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Pubag Copy: Download This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/copyright Author's personal copy Anaerobe 16 (2010) 74–82 Contents lists available at ScienceDirect Anaerobe journal homepage: www.elsevier.com/locate/anaerobe Ecology/environmental microbiology Spatial and temporal changes in the microbial community in an anaerobic swine waste treatment lagoon Kimberly L. Cook a,*, Michael J. Rothrock Jr. a,1, Nanh Lovanh a, John K. Sorrell b, John H. Loughrin a a USDA-ARS, AWMRU, Bowling Green, KY 42104, USA b Western Kentucky University, Bowling Green, KY 42104, USA article info abstract Article history: Microorganisms are central to both the beneficial (organic degradation, nutrient removal, biogas Received 3 December 2008 production) and detrimental (odor production, pathogen contamination) effects of swine waste storage Received in revised form systems. In this study, both quantitative (real-time polymerase chain reaction) and qualitative (dena- 3 June 2009 turing gradient gel electrophoresis, cloning, sequence analysis) molecular analyses were used to track Accepted 9 June 2009 spatial and temporal changes in the microbial community of swine slurry from a 0.4 ha anaerobic lagoon. Available online 16 June 2009 The lagoon, located in a region of western Kentucky which has a humid, subtropical environment, was sampled on a monthly basis (n ¼ 10) over a period of one year at four different depths (top, 51 cm from Keywords: Waste treatment the top, 152 cm from the top, and bottom >198 cm). The concentration and diversity of Bacteroides sp. Denaturing gradient gel electrophoresis was seasonal (up to 90% decrease between March and June). Hespellia sp. and other clostridial species, on 7 À1 (DGGE) the other hand, were endemic in the slurry (concentrations up to 1.0 Â 10 cells mL slurry) regardless Lagoon of time of the year or lagoon depth. Results suggest that there were seasonal effects on the microbial Odor community in the swine lagoon, while the effect of depth was not as pronounced. Seasonal changes in Swine the microbial community in stored wastes may be (directly or indirectly) correlated with changes in malodor emissions from lagoons. Published by Elsevier Ltd. 1. Introduction pathogen contamination. Land application of waste products may pose an additional risk to environmental resources through release Over the last 30 years animal production has become increas- of excessive nutrients, salts and organic materials, among others ingly intensive with fewer operations producing larger numbers of [2,3]. Additionally, long term or excessive application of manure animals. As a consequence, the animal waste is produced and may lead to eutrophication of water sources or soil deterioration. maintained in fewer facilities and in greater concentrations [1].In With the national swine inventory exceeding 65 million [4] and swine production systems, this mixture of feces, urine, and wash steadily increasing, there is greater need to improve swine waste water is stored in pits beneath the facility or in lagoons located storage and handling to maximize the benefits while mitigating any adjacent to confinement areas. The manure is a valuable resource negative consequences of animal waste utilization. Microorganisms for crop fertilization and soil conditioning. However, there are also are central to both the beneficial (organic degradation, nutrient significant concerns associated with the handling of increasing removal, biogas production) and detrimental (odor production, volumes of swine wastes including malodor emissions and pathogen contamination) effects of swine waste storage systems. Therefore, understanding more about how the microbial commu- nity functions in stored swine manure should aid in developing better means for management and usage of waste materials. Abbreviations: DGGE, Denaturing gradient gel electrophoresis; QRT-PCR, The microbial community in swine slurry has been charac- Quantitative, real-time PCR; CE, Clostridia/Eubacteria; BPP, Bacteroides/Prevotella/ terized by traditional culture methods [5], 16S rDNA clone Porphryomonas; PCA, Principle component analysis; UPGMA, Unweighted pair group method with arithmetic mean. sequence analysis [6,7] or other molecular microbial community * Correspondence to: Kimberly L. Cook, USDA-ARS, AWMRU, 230 Bennett Lane, analysis techniques [8–10]. These studies showed that the slurry Bowling Green, KY 42104, USA. Tel.: þ1 270 781 2579x232; fax: þ1 270 781 7994. community is dominated by low G þ C gram positive bacteria E-mail addresses: [email protected] (K.L. Cook), michael.rothrock@ars. (predominantly Clostridium–Eubacterium species) and Bacteroides usda.gov (M.J. Rothrock Jr.), [email protected] (N. Lovanh), john.sorrell@ from the gram-negative bacteria. This is similar to the population wku.edu (J.K. Sorrell), [email protected] (J.H. Loughrin). 1 Present address: Coastal Plains Soil, Water and Plant Research Center, 2611 in swine feces in which 81% of clones belonged to the low G þ C W. Lucas Street, Florence, SC 29501-1242, USA. gram positive bacteria and 11% to Bacteroides [11]. As in most 1075-9964/$ – see front matter Published by Elsevier Ltd. doi:10.1016/j.anaerobe.2009.06.003 Author's personal copy K.L. Cook et al. / Anaerobe 16 (2010) 74–82 75 other environments, culturability is very low in swine slurry (20% (top, 51 cm from the top, 152 cm from the top, and bottom or less) [5]. Most of the culturable organisms can be identified >198 cm) with a pulley system designed so samples can be (95–97% 16S rDNA sequence similarity to known organisms) retrieved at specific depths. The bottom of the lagoon ranged from while less than 50% of clones from 16S rRNA gene libraries are 250 cm to 300 cm, except at drawdown. Slurry from the lagoon was closely related to those of known organisms [6,7,10]. Others have land-applied during September 2006 and April 2007 resulting in found pathogenic organisms including Salmonella sp. and a drawdown of the lagoon depth to 124 cm and 170 cm, respec- Campylobacter sp. in slurries [12,13]. These recent studies have tively. Samples were taken from all depths approximately every provided insight into the dominant species in swine slurry and month (n ¼ 10) from June 2006 to May 2007. Lagoon temperature data on the occurrence of pathogens, but more information is was monitored with two HOBO water temperature probes (Onset needed to understand how the microbial community responds to Computer Corporation, Bourne, MA). One was floated on the chemical, physical and environmental fluctuations in these waste surface of the lagoon while the other was weighted with lead storage systems. sinkers and sunk to the bottom of the lagoon. Both probes were More than 150 chemical compounds can be emitted from waste moored to an anchor at the edge of the lagoon for retrieval. storage systems including small-chain volatile fatty acids, phenols, A combination electrode (Fisher Scientific, Hampton, NH) was indoles, ammonia and hydrogen sulfide [9,14,15]. Many of these used to determine pH. Chemical oxygen demand (COD) was compounds are problematic due to their low threshold for detec- determined using dichromate reactor digestion kit for high tion and difficulties involved in identifying any direct relationship strength wastewater (Chemetrics Inc., Calverton, VA). One mL between odor production from livestock facilities and concentra- samples were digested for 2 h at 150 C and after the samples tions in air emissions. For example, hydrogen sulfide and other cooled, absorbance was read at 620 nm. Total dissolved solids (TDS) sulfur-containing compounds are responsible for as much as half of were determined according to standard method [19]. TDS were the odorants in swine waste [15,16]. However, the form and filterable-solids fraction which consists of both organic and inor- concentration of the emissions depends on a complex interaction of ganic molecules and ions that are present in true solution in water. factors including transport efficiency, lagoon physical parameters Total organic carbon (TOC) was determined by combustion [20] of (temperature and pH) and differences in microbiological pop- the lagoon water using a Vario Max CN analyzer (Elementar ulations and activities [3,15]. Americas, Inc. Mt. Laurel, NJ). Many of these malodorous compounds are generated as a consequence of metabolism of waste components by microor- 2.2. DNA extraction and PCR amplification ganisms [3,14,17]. However, much less is known about the micro- bial community dynamics in waste storage systems than is known DNA was extracted from swine lagoon slurry samples (0.5 mL) in about the physical or chemical parameters within those systems duplicate for each stratum on each sampling day using the [9,18]. Merrill and Halverson [9] evaluated variations in microbial FastDNAÒ Spin kit for soils (MP Biomedical, Solon, OH, USA) communities associated with stored swine manure in lagoons. They according to manufacturer’s specifications. Specific PCR primer sets found that there were seasonal changes in community profiles that were used to target the 16S rRNA gene of the total microbial pop- may be linked with malodor emissions. However, the method they ulation, the Clostridia/Eubacteria (CE) group, or the Bacteroides/ used for community analysis (fatty acid methyl ester (FAME)) did Prevotella/Porphryomonas (BPP) group in the swine lagoon (Table 1). not permit identification of specific organisms that were associated One to 10 ng of DNA extract was used as PCR template with the with shifts in the community.
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