Influence of pharmaceutical residues on the structure of activated sludge bacterial communities in wastewater treatment bioreactors

Abstract

Concern is growing over contamination of the environment with pharmaceuticals because of their widespread use and incomplete removal during wastewater treatment, where microorganisms drive the key processes. The influence of pharmaceuticals on bacterial community structure in activated sludge was assessed in small-scale wastewater treatment bioreactors containing different concentrations (5, 50, 200 and 500 μg L⁻¹) of several commonly used pharmaceuticals (ibuprofen, naproxen, ketoprofen, diclofenac and clofibric acid). T-RFLP analyses of the bacterial 16S rRNA genes indicated a minor but consistent shift in the bacterial community structure in the bioreactor R50 supplied with pharmaceuticals at a concentration of 50 μg L⁻¹, compared to the control reactor R0, which was operated without addition of pharmaceuticals. In the reactors operated with higher concentrations of pharmaceuticals, a greater structural divergence was observed. Bacterial community composition was further investigated by preparation of two clone libraries of bacterial 16S rRNA genes from reactors R0 and R50. Most clones in both libraries belonged to the Betaproteobacteria, among which Thauera, Sphaerotilus, Ideonella and Acidovorax-related spp. dominated. Nitrite-oxidizing bacteria of the genus Nitrospira sp., which are key organisms for the second stage of nitrification in wastewater treatment plants, were found only in the clone library of the reactor without pharmaceuticals. In addition, diversity indices were calculated for the two clone libraries, indicating a reduced diversity of activated sludge bacterial community in the reactor supplied with 50 μg L⁻¹ of each of selected pharmaceuticals.

Introduction

‘New emerging contaminants’ have recently gained much public and scientific attention and represent an important issue for environmental pollution. They are manufactured continually, applied widely and released into the environment, thus contaminating groundwater and surface water bodies; however, their burden on the environment has not been comprehensively assessed yet. Among ‘new emerging contaminants’, pharmaceuticals are particularly interesting due to their pharmacological activity and consumption at rates of tons per year (Kasprzyk-Hordern et al., 2008, Daughton and Ternes, 1999). Moreover, it is expected that their worldwide production will increase, due to developing health care systems and higher life expectancies in industrial countries. Substantial amounts of pharmaceuticals can reach the environment, either through direct discharge into the water bodies or due to the inefficient elimination in wastewater treatment plants (WWTPs). As a result, numerous pharmaceuticals have been detected in surface waters, groundwater, wastewater and even in drinking water, in concentrations ranging from ng L⁻¹ to several μg L⁻¹ (Carballa et al., 2008, Gómez et al., 2007, Ashton et al., 2004, Farré et al., 2001, Daughton and Ternes, 1999).

Biological wastewater treatment is one of the most important biotechnological processes, in which microorganisms play a key role in removal of organic contaminants. However, biological WWTPs are primarily designed to remove easily degradable organic substances, whereas degradation efficiency of complex organic compounds is usually less efficient. Thus, several studies have demonstrated that some pharmaceuticals are efficiently eliminated (ibuprofen, naproxen and ketoprofen), while others are resistant to biodegradation (diclofenac and clofibric acid) (Kosjek et al., 2007, Quintana et al., 2005, Ashton et al., 2004). Various removal mechanisms (e.g. sorption, biodegradation and abiotic degradation) may contribute to the total elimination of organic contaminants; yet, for polar acidic pharmaceuticals, microbial degradation is believed to be the most important removal process in activated sludge wastewater treatment (Quintana et al., 2005). A broad bacterial consortium is required to achieve the desired biological conversions and the performance of wastewater treatment largely depends on the bacterial diversity present (Saikaly et al., 2005). Therefore, a fundamental understanding of the microbial community structure and stability as well as its response to different chemicals entering the wastewater, are desirable for stable and efficient WWTP operation.

The aim of this study was to evaluate the influence of commonly used pharmaceuticals on the structure of activated sludge bacterial communities in small-scale pilot wastewater treatment reactors. The compounds involved in the study were polar acidic pharmaceuticals: four non-steroidal anti-inflammatory drugs (NSAIDs: ibuprofen, naproxen, ketoprofen, and diclofenac) and clofibric acid (CLA), an active metabolite of blood-lipid regulators. Terminal restriction fragment length polymorphism (T-RFLP) analysis of 16S rRNA gene sequences was used to characterize and compare the community structure of the bioreactors. Furthermore, to facilitate a more detailed, sequence-based identification of the bacterial species, and to investigate the effect of pharmaceuticals on bacterial community composition, two 16S rRNA gene clone libraries were constructed from the sludge samples of two bioreactors, with and without pharmaceuticals.