 by sulfur-oxidizing bacteria detected in this study.
The inland sinkhole had a low concentration of sulfate and low abundances of SRB. The high abundance of methanogenic bacteria in the WM zone from the coastal sinkhole detected in the MFG analysis supports the previous hypothesis of acetoclastic methanogenesis due to high inputs of organic matter13. Methylotrophic bacteria were most abundant at the inland sinkhole in the WM zone, suggesting the presence of methyl compounds, such as methane or methanol which can be used as a source of carbon and energy32. High methane concentrations have been quantified in shallow water masses from the Yucatan aquifer system13, consistent with observations from this study. ‘Candidatus Methylomirabilis’ was identified in the sediment of the WM zone from Noh-Mozón and has been previously described as being able to perform nitrite-dependent anaerobic methane oxidation, using methane as electron donor and nitrate and nitrite as electron acceptors33, which would be possible in these sediments considering the low levels of oxygen (average of 2.3 mg/L) detected in the water column above them and assuming this would lead to lower levels of oxygen in the sediments. We hypothesize that bacteria from this genus could be using the nitrite produced by ammonia oxidizing bacteria and archaea observed in this zone (Nitrosomonadaceae, Nitrospiraceae, and Nitrosococcaceae). The low abundance of methanotrophic microbes in the coastal sinkhole (mainly the cavern and cave zones) could be derived from the high concentrations of hydrogen sulfide previously reported at this location, which have been suggested to be toxic to methane-oxidizing microbes34. Therefore, a decrease in the anaerobic oxidation of methane, and a poor methane removal capacity is hypothesized in the sediments from this coastal sinkhole. Further research could focus on the influence of the saline intrusion on methanotrophic microbes and methane levels in El Zapote sinkhole sediments. As expected, photosynthetic bacterial abundances differed with the type of sinkhole and sediment zone. Both sinkholes are so the presence of daylight can start the photosynthetic process which would occur most in the WM zone. However, only the inland sinkhole showed a high abundance of photosynthetic bacteria within its WM zone. The coastal sinkhole water column and sediments would be deprived of photosynthetic bacteria since the water source is the underground aquifer, lacks photosynthesizers. Ammonia oxidizing bacteria (AOB) and archaea (AOA), and nitrite-oxidizing bacteria (NOB) were relatively abundant in the three sediment zones from the inland sinkhole and in the WM from the coastal sinkhole, these observation at the WM from El Zapote agree with previous observations2. Anaerobic ammonium oxidation (anammox) uses nitrite (as a product of nitrate reduction), as electron acceptor35. The high levels of nitrate concentration in the water column from the three sediment zones at the inland sinkhole and at the WM from the coastal sinkhole may influence the abundance of AOB, AOA and NOB in the sediments from these zones, while NH4+ and nitrite values were below detection limit (< 0.2 mg/L and < 1.5 mg/L, respectively) in all sediment zones at both sinkholes, suggesting an oxidation ammonium pathway with N2 and 2H2O as products35, and a rapid cycling of ammonia and nitrite in the sinkhole sediments. Nitrite oxidation could also be responsible for the high NO3− concentrations in the water column from the sinkholes, however, we do not discard other sources of NO3− including the agricultural and wastewater infiltration into the groundwater aquifer ecosystem4, or the presence of abundant bat population which has been previously associated with higher nitrate concentrations in caves from the Yucatan peninsula8. Lower concentrations of nitrate (< 1.5 mg/L) in the sediments from caverns and caves correlate with low abundance of NOB. It is thus possible that continuous input of organic matter favors microbial groups associated with the nitrogen cycle (ammonia, nitrite, denitrification) in the sinkholes (mainly the inland sinkhole). Further research could investigate the effects of natural and anthropogenic sources of OM, on the sediment microbial communities in underground caves from the aquifer in the Yucatan peninsula.
When the microbial communities from the two sinkholes were compared to several environments from the Yucatan peninsula, there was a clear difference between communities from water, soil/sediment and speleothems. These differences between the water column and sediment communities have been previously reported in lakes22, karst caves6, and marine environments23,24. We were unable to perform a comparative analysis of environmental variables from sediments and water samples from our study areas since cations, anions, and in situ parameters were not measured in sediments, and TOC, TIC, and total N were only measured in the water column. However, comparisons to other environments suggest that the water and sediments from sinkholes are unique environments which can provide specific ecological niches and host distinctive microbes with specialized functions.
In this study, we successfully revealed differences in the sediment microbial community structure associated with physical and chemical characteristics within two types of sinkholes (coastal and inland) and within defined sediment zones (WM, cavern, and cave). Based on the analyses of environmental variables, taxonomic diversity, alpha and beta diversity, and the potential metabolic function of these microbial communities, we demonstrate that each sinkhole represents a unique environment harboring different microbial assemblages. Therefore, we plan to pursue future research that includes investigating a greater number of coastal and inland sinkholes to determine if the patterns found in this study apply to the majority of coastal and inland sinkholes in the Yucatan peninsula. We recognize the limitations of partial sequencing of the 16S rRNA gene and, therefore, all hypotheses about MFGs environmental functions should investigated by further experimental and metagenomic studies that shed more light on their actual roles in geochemical cycles occurring in the underground aquifer of the Yucatan peninsula.
Source: Ecology - nature.com
