Diversity,Abundance, and Spatial Distribution of Sediment Ammonia-Oxidizing Betaproteobacteria in Response to Environmental Gradients and Coastal Eutrophication in Jiaozhou Bay,China

Ongoing anthropogenic eutrophication of Jiaozhou Bay offers an opportunity to study the influence of human activity on bacterial communities that drive biogeochemical cycling. Nitrification in coastal waters appears to be a sensitive indicator of environmental change, suggesting that function and structure of the microbial nitrifying community may be associated closely with environmental conditions. In the current study, the amoA gene was used to unravel the relationship between sediment aerobic obligate ammonia-oxidizing Betaproteobacteria (Beta-AOB) and their environment in Jiaozhou Bay. Protein sequences deduced from amoA gene sequences grouped within four distinct clusters in the Nitrosomonas lineage, including a putative new cluster. In addition, AmoA sequences belonging to three newly defined clusters in the Nitrosospira lineage were also identified. Multivariate statistical analyses indicated that the studied Beta-AOB community structures correlated with environmental parameters, of which nitrite-N and sediment sand content had significant impact on the composition, structure, and distribution of the Beta-AOB community. Both amoA clone library and quantitative PCR (qPCR) analyses indicated that continental input from the nearby wastewater treatment plants and polluted rivers may have significant impact on the composition and abundance of the sediment Beta-AOB assemblages in Jiaozhou Bay. Our work is the first report of a direct link between a sedimentological parameter and the composition and distribution of the sediment Beta-AOB and indicates the potential for using the Beta-AOB community composition in general and individual isolates or environmental clones in the Nitrosomonas oligotropha lineage in particular as bioindicators and biotracers of pollution or freshwater or wastewater input in coastal environments.Nitrification, the oxidation of ammonia to nitrate via nitrite, plays a critical role in the biogeochemical cycling of nitrogen and the formation of the large deep-sea nitrate reservoir (37, 46, 51). Because the N cycle may affect the global C cycle, shifts in N transformation processes may also affect the climate (30, 40). Nitrification is an important bioremediation process in human-perturbed estuarine and coastal ecosystems, where it may serve as a detoxification process for excess ammonia (14). If coupled to classical denitrification or anaerobic ammonium oxidation (anammox), these processes may remove most of the anthropogenic N pollution (51, 81). Bacterial nitrifiers may also cooxidize a variety of xenobiotic compounds (3, 49). On the other hand, nitrification may lead to enhanced production of the potent greenhouse gases nitric oxide (NO) and nitrous oxide (N₂O) (15). Because the input of excess ammonia stimulates the growth of ammonia-oxidizing microorganisms, research in coastal environments and ecosystems increasingly includes the study of microbial communities involved in nitrification.Marine nitrification is performed by chemolithoautotrophic proteobacteria and the newly discovered ammonia-oxidizing archaea (AOA) (47); nevertheless, reliable information on the individual contributions of each cohort to the process is still lacking (72) and the contributions likely vary in different environments (33, 51, 93). Beta- and gammaproteobacterial aerobic obligate ammonia-oxidizing bacteria (AOB) are known to catalyze the oxidization of ammonia to nitrite, the first and rate-limiting step of nitrification (3). Because of their monophyletic nature, diversity, and important environmental functionality, the betaproteobacterial AOB (Beta-AOB) have served as a model system in the study of fundamental questions in microbial ecology, including microbial community structure, distribution, activity, and environmental response (9, 49, 92).The growth of AOB is slow, and present isolates represent only a fraction of their natural diversity. Culture-independent molecular methods provide a more convenient and accurate approach for community analyses (76, 92). All AOB genomes contain at least one cluster of amoCAB genes encoding functional ammonia monooxygenase (AMO), which catalyzes the oxidation of ammonia to hydroxylamine (4). Because AmoA- and 16S rRNA-based phylogenies are congruent (73), the amoA gene has been extensively used as a molecular marker to explore and characterize the structure and diversity of AOB communities in a variety of estuarine and coastal environments (9, 10, 11, 13, 27, 32, 36, 43, 68, 88). Some of these studies indicated that local environmental factors such as salinity, pH, ammonium, and O₂ concentrations might be drivers for the formation of distinct AOB assemblages, in which individual lineages may have evolved differential ecophysiological adaptivity (4, 73, 85). Furthermore, differences in AmoA sequences may correlate with differences in isotopic discrimination during ammonia oxidation, implicating function-specific ammonia monooxygenases (15). Therefore, the AmoA sequences may provide information about the structure and composition of the AOB communities and their ecological function and response to environmental complexity and variability. Despite long-standing efforts, a complete understanding of these relationships is still lacking (9), especially in complex environments such as anthropogenic activity-impacted coastal areas.China consumes more than 20 million tons of N fertilizer each year, leading to a significant increase of coastal N pollution (38). Thus, the China coast is an important location for intense N biogeochemical cycling. Jiaozhou Bay is a large semienclosed water body of the temperate Yellow Sea in China. Eutrophication has become its most serious environmental problem, along with red tides, species loss, and contamination with toxic chemicals and harmful microbes (21, 23, 24, 28, 82, 91). In similar environments with a high input of nitrogenous compounds, surface sediment is a major site for nitrification due to a relatively high AOB abundance and activity (79).Although the China coast is important in N cycling and in related environmental and climatic issues, surprisingly very little is known, especially about the microbial processes and functions involved. On a global scale, it is currently not well understood how the AOB community structure, abundance, and distribution respond to coastal eutrophication, though partial knowledge is emerging (36, 49, 89). Recent studies indicated that spatial distribution and structure of the sediment AOA community could be influenced by a variety of environmental factors, of which continental input may play important roles in estuary and continental margin systems (22, 26). The sediment AOA community may serve as useful biotracers and bioindicators of specific environmental disturbance. Likewise, the sediment AOB community may also serve as biotracers or bioindicators of continental influence, such as eutrophication in coastal environments. In this study, the bacterial functional marker gene amoA was employed to test this hypothesis in the eutrophied Jiaozhou Bay.