Monitoring the perturbation of soil and groundwater microbial communities due to pig production activities

Abstract

This study aimed to determine if biotic contaminants originating from pig production farms are disseminated into soil and groundwater microbial communities. A spatial and temporal sampling of soil and groundwater in proximity to pig production farms was conducted, and quantitative PCR (Q-PCR) was utilized to determine the abundances of tetracycline resistance genes (i.e., tetQ and tetZ) and integrase genes (i.e., intI1 and intI2). We observed that the abundances of tetZ, tetQ, intI1, and intI2 in the soils increased at least 6-fold after manure application, and their abundances remained elevated above the background for up to 16 months. Q-PCR further determined total abundances of up to 5.88 × 10(9) copies/ng DNA for tetZ, tetQ, intI1, and intI2 in some of the groundwater wells that were situated next to the manure lagoon and in the facility well used to supply water for one of the farms. We further utilized 16S rRNA-based pyrosequencing to assess the microbial communities, and our comparative analyses suggest that most of the soil samples collected before and after manure application did not change significantly, sharing a high Bray-Curtis similarity of 78.5%. In contrast, an increase in Bacteroidetes and sulfur-oxidizing bacterial populations was observed in the groundwaters collected from lagoon-associated groundwater wells. Genera associated with opportunistic human and animal pathogens, such as Acinetobacter, Arcobacter, Yersinia, and Coxiella, were detected in some of the manure-treated soils and affected groundwater wells. Feces-associated bacteria such as Streptococcus, Erysipelothrix, and Bacteroides were detected in the manure, soil, and groundwater ecosystems, suggesting a perturbation of the soil and groundwater environments by invader species from pig production activities.

Fig 1

Soil samples were collected in 2005 to 2007 from three agricultural crop fields that applied manure supplied by sites A, C, and E (pig production farms). The first sampling point of each time series is highlighted in bold, which also denotes that soil samples were collected before manure application. The subsequent sampling points and the spatial distance from the first sampling point denote the duration that passed after manure was applied. Manure was applied as a single dose, and no subsequent application was carried out at that particular field throughout the sampled time series.

Fig 2

Map of groundwater wells at the site E pig production farm. The locations of groundwater wells are indicated by black circles, and the numbers in parentheses are well depths (m). An animal confinement building is situated above the waste lagoon. The direction of groundwater flow is indicated by large open arrows. Based on the direction of the groundwater flow and proximities to the waste lagoon, monitoring well E1 and the facility well served as background wells, where contamination was least anticipated. A corresponding stratigraphic column on the right indicates the characteristics of sand layers.

Fig 3

Summed abundances of 16S rRNA genes, tetracycline resistance genes, and integron genes detected in soil samples from agricultural crop fields that applied manure from site A (A), soil samples from agricultural crop fields that applied manure from site C (B), soil samples from agricultural crop fields that applied manure from site E (C), and groundwater samples from site E (D). Soil samples collected before manure application are highlighted in bold. The duration that passed after manure was first applied is denoted after each sample name. Groundwater samples from each well were collected on June and July 2010 at site E and are labeled with the name of the well followed by “a” and “b,” respectively.

Fig 4

Percent abundances of bacterial phyla present in soil (A) and groundwater (B) samples. Soil samples collected before manure application are highlighted in bold. The duration that passed after manure was first applied is denoted after each sample name. Groundwater samples from each well were collected on June and July 2010 and are labeled with the name of the well followed by “a” and “b,” respectively.

Fig 5

Heat map of bacterial populations present in the groundwater at site E. Groundwater samples were clustered into three groups. Highly abundant bacterial populations associated with each group are boxed. Groundwater samples from each well were collected on June and July 2010 and are labeled with the name of the well followed by “a” and “b,” respectively.