Effect of Alum Amendment on the Bacterial and Fungal Populations in Poultry Litter Michael J Rothrock Jr., Jason G. Warren, Kimberly L. Cook, Karamat Sistani USDA-ARS Animal Waste Management Research Unit Bowling Green KY 42104 Special Thanks to: John Sorrell (USDA-ARS AWMRU) Tyler Allen (Vanderbilt University) For More Information Contact: Michael J Rothrock Jr, Ph.D. USDA-ARS AWMRU 230 Bennett Lane Bowling Green, KY 42104 Phone: 270-781-2260 ext. 223 E-mail: mrothrock@ars.usda.gov BACKGROUND AND OBJECTIVES Alum (Al2(SO4)3 · 14 H2O) is a commonly used acidifier amendment added to poultry litter in houses, which has a variety of beneficial effects: Decreases ammonia volatilization Increase P availability to plants, by decreasing water soluble P Reduces estrogenic compounds, heavy metals, and pathogens in soils (when applied as alum amended poultry litter) Ammonia volatilization is of key importance due to the detrimental effects on flock health and performance, as well as the environmental issues with the emissions of large amounts of ammonia from the houses The chemical and physical affects of alum addition on poultry litter, and its affect on ammonia volatilization, has been extensively studied, but very little work has been performed on the biological effects of alum addition Our goals were to: Monitor the changes in major microbial communities in acidified versus normal litter Determine which group(s) of microbes were most affected by alum addition, Compare group specific PCR primer sets to general primer sets MATERIALS AND METHODS Incubation chambers, in triplicate, contained either 10% (w/w) alum treatment (ATPL) or no treatment (NTPL), and all litters were normalized to a moisture content of 30%. Lids with a small air hole were placed on the chambers, and they were incubated at 25 °C for 4 months (typical grow-out is 6 weeks) Every 2-4 weeks, litter samples were taken for microbiological and physiochemical analyses For the microbiological analyses, total DNA was extracted from a litter samples and this DNA was used for two different types of molecular analysis: Denaturing Gradient Gel Electrophoresis (DGGE) – For Community Fingerprints Quantitative Real-time PCR (QRT-PCR) – To determine concentrations of certain microbial groups For DGGE, general PCR primers were used to amplify the total bacterial and fungal communities, whereas group specific primers were used to target the High %GC Gram positives (Actinomycetes) and Low %GC Gram Positives (Clostridia/Eubacteria and Bacillales) For QRT-PCR, general PCR primers were used to amplify the total bacterial and fungal communities, whereas group specific primers targets the dominant urease producers in poultry litter (PLUP) as well as three known pathogens (E. coli, Campylobacter jejuni, and Salmonella sp.) BACTERIAL 16S DGGE (GENERAL AND GROUP SPECIFIC) Total bacterial community fingerprint was dominated by 4 bands (Fig. 1A; BacDGGEBand1, 2, 12, 13). 3 matched Actinomycetes, 1 matched a Low % GC Gram Positive (Fig. 1B) Overall, minimal differences were observed throughout the experiment For NTPL samples: 4 bands (BacDGGEBand3, 4, 5, 9) that are present at T=0 are reduced/disappear between weeks 8-12, if not sooner. All matched Low %GC Gram Positives For ATPL samples: 4 bands (BacDGGEBand3, 4, 5, 9) that are present at T=0 are reduced/disappear by week 4 Bands matching the Cytophaga/Flavobacterium/Bacteroides cluster appeared starting at week 12 POTENTIAL PROBLEM: If the community is dominated by few members, then general PCR primer sets reveal a less diverse community than there is SOLUTION: Use PCR primers that target specific groups Actinomycetes • Clostridia/Eubacteria Lactobacilli/Bacilli/Streptococci/Staphylococci (Low %GC Gram Positive Group) Low %GC Gram Positives Acidified litter (ATPL) resulted in a 1 month quicker reduction in community diversity and fingerprint intensity in both the C/E and LGC groups, as compared to the normal litter (NTPL; Fig. 2A,B) Sequences from these primer sets (LGC DGGEBands and C/E DGGEBands) matched similar genera that were found from the sequence analysis of the general primer set bands (BacDGGEBands; Fig. 2C) Much higher diversity in both groups was observed as compared to their diversity using the general bacteria primer set (Fig 1A) Fig. 1: Total Bacteria Fig. 2: Low %GC Gram Positives Fig. 3 Actinomycetes Actinomycetes Acidified litter (ATPL) did not reduce the diversity or intensity of the Actinomycetes community fingerprint as compared to the normal litter (NTPL; Fig. 3A) A shift in community fingerprints for both litter types was observed at week 8, but it appears to a temporal shift, not a treatment based shift Sequences from this primer set (ActDGGEBands) generally matched similar genera that were found from the sequence analysis of the general primer set bands (BacDGGEBands; Fig. 2C) Again, a much higher diversity was observed with the group specific, rather than general, primer set. CONCLUSIONS The use of group specific PCR primers targeting known relevant groups allowed for a much greater assessment of diversity, as compared to using general PCR primer sets Alum amendment reduced groups known to dominate poultry litter (Low %GC Gram Positives, specifically Clostridia/Eubacteria), while having very little effect the Actinomycetes. It was also shown to significantly reduce common pathogens, such as C. jejuni and E. coli The most significant change to the poultry litter microbial communities as a result of alum amendment was fungal bloom (shown by both DGGE and QRT-PCR) When the uric acid (i.e. organic-N) content was measured, most of the uric acid hydrolysis coincided with the PLUP peak in the NTPL samples, but coincided with the fungal peak in the ATPL samples. Considering numerous fungal taxa possess ureolytic capabilities (including. Aspergillus sp.), their influence on N content in acidified litter may be considerable FUNGAL 18S DGGE Fig. 4: Total Fungi The fungal populations exhibited the largest shift of any of the microbial groups observed, and represented the only group that exhibited an increase in intensity and diversity with the alum addition to poultry litter (Fig. 4A) While diversity was low in the original litter, by week 4 in the ATPL litter there was a vast increase in the diversity and richness of the fungal community. This diversity was constant throughout the remaining three months of the experiment Acidifying the litter may be selecting for fungal growth, since they are known to be acid tolerant/acidophilic and are able to decompose the organic matter (bedding materials) in poultry litter In the NTPL samples, the fungal intensity was greatly reduced by week 4, and the fungal bands were absent for the remainder of the experiment. Phylogenetic analyses of the fungal bands (Fig. 4B) revealed that a majority of the bands that dominate the fungal community in the ATPL samples were similar to Aspergillus sp. (FungDGGEBand5, 6, 9), whereas the remaining two dominant bands (FungDGGEBand7, 8) represent the two fungi that were present in both litter types at T=0. Chemical analyses of the litter showed a large amount of hydrolysis of uric acid (precursor to urea, which is hydrolyzed to produce the ammonia) occurred in the ATPL samples between weeks 4-8, which coincided with this explosion in fungal diversity. FUTURE DIRECTIONS Determine the ureolytic potential of this dominate fungal community in acidified litter, and test the use of fungicides (i.e. Clinafarm®) to inhibit the hydrolysis of the uric acid/urea in the litter Aid in reduction of ammonia • Retain litter N in an organic form Isolate and characterize the fungi that produced from alum addition to poultry litter Inoculate fresh litter with these fungi to determine their growth on the litter, and determine their ability to transform N compounds within the litter Determine how a variety of commonly used litter amendments (Al+Clear®, PLT®, Agrotain®) effect microbial communities using DGGE and QRT-PCR analyses QRT-PCR ANALYSES (16S, 18S, ureC) QRT-PCR was used to determine cell concentrations of pertinent microbial groups in both NTPL (Fig.5 top) and ATPL (Fig.5 bottom) litters Total Bacteria (16S) • Total Fungi (18S) Dominant poultry litter urease producing bacterial group (PLUP) In the NTPL samples (top): Both the total bacteria and PLUP groups exhibited a peak in concentration at week 4 Cellular concentrations for these two groups fell below T=0 by week 8 for PLUP and week 16 for total bacteria Fungal populations steadily decreased from the beginning of experiment In the ATPL samples (bottom): Both the total bacteria and PLUP populations steadily decreased from the beginning of the experiment Fungal populations significantly increased by week 4 of the experiment, and peaked at week 8 (~1.5 log increase). Fungal concentrations decreased by the end of the experiment, but they were still more than 1 log greater than T=0. Fig. 5: Changes in pertinent microbial concentrations throughout the incubation for NTPL (top) and ATPL (bottom) litters 50 100 150 200 250 300 ureC (PLUP) 16S (Total Bacteria) 18S (Total Fungi) 2 4 6 8 10 12 14 16 25 75 1000 2000 3000 4000 Sampling Week Percent Change from T = 0 QRT-PCR ANALYSES (PATHOGENS) Acidified litter (ATPL) resulted in a ≥ 3 log reduction of C.jejuni by week 4, whereas a < 1 log reduction was seen in the normal litter (NTPL). At week 8, C. jejuni levels were below detection (< 104 cells/g) in both litter types For E. coli, minimal reduction was seen in the NTPL litter by week 4, while there was ~1.5 log reduction in the ATPL samples. By week 8, E.coli levels were still 1 log lower in the ATPL, as compared to NTPL. Salmonella sp. was below the detection limit (≤ 5x103 cells/g) throughout the entire incubation NTPL ATPL