Microbiology and Nitrogen Mineralization in Composted Poultry Litter Amended with Biodiesel ...

185 Pages·2014·2.85 MB·English

Microbiology and Nitrogen Mineralization in Composted Poultry Litter
Amended with Biodiesel Wash Water

by

Jonathan Robert Gaiero

A Thesis
Presented to
The University of Guelph

In partial fulfilment of requirements
for the degree of
Master of Science
in
Environmental Biology

Guelph, Ontario, Canada
© Jonathan R. Gaiero, April, 2014
i
ABSTRACT

MICROBIOLOGY AND NITROGEN MINERALIZATION OF COMPOSTED
POULTRY LITTER AMENDED WITH BIODIESEL WASH WATER

Jonathan R. Gaiero Advisors:
University of Guelph, 2014 Professors M. B. Habash, R. Nicol

Committee member:
J. T. Trevors

Composting of poultry litter is an important method to increase the retention of nutrients
such as nitrogen (N) prior to its use as an agricultural soil amendment. Biodiesel wash water
(BWW) was used as a treatment during composting and compared to municipal water as a
control. Molecular analyses examined the effects of BWW on the microbial communities (via
denaturing gradient gel electrophoresis) and the abundance of bacteria, fungi, pathogens, and
ureolytic/uricolytic N-cycling microorganisms (via quantitative real-time PCR). The lack of
large community perturbence and variable abundance of N-cycling microbes supports the use of
BWW, but higher levels of Campylobacter in the mature BWW compost will need to be
examined further. Ureolytic screening of bacterial isolates identified the dominant group PLUP
(poultry litter urease producers). Incubation of PL with BWW and PLUP microbial inoculant,
revealed active ureolysis by PLUP, and inhibition of the enzymes involved in N-mineralization
by BWW.

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ACKNOWLEDGEMENTS

Firstly, I would like to greatly thank my co-advisors Dr. Marc Habash and Dr. Rob Nicol, and
committee advisor Dr. Jack Trevors for their continued support during my project. My Master’s
education would not have been the same without the insightful conversations I had with Marc on
many occasions.

Family, friends and fellow office-mates were incredibly helpful and supportive during my
Master’s. My parents (Robert and Dianne Gaiero), brother (Andrew) and sister (Angela) were
very encouraging and I’m glad to make them proud. I had the privilege of meeting many students
during my graduate studies. Their friendship and help during my thesis will not be forgotten.

The staff at the Ridgetown biodiesel facility, and Cold Springs composting farm, were
instrumental in preparing the samples required for this project. Peter Smith was very considerate
and timely in his chemical analyses for which I am grateful. I am also grateful to Dr. Lee, Dr.
Trevors, Dr. Kari Dunfield, and Dr. Susan Glasauer for the use of their labs and equipment.

Finally I would like to thank the Government of Ontario, the University of Guelph and private
donors, and the Natural Sciences and Engineering Research Council (NSERC) of Canada for
their generous funding.

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TABLE OF CONTENTS

ABSTRACT .................................................................................................................................... ii
LIST OF TABLES ......................................................................................................................... ix
LIST OF FIGURES ........................................................................................................................ x
LIST OF ABBREVIATIONS AND ACRONYMS ..................................................................... xii
CHAPTER 1: INTRODUCTION ................................................................................................... 1
1.1 Poultry litter (PL) .................................................................................................................. 1
1.2 Potential economic cost of N loss from PL ........................................................................... 2
1.3 PL Composting ...................................................................................................................... 3
1.4 Minimizing NH volatilization from PL ............................................................................... 4
3
1.5 General microbiology of PL .................................................................................................. 7
1.6 Microbial N dynamics in PL ................................................................................................. 9
1.6.1 Uric acid mineralization to NH (Fig 2) ...................................................................... 10
3
1.6.1.1 Uricase in microorganisms ................................................................................... 12
1.6.1.2 Urease in microorganisms..................................................................................... 12
1.6.2 Diversity of ureolytic and uricolytic microbes in PL ................................................... 14
1.6.3 Ureolytic activity in a compost environment ............................................................... 16
1.6.4 Treatments used to minimize NH loss from PL by inhibiting mineralization............ 17
3
1.7 Molecular microbial analysis .............................................................................................. 18
1.7.1 Molecular versus culture based methods ..................................................................... 18
1.7.2 Extraction of nucleic acids ........................................................................................... 21
1.7.3 PCR inhibition limits effectiveness of molecular techniques ...................................... 22
1.7.4 Sensitivity of PCR-based assays and safety of composts ............................................ 23
1.7.5 Molecular methods used to study compost microbial communities ............................ 24
1.7.5.1 Molecular ecology of N mineralizing microbes ................................................... 29
1.7.5.2 Poultry litter urease producer (PLUP) .................................................................. 30
1.7.5.3 Genomic studies of ureolytic microbes ................................................................. 30
1.8 Biodiesel production and biodiesel wash water (BWW) .................................................... 32
1.8.1 BWW and inhibition of microbial growth ................................................................... 33
1.9 Summary ............................................................................................................................. 34
1.10 Research objectives ........................................................................................................... 34
CHAPTER 2: MATERIALS AND METHODS .......................................................................... 36
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2.1 Pilot project: large scale composting of PL treated with BWW or MW............................. 36
2.1.1 Materials ...................................................................................................................... 36
2.1.2 General method ............................................................................................................ 36
2.1.3 Temperature measurements ......................................................................................... 37
2.1.4 Sampling ...................................................................................................................... 37
2.2 Optimization of genomic DNA extraction from PL ............................................................ 38
2.2.1 Initial comparison of commercial gDNA extraction kits ............................................. 38
2.2.2 Pseudomonas sp. UG14Lr ........................................................................................... 38
2.2.3 UG14Lr growth conditions .......................................................................................... 39
2.2.4 qPCR inhibitors from PL gDNA extracts .................................................................... 39
2.2.5 Extraction efficiency of the FASTDNA kit ................................................................. 40
2.2.6 Sensitivity of gDNA extractions and detection by PCR .............................................. 40
2.3 DNA extraction methods used in this study ........................................................................ 41
2.3.1 Final protocol for genomic DNA extractions from PL ................................................ 41
2.3.2 From pure cultures ....................................................................................................... 42
2.3.3 Isolation of plasmid vectors from transformed E. coli ................................................ 42
2.4 General molecular analyses ................................................................................................. 43
2.4.1 Determining DNA purity by spectrophotometry ......................................................... 43
2.4.2 Agarose gel electrophoresis ......................................................................................... 43
2.4.3 Cloning ......................................................................................................................... 43
2.4.4 Sequencing and phylogenetic analysis......................................................................... 44
2.5 Molecular analysis of PL by PCR, DGGE, and qPCR........................................................ 45
2.5.1 Primers used in this study ............................................................................................ 45
2.5.2 Quantitative real-time PCR (qPCR) of nitrogen cycling microbes and pathogens ..... 47
2.5.2.1 General procedure of qPCR .................................................................................. 47
2.5.2.2 Plasmid standard curves used for quantification of samples ................................ 47
2.5.2.3 Cell abundances and copy number calculations ................................................... 48
2.5.3 General end-point PCR ................................................................................................ 48
2.5.4 End-point PCR for DGGE ........................................................................................... 49
2.5.5 DGGE .......................................................................................................................... 49
2.5.5.1 General procedure ................................................................................................. 49
2.5.5.2 Band identification: extraction, cloning, and sequencing ..................................... 50
2.5.5.3 Analysis of DGGE profiles ................................................................................... 51
v
2.6 Isolation of culturable ureolytic microbes from composted PL .......................................... 52
2.6.1 Extracting viable microorganisms from PL ................................................................. 52
2.6.2 Media and culture conditions ....................................................................................... 53
2.6.3 Testing ureolytic capacity of cultured microbial isolates ............................................ 54
2.6.4 Identification of positive ureolytic bacterial isolates ................................................... 54
2.6.5 Storage of cultured microorganisms ............................................................................ 55
2.7 Microcosm incubation experiments .................................................................................... 55
2.7.1 Materials ...................................................................................................................... 55
2.7.2 PLUP cultures used for inoculations ............................................................................ 55
2.7.3 Experimental design..................................................................................................... 56
2.7.4 Sampling from the microcosms ................................................................................... 56
2.7.5 Urea extraction and quantification ............................................................................... 57
2.7.6 Urease activity assay .................................................................................................... 57
2.8 Statistical Analysis .............................................................................................................. 58
CHAPTER 3: RESULTS .............................................................................................................. 59
3.1 Optimization of gDNA extraction and qPCR sensitivity of detection from extracts .......... 59
3.1.1 Testing commercial gDNA extraction kits .................................................................. 59
3.1.2 Improved extraction efficiency from PL using the FASTDNA kit ............................. 60
3.1.3 Sensitivity of DNA extraction and detection by qPCR ............................................... 62
3.2 Pilot project: large scale composting of PL treated with BWW or MW............................. 64
3.2.1 Compost temperature ................................................................................................... 64
3.2.2 Quantification of microbes in pilot scale composting ................................................. 66
3.2.2.1 General and nitrogen cycling microbes ................................................................ 66
3.2.2.2 qPCR – Pathogens................................................................................................. 68
3.2.3 Denaturing gradient gel electrophoresis (DGGE)........................................................ 69
3.2.3.1 DGGE - Fungal 18S rRNA gene (Fig 8) .............................................................. 70
3.2.3.1.1 18S rRNA gene DGGE analysis .................................................................... 70
3.2.3.1.2 Band isolations and sequencing ..................................................................... 72
3.2.3.1.3 18S phylotype richness .................................................................................. 74
3.2.3.2 DGGE - Bacterial 16S rRNA gene (Fig 12) ......................................................... 76
3.2.3.2.1 16S rRNA gene DGGE analysis .................................................................... 76
3.2.3.2.2 Band isolations and sequencing ..................................................................... 78
3.2.3.2.3 16S phylotype richness .................................................................................. 80
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3.2.3.3 DGGE - bacterial partial ureC gene (Fig 10a) ...................................................... 82
3.2.3.3.1 PCR of ureC for DGGE ................................................................................. 82
3.2.3.3.2 Partial gene ureC DGGE analysis .................................................................. 82
3.3 Isolation of culturable ureolytic bacteria from poultry litter ............................................... 83
3.3.1 Media and isolate naming ............................................................................................ 83
3.3.2 General culture characteristics ..................................................................................... 84
3.3.3 Testing isolates for ureolytic activity ........................................................................... 84
3.3.4 ureC partial gene analysis of ureolytic bacterial isolates ............................................. 87
3.3.4.1 Preliminary isolate screening by PCR .................................................................. 87
3.3.4.2 ureC partial gene sequencing and sequence alignment......................................... 87
3.3.4.3 Isolation of novel bacterial isolates containing PLUP ureC sequences ................ 93
3.3.4.4 Translated protein vs. nucleotide ureC sequences ................................................ 94
3.3.4.5 Multiple copies of ureC gene per genome ............................................................ 94
3.3.4.6 ureC phylogenetic analysis ................................................................................... 95
3.3.5 16S rRNA gene analysis of ureolytic bacterial isolates ............................................... 97
3.3.5.1 16S rRNA gene sequencing .................................................................................. 97
3.3.5.2 16S rRNA gene phylogenetic analysis ............................................................... 100
3.3.5.3 Comparing 16S rRNA gene identities to ureC BLASTn and BLASTp ............. 102
3.4 Microcosm incubation experiments .................................................................................. 104
3.4.1 PLUP inoculants ........................................................................................................ 105
3.4.2 Moisture content (MC%) and pH .............................................................................. 106
3.4.3 Urea levels in the microcosm ..................................................................................... 107
3.4.4 Urease activity in the microcosms ............................................................................. 109
CHAPTER 4: DISCUSSION ...................................................................................................... 114
4.1 Large scale composting ..................................................................................................... 114
4.1.1 Differences in compost process between BWW and MW treatments ....................... 114
4.1.2 Conclusions ................................................................................................................ 116
4.2 Molecular analysis of microorganisms during PL composting ......................................... 116
4.2.1 Sampling procedure ................................................................................................... 116
4.2.2 Optimization of DNA extraction and detection by qPCR ......................................... 117
4.2.2.1 Extraction efficiency ........................................................................................... 117
4.2.2.2 Sensitivity of detection by qPCR ........................................................................ 118
4.2.2.3 Conclusions ......................................................................................................... 120
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4.2.3 Determining the abundance of microbial groups by qPCR ....................................... 120
4.2.3.1 Pathogens ............................................................................................................ 121
4.2.3.2 Nitrogen cycling microbes .................................................................................. 123
4.2.4 Community analysis by DGGE ................................................................................. 125
4.2.4.1 Bacteria ............................................................................................................... 126
4.2.4.2 Fungi ................................................................................................................... 128
4.2.4.3 ureC DGGE ......................................................................................................... 129
4.3 The isolation and analysis of ureolytic microorganisms from PL .................................... 130
4.3.1 Culturing of ureolytic microbes ................................................................................. 130
4.3.2 Phylogenetic analysis of isolates ............................................................................... 131
4.3.3 Discovery of novel ureolytic bacteria ........................................................................ 131
4.3.4 Variable ureolytic activity of isolates ........................................................................ 133
4.3.5 Identification of PLUP ureC in several isolates ........................................................ 134
4.3.6 Evidence of horizontal gene transfer (HGT) among PL bacteria .............................. 135
4.3.7 Conclusions ................................................................................................................ 137
4.4 Microcosm incubation experiment comparing BWW and MW treatments for N-
mineralization and microbial ureolytic activity ...................................................................... 137
4.4.1 Moisture content (MC%) and aerobicity of microcosms ........................................... 138
4.4.2 pH and enzymatic activity within microcosms .......................................................... 139
4.4.3 N mineralization to urea............................................................................................. 140
4.4.4 Explanations for the minimal accumulation of urea in BWW treated PL microcosms
............................................................................................................................................. 142
4.4.5 Urease activity ........................................................................................................... 143
4.4.6 Conclusions ................................................................................................................ 145
CHAPTER 5: SUMMARY AND CONCLUSIONS .................................................................. 146
CHAPTER 6: LITERATURE CITED ........................................................................................ 150

viii
LIST OF TABLES
Table 1: Meta-analysis of poultry litter properties from literature findings. .................................. 2

Table 2: Summary of microbial surveys of poultry litters from diverse regions, by culture-based
(cult) or molecular analyses. ........................................................................................................... 8

Table 3: Several of the more common molecular methodologies and their intended purpose in the
analysis of microbial communities in composts. .......................................................................... 28

Table 4: List of primer sets used for PCR and sequencing, including the sequence of the forward
and reverse primers (listed first and second, respectively), the target and size of the amplicon,
and the primer anneal temperature and final concentration (conc) used. ..................................... 45

Table 5: Absorbance measurements of DNA extracts from a number of commercial kits…. ..... 60

Table 6: Quantification of luxAB copy number from gDNA extractions from 100 mg of PL
inoculated with increasingly diluted Pseudomonas sp. UG14Lr stock culture.. .......................... 63

Table 7: Sensitivity of gDNA extraction protocol and qPCR to detect bacterial cells at
concentrations similar to the pathogen detection limits for E. coli in compost (1 x 103 CFU(g)-1;
CCME 2005). ................................................................................................................................ 64

Table 8: Quantification of pathogenic groups at three time points (Initial, PT – post-treatment,
and final) from the pilot scale study of BWW and MW amended composted poultry litter. Genus
or species specific primers were used to target a number of different pathogens. ....................... 69

Table 9: Summary of total culturable microbe counts and number of ureolytic and PLUP isolates.
Media types (NA, nutrient agar; Enrich, urea enrichment culture followed by NA with added
urea; PLA, poultry litter extract agar). Bacteria (bac) and fungi are both included. .................... 86

Table 10: Translated ureC sequences for the isolates found in composted PL.. .......................... 90

Table 11: 16S sequences from the bacterial isolates cultured from composted PL, and closest
matches found on Genbank via BLASTn ..................................................................................... 98

Table 12: PLUP cultures used to inoculate the PL for incubation microcosms. ........................ 105

Table 13: Moisture content (MC%) averaged across three replicates for each treatment used in
microcosm incubation experiment. ............................................................................................. 106

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LIST OF FIGURES

Figure 1: Nitrogen flow of microbially-mediated processes relating to the production of
ammoniacal-N species.. ................................................................................................................ 10

Figure 2: Generalized pathway of uric acid mineralization to ammonia ...................................... 11

Figure 3: Flow chart of the steps required for appropriate analysis of microbial communities in
diverse environments .................................................................................................................... 20

Figure 4: Flow diagram of the general processing of biodiesel .................................................... 33

Figure 5: Efficiency of gDNA extraction from 100 mg litter spiked with 8log Pseudomonas sp.
UG14Lr (+ PL) using the FASTDNA kit for soil, compared to UG14Lr pure culture (no PL) of
the same quantity of bacteria. ....................................................................................................... 62

Figure 6: Temperature profiles of poultry litter during composting from the start July 14 2011 to
Sept 20 2011.. ............................................................................................................................... 65

Figure 7: Cell abundances of nitrogen cycling microbes found in composted poultry litter
amended with either BWW or MW.. ............................................................................................ 67

Figure 8: Fungal 18S rRNA gene DGGE ..................................................................................... 71

Figure 9: Fungal 18S rRNA gene DGGE analysis. ...................................................................... 72

Figure 10: Dendrogram of 18S sequences obtained from DGGE and relevant Genbank results
from BLASTn searches................................................................................................................. 74

Figure 11: Phylotype richness (R) from the fungal 18S rRNA gene DGGE profiles. .................. 75

Figure 12: Bacterial 16S rRNA gene DGGE. ............................................................................... 77

Figure 13: Bacterial 16S rRNA gene DGGE analysis. ................................................................. 78

Figure 14: Dendrogram of 16S sequences obtained from DGGE and relevant Genbank results
from BLASTn searches................................................................................................................. 80

Figure 15: Phylotype richness (R) values from the bacterial 16S DGGE profiles. ...................... 81

Figure 16: Bacterial partial ureC DGGE ...................................................................................... 83

Figure 17: Bacterial isolates growing on PLA (A), isolation onto master plates (B), and testing
on Christensen’s urea agar: Proteus vulgaris positive control (C), and bacterial isolates (D). .... 85
x