EFFECTS OF TURNING FREQUENCY, PILE SIZE AND SEASON ON PHYSICAL, CHEMICAL AND BIOLOGICAL PROPERTIES DURING COMPOSTING OF DAIRY MANURE/SAWDUST (DM+S) M.S Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master in Food, Agricultural, and Biological Engineering in the Graduate School of The Ohio State University By Sandra M. Tirado, B.S. **** The Ohio State University 2008 Dissertation Committee: Approved by Dr. Frederick C. Michel, Jr, Adviser Dr. Harold M. Keener Advisor Dr. Brian McSpadden Gardener Food, Agricultural and Biological Dr. Warren A. Dick Engineering Graduate Program
Transcript
EFFECTS OF TURNING FREQUENCY, PILE SIZE AND SEASON ON PHYSICAL, CHEMICAL AND BIOLOGICAL PROPERTIES DURING
COMPOSTING OF DAIRY MANURE/SAWDUST (DM+S)
M.S Thesis
Presented in Partial Fulfillment of the Requirements for the Degree Master in
Food, Agricultural, and Biological Engineering in the Graduate School of The
Ohio State University
By
Sandra M. Tirado, B.S.
****
The Ohio State University
2008
Dissertation Committee: Approved by
Dr. Frederick C. Michel, Jr, Adviser
Dr. Harold M. Keener Advisor
Dr. Brian McSpadden Gardener Food, Agricultural and Biological
Dr. Warren A. Dick Engineering Graduate Program
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ABSTRACT
Composting offers the potential to significantly reduce problems
associated with manure management including odors, pathogens, ground water
pollution, and utilization costs. Two variables that directly affect on-farm
composting costs are windrow size and windrow turning frequency. However the
size of a windrow is limited by the depth of penetration of oxygen and high
temperatures as well as available equipment. In this study three full scale
compost sets were set-up at the Ohio Agricultural Research and Developing
Center (OARDC) compost pad to evaluate the effects of turning frequency, pile
size and seasonal variability on physical (temperature, oxygen, bulk density,
moisture and weigh loss), chemical (volatile solid loss, pH, Carbon and Nitrogen
concentrations) and biological (plant growth bioassays and microbial community
structure) parameters during dairy manure/sawdust composting (DM+S). Based
on these data the operational costs for producing and transporting compost were
estimated and compared to those for liquid manure and fertilizer.
The three treatments consisted of a set of windrows (A) which were
turned using a self propelled and tractor drawn windrow turner every three days
for a total of 32 turns during 16 weeks, a second set (B) that was turned once
every ten days and a third set (C) consisting of much larger piles turned that was
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also turned every ten days with a loader. All three sets were composted in both
winter and summer for 120 days.
The hypotheses of the study was that: “turning frequency, pile size and
season do not significantly affect compost process parameters or the final
chemical, physical or biological properties of cured composts”
Results showed that neither physical chemical nor biological properties of
the final cured composts were significantly affected by turning frequency, season
or pile size (p> 0.05). During composting, he the surface area, oxygen
concentrations and Total nitrogen losses were significantly affected by pile size
(p < 0.05). Turning frequency affected (p < 0.05) mass losses, bulk density and
total nitrogen losses. The seasonal effects on composting during the process
were primarily related to moisture (p < 0.05), mostly due to ambient temperatures
which affects water holding capacity of air. Despite these process differences,
the final cured composts from all treatments and seasons had similar properties
(p > 0.5).
Plant growth bioassays showed a high emergence percentage (> 80%).
The fertilized cucumber plants grown in composts from the various treatments in
summer had higher shoot dry weights than peat controls ( ≥ 100%) except for
day 30 in pile C (89%). The unfertilized cucumbers plants showed an increase of
shoot dry weight at the end of the composting process (day 120) except for
windrow A in summer. However the bioassay tests were inconclusive.
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Microbial Community analysis, based on Terminal Restriction Fragment
Length Polymorphisms (T-RFLP), showed that management differences (turning
frequency, pile size and season) did not significantly affect (p > 0.05) microbial
community structure. Clustering, pairwise comparison, principal component
analysis (PCA) and Kruskal Wallis tests were used to determine the similarities
and differences between microbial communities in the different treatments. In
each treatment a different subset of TRFs were present suggesting that different
classes of organisms predominate during different stages of composting..
However, one terminal restriction fragment H371 contributed significantly (p< 0.1)
to the observed variation as a function of compost age
The Restriction Fragment (TRF) sizes obtained in the different treatments
were compared to fragment sizes predicted by in silico amplification and
digestion (RDP v.9.0) to characterize the microbial community in the composts.
TRFs fragments sizes were also compared to a clone library of 263 sequences
215, 227, 365, 373, 437 and 481) in the compost samples were consistent with
the predicted TRFs of Proteobacteria, Firmicutes, Bacteroidetes and
Actinobacteria.
The main factor affecting total compost production operational cost was
the cost of the bulking agent. Operational costs for frequently turned windrow
were higher ($109/Mg) compared to the infrequently turned windrow ($95/Mg),
and the infrequently turned piles ($93/Mg). These differences are due to the time
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that is needed to turn and the equipment fuel costs. Thus, infrequent turning
(every ten days) with larger windrow sizes reduced the operating costs
associated with unseparated dairy manure composting compared to more
frequently turning windrows. It is recommended for the farmers to use a turning
frequency of ten days and piles with a surface to volume ratio of 0.9-1.2 m2/m3 to
minimize operational costs. If composting is performed in temperate climates
there is a need to consider the moisture content at the beginning of the process
to prevent moisture irregularities during the process.
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ACKNOWLEDGMENTS
I would like to express my deep appreciation to Harold M. Keener, Warren
A. Dick and Brian McSpadden Gardener, my academic committee, for all the
advice and support in every aspect of my graduate school experience; also for
encouraging me and showing me the right way for success. I would like to thank
Dr. Frederick C. Michel, my academic advisor, for showing me that
independence, patience and understanding are also essential to achieve any
goal.
I also would like to thank Dr. Jerome F. Rigot, Michael Klingman, Michael
J. Sciarini and the entire department in Wooster for the support, advice and help
during the study. A special thanks to Gerald L.Reid, Richard Franks and all the
crew of the Farm Operations at the OARDC; without them this study could not be
possible. Thanks also to Nathan Smith and my family in Colombia, for the
support and the concerned about my academic career and my life.
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VITA
August 12, 1980……………………………..Born in Manizales, Caldas, Colombia 2000 - 2005…………………………………. B.S., Pontificia Universidad Javeriana, Bogotá, Colombia 2005- 2006…………………………………..Researcher, Department of Food, Agricultural and
Biological Engineering The Ohio State University, Columbus,
Ohio
2006-Present………………………………..Graduate Research Associate- Student
Department of Food, Agricultural and Biological Engineering
The Ohio State University, Columbus, Ohio
PUBLICATIONS
Tirado, S. M., J. Rigot, Michel F.C. (2007). Analysis of bacterial community structure in dairy manure composts. Abstracts of the General Meeting of the
American Society for Microbiology. Washington, DC. p468-469.
Tirado, S.M., Michel F.C. (2008) Seasonal Effects on the composting of Dairy Manure/Sawdust (DMS)” Paper No 083671 Annual Meeting American Society of
Agricultural and Biological Engineers (ASABE)- Providence, R.I
FIELDS OF STUDY
Major Field: Food, Agricultural and Biological Engineering Studies in: Environmental Biology Environmental Microbiology
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TABLES OF CONTENTS
Page
ABSTRACT ...........................................................................................................ii ACKNOWLEDGMENTS .......................................................................................vi VITA….................................................................................................................vii LIST OF TABLES .................................................................................................xi LIST OF FIGURES............................................................................................. xiii LIST OF ABBREVIATIONS .................................................................................xv CHAPTERS 1.INTRODUCTION ............................................................................................... 1
2.3 Microbiology of composting ............................................................... 21
2.4 Current Research Interest on Composting......................................... 30
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3.EFFECTS ON TURNING FREQUENCY, PILE SIZE AND SEASON VARIABILITY ON THE COMPOSTING OF DAIRY MANURE SAWDUST (DMS) ................................................................................... 323.1 ABSTRACT........................................................................................ 32
4.3 Materials and methods....................................................................... 81
4.4 Results and discussion ...................................................................... 88 4.5 Summary and conclusions............................................................... 109
APPENDICES Appendix A. Physical, Chemical, Biological and Molecular parameters
analyzed during the composting process............................................... 110 Appendix B. Physical, Chemical, Biological and Molecular Parameters
Analyzed during the Composting Process in Summer........................... 114
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Appendix C. Operation Costs Equation in Dairy Manure Composting.............. 119 Appendix D. Potential Classes of Bacteria for samples I (Day 50), II (Day
155) and III (Day 330)-Clone Bank ........................................................ 122 REFERENCES................................................................................................. 124
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LIST OF TABLES
Table ..............................................................................................................Page 3.1 Summary of weather data for study period summer and winter 2007........... 37 3.2 Machinery used to build, turn and composite frequently turned windrows
(A), infrequently turned windrows (B) and piles (C) during this study and its respectively fuel efficiency (Grisso R.D., 2004)............................ 42
3.3 Initial and Final compost properties performed in this study, for
3.4 Effect of depth on temperature (°C), pH and oxygen concentrations (%)
for winter and summer on day 30. In frequently turned windrow (A-Every three days), infrequently turned windrow (B-Every 10 days) and infrequently turned pile (C-Every 10 days). ** Missing data .............. 55
3.5 Effects of Management practices (pile size, turning frequency and
season) during the composting process (from day zero through day 120) with p values (α= 0.05) and correlation coefficients......................... 65
3.6 Estimated costs per Mg of cured composts (produced in this study) in
US dollars for DM+S compost managed with different turning frequencies and pile sizes........................................................................ 68
3.7 Nutrient concentrations, values and costs where transportation costs
equal the nutrient value in miles for dairy manure (Heifer barn), composts (DM+S, produced in this study) and fertilizers (15:15:15)........ 71
4.18Biochemical changes of composite samples in frequently turned
windrows (A), infrequently turned windrows (B) and infrequently turned piles (C) for the full scale study..................................................... 91
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4.29Concentration of genomic DNA and conditions for the frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C) compost treatments. ....................................................... 96
4.310Similarity coefficients between the TRFs from the middle of the pile (120cm) on day 30 of frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C) compost.............. 98
4.41116S rDNA terminal restriction fragment with factor loadings |x|>0.60 on
the four principal components (PC) for each experimental treatment .... 103 4.512Predicted bacterial genera to generate a terminal restriction fragments
(TRFs) with factor loadings |x| ≥ 0.60 on the PCA for each experimental treatment .......................................................................... 107
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LIST OF FIGURES
Figure………………………………………………………………………………..Page
3.1 Experimental treatments and dimensions for winter (w) and summer (s). In frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C). w= Winter, s= Summer........................ 38
3.2 Oxygen concentrations in frequently turned windrows (A), infrequently
turned windrows (B) and piles (C), before, immediately after and 2 hours after turning (120cm depth)............................................................ 51
3.3 Temperatures (°C) in frequently turned windrows (A), infrequently turned
windrows (B) and piles (C), before, immediately after and 2 hours after turning (120 cm depth)..................................................................... 52
3.4 Moisture Content and cumulative precipitations during winter (w) and
summer (s) for frequently turned widrows (A), infrequently turned windrows (B) and piles (C)....................................................................... 62
3.5 Day-by-day average daily temperatures during the composting process
(Wooster Experimental Station, OSU/OARDC). ...................................... 63 3.6 Revenues of compost in $/yd3 (produced in this study) when selling
compost in fertility-based (same product category such as soil amendments and fertilizer) and nonfertility-based (erosion control, disease suppression, bioremediation, storm water management) markets. ................................................................................................... 75
4.17Effects of compost age on Total N supplied by compost and shoot dry
weight of cucumber plants (C.sativus. L.cv) produced in the three different compost amended potting mixes treatments. Compost physical and chemical conditions are shown in previous results. ............ 92
4.28Dendogram-Relatedness of T-RFs profiles of HhaI-digested of 16S
rDNA from frequently turned windrows (A), infrequently turned
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windrows (B), infrequently turned piles (C) and clone compost samples (I, II, II). (The UPGMA, single linkage, was used to performed the cluster patterns and obtain the similarity dendogram) ...... 97
4.39Effects of composting age, turning frequency and pile size during winter
and summer in bacterial community structure for the frequently turned windrows (A), infrequently turned windrows (B), infrequently turned piles (C) and clone compost samples (I, II, II). Ordination plots from the first two principal components (PC) are shown with the corresponding standard error bars. The PCA was performed using the 16S rDNA terminal restriction fragment (HhaI) relative abundance data obtained from composts collected on day zero, 30, 60, 90 and 120 exposed to different management practices. ................ 101
4.410Effects of season variability, turning frequency, depth and pile size in
day 30 on bacterial community structure for the frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C). ..................................................................................... 102
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LIST OF ABBREVIATIONS
AFLP………………………..Amplified Fragment Length Polymorphisms
* I Medium Duty Dump truck Class 1-3 GVW, II Aereomaster MidWest PT120+Truck Hydrostatic drive Farmall 1026, III CASE 1840 Wheel Skid Steer Loader, IV Butler 3340 ensilmixer+Truck Hydrostatic drive Farmall 1026 *** Average price of gas (May, 2008) Table 3.2 Machinery used to build, turn and composite frequently turned windrows (A), infrequently turned windrows (B) and piles (C) during this study and its respectively fuel efficiency (Grisso R.D., 2004) 3.3.6 Statistical Analysis
All statistical analyses were performed using MINITAB (ver 15.1) from
MINITAB, Inc. Plots were made using SIGMAPLOT (ver. 10.0) from TE Sub
Systems Inc. Standard one way analysis of variance was used to determine
differences in treatments, while mean comparisons among treatments and
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seasons were performed using Fisher’s protected least significance difference
test (5% level). Correlation analysis between random variables was performed
using Pearson product moment and Spearman R for categorical variables.
Compost heat is produced as a by-product of the microbial breakdown of
organic material. The heat production depends on the size of the pile, its
moisture content, aeration, and C/N ratio. Additionally, ambient temperature
affects compost temperatures. According to Keener et al (2005) consistent
performance of composting systems to generate high quality compost requires
controlled process conditions, such as temperature (ranges vary from 35 to
60°C), oxygen (> 5%) and mixing. The optimal compost temperatures from the
standpoint of pathogen destruction and organic matter decomposition are 55-
60°C (Hoitink et al., 1986; Dick et al., 1993; Inbar et al., 1993; Alexander, 2007;
Tateda et al., 2005; Michel et al 2004; Hogland et al., 2003; Grewal et al., 2007).
According to these parameters, windrows and piles in this study were
grouped into six different temperature ranges (from -1 to 15°C, 15.1 to 31.1°C,
31.2 to 47.2°C, 47.3 to 63.3°C, 63.4 to 79.3°C, 79.4 to 95.4°C) and groups with
oxygen concentrations greater than 5%. Optimal composting conditions were
considered to be temperatures of 35°C to 60°C and oxygen concentrations
greater than 5%. These, to determine the frequency of dates that had optimal
composting conditions. Optimal temperatures were reached in the majority (>
70%) of the composting periods in all treatments for both seasons.
Turning frequency had little effect on windrow temperature and oxygen
gradients. Compost temperatures at the center of the windrows (120 cm) rose to
greater than 55°C after 10 days in both treatments (Figure 3.2). At depths closer
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to the surface (5 and 60 cm) temperatures were lower and appeared to be
influenced by turning frequency. Temperatures at these levels were higher in the
infrequently turned windrows.
There was a significant relationship (r > 0.80) between oxygen
concentration at the center of the windrows and the number of turnings during
summer (Table 3.5). However, overall, turning had only a transitory effect on
compost oxygen concentrations; two hours after turning oxygen concentrations
were similar to levels before turning (Figure 3.3). Overall, the different turning
frequencies used in this study did not appear to have a great impact on compost
temperatures or oxygen concentrations during the process (p > 0.05).
Bulk Density
Initial bulk density varied between 117-142 kg/m3 and rose during
composting to 143 to 182 kg/m3. There was a small difference in final bulk
densities between frequently turned windrows as compared to the infrequently
turned (Table 3.3); this difference can be attributed to the chopping and mixing
action of the windrow turner which may have accelerated the breakdown of straw
fragments, hence reducing air space and increasing bulk density. Our values are
similar to those reported by Larney et al., (2000) who reported final bulk density
values ranging from 170 to 290 kg/m3 for the composting of similar feedstocks.
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A higher correlation between bulk density in windrows (A and B) and
turning frequency was observed during winter (r > 0.85) than between windrows
in summer (r < - 0.75) (Table 3.5).
Moisture Contents
Initial moisture contents were 62-68% (Table 3.3). Final moisture contents
for the frequently turned windrows (≤ 70%) where higher than those observed for
the infrequently turned windrows (≥ 59%). However these differences did not
appear to be significant (p > 0.5) (Table 3.5).
Particle Size
Particle sizes during composting (day zero thorough day 120) were 2.15 ±
0.82 mm in the frequently turned windrows and 2.25 ± 0.81 mm for the
infrequently turned windrows. The particle size in the frequently turned windrows
showed lower values during the composting process (day zero thorough day
120) than those observed in the infrequently turned windrows (Table 3.3).
No correlation was observed between particle size and turning frequency
with an r= 0.19 for frequently turned windrows and r= -0.12 for infrequently turned
windrows (Table 3.5).
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A* Frequently turned
windrow
B* Infrequently turned
windrow
C* Infrequently turned
pile Winter Summer Winter Summer Winter Summer Surface Initial 69.3±5.5 48 51.6±14.2 51 42.7±0.7 34.4 Area (m2) Final 33.6 43 46.9 38 30.6 29.3 Reduction 51% 10% 19% 25% 28% 15% Volume Initial 38.8±2.8 24.40 37.5±7.2 23.70 43±1.2 31.20 (m3) Final 14.3±0.9 15.40 21.80±0.5 12.50 25.50±0.5 24.50 Reduction 63% 37% 21% 47% 41% 21% Surface Area Initial 1.78 1.96 1.87 2.15 0.99 1.1 to volume Ratio Final 2.35 2.79 2.15 3.02 1.2 1.2 Wet Mass Initial 6145±522 3992 6152±501 3919 6424±29 3909 (kg) Final 2026±200 2531 2331±527 1923 3115±141 2195 Loss 67% 37% 62% 51% 52% 44% Dry Mass Initial 2129±181 1709 1951±159 1697 2177±10 1732 (kg) Final 643±63 734 691±156 785 1013±46 676 Loss 70% 57% 65% 54% 53% 61% Bulk Density a,b Initial 125 127 122 135 117 143 (kg/m3) Final 176 146 170 151 143 182 Moisture Initial 65.4±2.2 61.9 68.3±4.4 60.3 66.1±2.0 58.2 (%) Final 68.8±4.4 71.0 70.4±5.9 59.2 67.5±7.8 69.2 pH Initial 8.25±1.07 7.85 8.59±0.01 8.1 8.35±0.29 8.2 Final 8.62±0.17 7.24 8.17±1.00 7.2 7.94±0.09 7.4 Carbon b, c Initial 52.75±3.33 50.56±2.60 52.75±3.33 50.56±2.60 52.75±3.33 50.56±2.60(%) Final 41.35 44.50 45.01 43.09 44.71 44.16 loss 76% 62% 70% 61% 61% 66% C:N Initial 37.61 37.31 37.61 37.31 37.61 37.31 Final 15.1 24.4 17.6 21.4 17.4 21.7 Nitrogen b, c Initial 1.40±0.18 1.35±0.10 1.40±0.18 1.35±0.10 1.40±0.18 1.35±0.10 % g/g dw Final 2.74 1.82 2.55 2.01 2.57 2.03 Loss 41% 42% 35% 31% 15% 41% Volatile Initial 94.2±1.5 93.10 92.8±2.8 93.80 93.8±0.4 93.5 Solids a Final 76.5±3.4 80.10 76.2±10 72.20 69.1±22.5 76.2 (g/ginitial) Loss 75% 63% 71% 64% 66% 68% Particle size Initial 2.4±1.0 1.7 3.2±1.5 1.6 1.7±0.2 0.9 (mm) Final 2.6±0.7 1.9±0.2 1.9±0.6 2.0±0.3 1.9±1.7 2.9±0.9
a Dry weight basis b Composite samples c Analysis performed in StarLab * A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days Table 3.3 Initial and Final compost properties performed in this study, for frequently turned windrows (A), infrequently turned windrows (B) and piles (C).
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Volatile Solid Loss, Nitrogen Loss and pH
The volatile solid losses, calculated assuming constant ash, reached 25%
loss on day 30 (for frequently and infrequently turned) and increased to 63-75%
in the final cured compost for both windrows, regardless of turning frequency (p >
0.4) .
Frequently turned windrows had higher N losses (40%) than those
windrows turned infrequently (30%) (Table 3.3). Turning exposes fresh material
to microbial colonization and leads to the release of NH3 that has accumulated in
the internal void spaces of the compost (Parkinson et al., 2004; Ogunwande et
al., 2008).
According to Wu et al., (2000), Inbar et al., (1993), Alexander et al., (2007)
and Michel et al., (1996), compost pH varies between 7.0 to 9.2. There was no
significant pH difference among turning frequencies (p > 0.5) (Table 3.3 and 3.4).
For the frequently turned windrows, pH during composting was 8.2 ± 0.4 and for
infrequently turned 8.1 ± 0.5. The alkaline values obtained in this study may
contribute to nitrogen losses and ammonia odors during composting because
Figure 3.3 Temperatures (°C) in frequently turned windrows (A), infrequently turned windrows (B) and piles (C), before, immediately after and 2 hours after turning (120 cm depth).
Table 3.4 Effect of depth on temperature (°C), pH and oxygen concentrations (%) for winter and summer on day 30. In frequently turned windrow (A-Every three days), infrequently turned windrow (B-Every 10 days) and infrequently turned pile (C-Every 10 days). ** Missing data Particle Size
During composting (day zero thorough day 120), windrows had similar
particle sizes (2.2 ± 0.8mm) as piles (2.0 ± 0.7mm) (Table 3.3), There was no
significant difference between pile size and particle size (p > 0.150) in the final
cured composts. However there was a greater range of particle sizes (higher
heterogeneity) in the piles as compared to the windrows (Table 3.3, Table 3.5).
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Volatile Solid Loss, Nitrogen Loss and pH
Volatile solid loss did not vary significantly between windrows (60-75%)
and piles (66-68%) during the composting process. The volatile solids content
loss from the composting material during the composting process reflects the
amount of organic material converted to CO2 during composting.
Nitrogen losses during both seasons for windrows varied from 31-35%, for
piles it was significantly different between seasons with 41% and 15% in
summer and winter respectively; these differences suggest an effect of season
but not pile size (Table 3.3, Table 3.5).
In the thermophilic phase of composting (day 30), pH varied (8.43 ± 0.34)
between depths 5cm, 60cm and 120 cm (Table 3.3). However these variations
were not significant (p > 0.05) (Table 3.5)
3.4.3 Seasonal Effects
The effects of season variability during composting was determined by
comparing compost characteristics in winter and summer in frequently turned (A)
infrequently turned (B) windrows and piles (C).
Total Mass
In summer, the greatest dry mass loss was in the infrequently turned pile (61%);
followed by frequently turned windrow (57%) and the infrequently turned windrow
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(54%). During winter, the greatest dry mass loss was observed for the frequently
turned windrow (70%). Dry and wet weigh losses are shown in Table 3.3.
The greater dry mass loss observed in the summer pile compared to the
pile in winter may be due to the effect that in summer the piles were wetter than
the windrows in summer, allowing more extensive degradation (Table 3.3, Table
3.5). In winter, the piles and windrows all had similar higher moisture contents
(Figure 3.4). The piles also had lower oxygen concentrations (Figure 3.3) during
winter which may have limited decomposition.
Temperature and Oxygen gradients
On day zero of the winter season the average daily ambient temperature
was -0.6°C; during composting in the winter season daily average temperatures
varied from -23.4 (day 38) to 25°C (day 86) with an average relative humidity of
65%. Temperatures below freezing were present during days 10-60 (winter-
spring, January through March); after day 60 (until day 90) daily ambient
temperatures rose to levels similar than those in summer-autumn (Figure 3.5);
from day 90 (March-April) until day 120 (May) daily ambient temperatures in
winter-spring treatment were higher than those in summer-autumn treatments.
For the summer study initial ambient temperature was 26°C and varied from
34°C (day 1) to -7.8°C (day 119) with an average relative humidity of 75%. On
day 60 (August) until day 90 (October), summer-autumn ambient temperatures
decreased to temperatures similar to those in winter (10-0°C).
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Compost temperatures rose to greater than 50°C after 30 days in both
winter and summer treatments. Final temperatures in summer (15 ± 11°C) were
somewhat lower than in winter (32 ± 17°C). Even though average daily
temperatures varied between seasons, windrow and pile temperatures during
winter and summer rose to levels higher than 30˚C after day 5, increasing
thereafter (52 ± 10˚C) until day 90 in winter and day 110 in summer. Final
temperature (day 120) in the frequently turned windrows in winter-spring
(05/10/07) was 13˚C and in summer-autumn 5.2˚C (12/19/07). The piles seemed
to maintain higher temperatures at the end with 47˚C and 31˚C for winter and
summer respectively.
For the summer-autumn study there was a significant difference in
compost temperatures (p < 0.05), whereas there was no difference between
treatments for the winter-spring study (p > 0.3) (Table 3.5).
Bulk Density
For the winter study, bulk density on day zero varied from 117 kg/m3 to
122 kg/m3. It was necessary to construct compost piles at different times during
the winter treatment and the bulk density variation can be explained by the
amount of straw bedding that was included during construction. Average bulk
density during winter composting varied from 119 kg/m3 in the pile to 131 kg/m3
in the windrows. By the end of the curing phase, bulk density had increased to
greater than 160 kg/m3 in all treatments.
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For the summer study, initial bulk densities in all treatments were similar
(135 ± 8 kg/m3). Average bulk density during summer composting varied from
100 to 190 kg/m3 (Table 3.3). Final values ranged between 140 and 180 kg/m3.
Moisture Contents
The initial moisture contents of the compost treatments were 60-70% in
both seasons which are optimal for composting (Rynk, 1992). At the end of the
composting period, of both seasons, moisture reached values of 67.6 ± 4.32 %.
A winter storm (Table 3.1) on day 20-30 increased compost moisture to 70%. A
small amount of sawdust (Approximately 130kg per treatment) was added to the
windrows on day 28 which reduced the moisture content from 70% to 69%.
Due to high precipitations during winter (recorded precipitation of the
weather conditions showed an increase during dates 60-65), moisture content
rose similarly in all three treatments to approximately 75% after 60 days (Figure
3.4).
Geotextile (Midwest Biosystems) covers were used after the winter storm
to reduce water infiltration from precipitation. This material is permeable to air
and gas, but water-repellent. Covers were left on the compost the rest of the
cycle, being removed only for turning operations. Moisture content during the
summer replicate decreased to levels below 45% indicating a need for addition of
water in all treatments. On day 64 water addition of 130, 95 and 62 gal were
added to A, B and C respectively to maintain adequate moisture (60%) (Keener
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et al., 2005). The amount of water was added according to a mass balance
(Figure 3.4).
A medium linear relationship (r > 0.5) between the number of turnings and
moisture was observed indicating an effect of turning on moisture content.
Moisture content was highly correlated with windrow temperature during winter (r
> 0.7). During the curing phase, approximately on day 90, moisture contents in
both seasons dropped from 70% to 55%. Even though different moisture
contents were observed during the composting period, cured compost of more
than 120 days for all treatments showed similar values (45 ± 4.5%) (p > 0.6)
(Data not shown).
The average pile moistures during winter were slightly lower (68.57% ±
2.6) for piles compared to the windrows (70.3 ± 3.0%) but not significantly
different (p > 0.5) (Figure 3.4, Table 3.5). During summer the variation was
opposite. Piles maintained a higher moisture content (60.6 ± 7.8%) than
windrows (57.0 ± 9%). These variations may be correlated to weather conditions
and surface to volume area ratios (Table 3.3, Figure 3.4).
The total cumulative precipitation during winter was 33.81cm (Table 3.1).
The highest recorded precipitation occurred in March (11cm) followed by January
(9.8cm). During the summer treatment the total cumulative precipitation reached
45.69 cm with a highest precipitation of 14.4 cm during August and a lowest
precipitation of 6.4 cm in September (Figure 3.4). Although precipitation during
60
the summer study was higher (50cm) moisture contents were lower due to higher
evaporation rates.
The temperature at any point during composting depends on how much
heat is being produced by microorganisms, balanced by how much is being lost
through conduction, convection, and radiation (Richard et al., 1996). Conduction
occurs at the bottom of the compost pile into the concrete pad. Convection refers
to transfer of heat by movement of a fluid such as air or water. When compost
gets hot, warm air rises within the system, and the resulting convective currents
cause a steady but slow movement of heated moist air upwards through the
compost and out the top. In addition to this natural convection, turning adds a
forced convection (Richard et al., 1996).
In winter moisture in the air leaving the compost will condense as it leaves
the windrow. In summer high ambient temperatures allow much greater amounts
of water vapor to escape by convection. However according to Richard et al.,
(1996) the heat removal due to water evaporation (about 70%) is the largest heat
removal source, radiation (about 20%) is the second, and convection (about
Figure 3.4 Moisture Content and cumulative precipitations during winter (w) and summer (s) for frequently turned widrows (A), infrequently turned windrows (B) and piles (C).
62
-20
-15
-10
-5
0
5
10
15
20
25
30
0 30 60 90 120
Time (days)
Tem
pera
ture
( C
)
Summer Winter
Winter Day zero =Julian day 008, day 120= Julian date 125; Summer day zero = Julian date 220, day 120 = Julian date 340 (2007)
Figure 3.5 Day-by-day average daily temperatures during the composting process (Wooster Experimental Station, OSU/OARDC).
Particle Size
There was a significant difference between season and particle size (p <
0.05) during the composting process. These differences can be attributed to the
amount of straw fragments incorporated in the initial mixture. Initial particle size
for winter (2.4 ± 0.7mm) was higher than summer (1.4 ± 0.3 mm). Average final
particle sizes varied from 2.62 ± 0.74 mm in winter and 1.67 ± 0.46 mm in
summer.
63
Volatile Solid Loss, Nitrogen Loss and pH
Volatile solids did not appear to be influence by season (p = 0.9). Volatile
solid losses at the end of the composting process were 60 ± 7% in winter and
summer.
Infrequently turned windrows, during winter, had higher nitrogen losses
(35%) compared to those in summer (31%). For the frequently turned windrows
and the piles nitrogen losses were higher in summer compared to those in winter
which may be correlated to the exposure of the piles to direct sunlight which may
have accelerated the decomposition and loss of valuable nutrients (Ogunwande
et al., 2008).
Initial pH values were similar between winter (8.39 ± 0.17) and summer
(8.04 ± 0.11) treatments, but final pH values were significantly (p < 0.05) higher
in winter (8.2 ± 0.34) than in summer (7.28 ± 0.12). These differences can be
attributed to ambient and compost temperatures (Barron and Geary 2008) as
discussed previously (pile size effects). According to Barron and Geary (2008),
pH is a measure of the hydrogen ion concentration, and a change in the
temperature will be reflected by a subsequent change in pH. In this study there
was no significant correlation between pH and compost temperature of windrows
and piles during winter (r < 0.40). However, there was a slight correlation of pH
and temperatures in piles and windrows during summer (r > 0.50) (Table 3.5).
*Values during the composting process, final cured compost significance are different (p > 0.05). Table 3.5 Effects of Management practices (pile size, turning frequency and season) during the composting process (from day zero through day 120) with p values (α= 0.05) and correlation coefficients.
The management practices did not appear to significantly affect final cured
compost properties (p > 0.05).
65
3.4.4 Energy Inputs and Farm Composting Economics
The costs of producing compost can be offset by the value of composts in
the marketplace. This value is due not only to nutrients but the ability of compost
to reduce plant diseases and improve soil physical properties (Michel, 2002;
Hoitink et al., 1993).
In this study the size and the turning frequency of the composting
treatment affected total operating costs. Capital costs would add to total compost
costs but were not considered in this study.
Approximately $ 99.29 ± 8.57 per Mg (value depends on turning
frequency) U.S dollars were spent on operational costs to produce final cured
compost (Table 3.6).
The costs of transporting and application of solid or semi-solid manure
and composts vary greatly within the different states and between countries.
Custom haulers usually charge by load regardless of tonnage. A common
practice is to charge by load up to one or two miles radius and from there charge
on a per-mile bases. When custom haulers or farm owners haul compost their
major limiting factor is volume. So haulers usually charge by cubic yard of
compost regardless of tonnage. In this study operational cost calculations were
made according to the costs of amendments (sawdust/manure 3:1), rental of
agricultural machinery, size and load per machine, type of machinery, fuel
efficiency (Table 3.2), average local fuel prices, travel speed, distance hauled,
time of turning, and labor; assuming an initial compost moisture of 60% a final
66
moisture of 40% and mass weight loss of 70%. An average hauling cost on a per
mile basis was determined (Table 3.7).
When hauling solid stockpiled manure or semi-solid manure the moisture
content will vary according to the manure handling system, bedding material
used, meteorological conditions, storage type, how long that manure has been
stored, etc. In most cases trucks will be hauling a considerable amount of water.
Liquid manure, because of its high water content, cannot be transported as far
but low cost irrigation systems can be used to distribute it relatively
inexpensively.
An additional analysis of the real value of transporting and spreading
manure, compost and fertilizer was based on the nutrient value (Table 3.7). The
nutrient concentration was expressed as Total N- P2O5-K2O5, which are the
primary forms in the market. Even though there is variation between
concentrations, primary nutrients (Total N- P2O5 - K2O5) in this study were
unaffected (p > 0.05) by pile size, turning frequency and season. The average
percentage of total N in the final cured compost (regardless of season- final
cured composts were composite piles) was 2.25 ± 0.63%, 2.30 ± 0.42% and 2.30
± 0.41% for the frequently turned windrow (A), infrequently turned windrow (B)
and the infrequently turned pile (C), respectively. Phosphoric acid (P2O5)
concentrations were 0.60%, 0.59% and 0.58% for A, B and C respectively. Cured
composts had 2.66% (A), 2.76% (B) and 2.39% (C) of potash (K2O5). Table 3.7
67
shows total N: P: K per Mg of cured compost (regardless of turning frequency,
pile size or season) produced in this study (Assumes 40% moisture).
Compost *Amendment Machinery used-Costs ($/Mg) b Labor Total ** $/y3
Treatment Costs $U.S I II III IV Costs c ($/Mg)
$ U.S Mg
A a 88.47 0.02 10.2 8.62 0.01 1.8 109.13 20.6
B a 88.47 0.02 3.4 2.87 0.01 0.6 95.38 17.89
C a 88.47 0.02 0 4.33 0.01 0.54 93.37 17.74 * Prices include hauling to site (Sawdust: Dalton Wood Products Inc, Orville, OH (January, 2007), Manure from OARDC Heifer Barn (No cost) ** Total costs does not consider transportation or application costs (for final hauling, refer Table 3.7). All values were divided by the total mass produced in this study (15 Mg) a A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days). b I Medium Duty Dump truck Class 1-3 GVW, II Aeromaster MidWest PT120+Truck Hydrostatic drive Farmall 1026, III CASE 1840 Wheel Skid Steer Loader, IV Butler 3340 ensilmixer+Truck Hydrostatic drive Farmall 1026 c Labor costs were calculated with a rate of $15/h; 5 people on the day of construction (3h) and 2 people for each turning (approximately 1minute/windrow and 3.5minute/pile)
Table 3.6 Estimated costs per Mg of cured composts (produced in this study) in US dollars for DM+S compost managed with different turning frequencies and pile sizes.
Tables 3.5 show the costs of making and transporting frequently and
infrequently turn windrows and piles. Results show that turning frequency and
size are major compost production expenses. Operational costs for frequently
turned windrows were higher ($109/Mg) compared to the infrequently turned
windrows ($95/Mg). The lowest cost was observed for the infrequently turned pile
($93/Mg). These differences are due to the time that is needed to turn and the
68
equipment necessary (Table 3.6). Operational costs are affected greatly by the
type of amendment used.
Table 3.7 describe the costs and nutrient values per Mg of manure,
fertilizer (15:15:15) and composts (produced in this study) and shows the
distance where the transportation costs equal the nutrient value of each
amendment. Hauling costs were calculated assuming a labor rate of $15.00/hour,
a travel speed of 30mph, a rate of rent of $60.00/h plus additional mileage
($0.50/mile) and gas ($1.92/mile for a medium dump truck according to Grisso et
al., (2004)) for a total hauling costs of $4.90/mile per load in a medium duty truck
for materials with a moisture content of 40%. The total costs were divided by the
amount of material a medium duty dump truck class 1-3 carries (from 4.5 to 9.0
Mg per load); in this study 7.0 Mg was assumed per load.
The total nutrient values per kilogram of the composts generated in this
study were compared to the nutrient value of commonly used fertilizer (Total N,
Phosphoric acid and potash 15-15-15) and manure (Alexander, 2004; James et
Results showed that fertilizer had the greatest nutrient value per kg followed by
compost and manure. However to take into account the potential sale of compost
in the market, the value should be considered as the potential to suppress
pathogens, the slow release of nutrients and organic matter, the potential to
69
reduce erosion and increase water holding capacity, and other properties that
can off set the expense of creating compost in addition to the amount of nutrient
present.
70
Costs $ U.S/Mg,Nutrient Values (kg/Mg) and Transportation (miles)
kg/Mg $/Mg
Distance where transportation costs = value (miles)
Fertilizer a N 150 216 309 P2O5 150 252 360 K2O 150 159 227 Total 627 896 N 18.20 26.2 37.44 P2O5 5.77 9.7 13.85 Compost b K2O 26.03 27.6 39.42 Total 63.5 90.70 N 3.55 5.1 7.30 P2O5 1.10 1.8 2.64 Manure c K2O 3.37 3.6 5.10 Total 10.5 15.05
a= Source Town & Country Co-Op, Ashland, OH (April 21, 2007). Price is at the point of sale and does not consider application costs. b= Assume 70% wet mass loss, and moisture content 40%, Average operational cost $99/Mg, Results from this study c= Source Ohio Livestock Management guide. Table 3.7 Nutrient concentrations, values and costs where transportation costs equal the nutrient value in miles for dairy manure (Heifer barn), composts (DM+S, produced in this study) and fertilizers (15:15:15).
71
Fecal N is approximately 40% available (NH4, nitrate, nitrite) and only 50%
reacts (bacteria, sloughed digestive tract cells) generating a product potentially
available to plants. Significant amounts of N can be lost by volatilization of
ammonia, nitrous oxides and N2 (N2, N2O, NH3) (Martins and Dewes, 1992).
Fresh manure can harm plants due to elevated ammonia levels (Walker et al.,
2001). Composting can address this problem as composting accumulates N. N
that is not lost to the environment is assimilated in the microbial biomass and
incorporated into the organic compounds to give immobilized organic N, and a
highly stable end-product (Keeling and Cook, 1998). According to Keeling and
Cook (1998) during composting, ammonia gas is lost from the manure.
Therefore, nitrogen levels may be lower in composted manure than in raw
manure (Different from our results). On the other hand, the phosphorus and
potassium concentrations will be higher in composted manure. Salt levels also
will be higher in composted manure than in raw manure (Jeong et al., 2001).
Chemical fertilizers have been the principal source of N in conventional
agricultural systems, but the prices are increasing and the demand for
biofertilizers in the form of compost and manure has increased rapidly (Garnier et
al., 2003). New organic standards (Fed Reg. No 49) require the use of compost
or manure for Organic agricultural systems. Excess animal wastes have become
an endemic problem at large scale animal production facilities (Inbar et al.,
72
1993). Composting can address these problems by reducing the weight or
manure by up to 70% (found in this study), enabling sales in value-added off-
farm markets and by sequestering manure N (Michel et al., 2004).
From the agricultural point of view, the challenge persists on how to
produce nutrient-rich compost at the lowest cost, which can justify a price high
enough to cover (at least) the operating costs of a compost station and the
transportation costs for fertility sources. Operational costs are highly affected by
the bulking material used; however these costs can be offset when the compost
is sold off farm.
When assessing exactly how to price a high quality compost there is a
need to recognize two distinct markets: 1) Fertility based, same product category
such as soil amendments and fertilizers; and 2) Non-fertility based such as
erosion control, disease suppression, bioremediation, storm water management
(Alexander et al., 2004). Typically, there is little price elasticity between products
in fertility based markets, even when the benefits that compost adds are factored
in.
Non fertility based markets, on the other hand, are outside of the soil
amendment and fertilizer category. Therefore the price point is fixed by the most
competitive products in that category (i.e., fertility based products for that industry
or service sector such as mulches).
Composts can be priced based on the nutrients it contains, or based on
the typical selling price of composts in a market area. A typical price for compost
73
is in the range of $25-50 yd3 (Alexander et al., 2004). Figure 3.5 shows the
revenues of compost (produced in this study) when selling compost for nutrient
value or assessing its costs for its complete benefits.
Results showed that infrequently turned piles may produce higher
revenues (selling composts for nutrient value) than frequently turned windrows.
Figure 3.6 Revenues of compost in $/yd3 (produced in this study) when selling compost in fertility-based (same product category such as soil amendments and fertilizer) and nonfertility-based (erosion control, disease suppression, bioremediation, storm water management) markets.
75
3.5 RECOMMENDATIONS FOR FUTURE RESEARCH AND APPLIED
AGRICULTURE
As more farmers adopt composting to reduce the environmental impacts
of manure management, a wider spectrum and greater quantity of organic
materials are composted, and different management practices are developed,
the need to optimize and suggest ways to minimize compost production costs,
will become critical to the future growth of composting.
The results of this study indicate that even though different management
practices are employed, final properties of composts did not vary considerably.
However operational costs can differ. It is recommended for farmers not to use
frequent turning, a frequency of ten days rather than many times a week can
reduce operational costs. If composting is performed in temperate climates there
is a need to take into account the moisture content at the beginning of the
process. Composting in winter (January) can start with lower moisture contents
(45 - 50%) and if summer (July-August) composting is performed, additional
water addition may be necessary to maintain adequate moisture contents of 50-
60%.
Future research in composting is recommended in order to establish the
effects of amendment types on these management practices (e.g straw, leaves,
paper, woodchips, etc; and different manures pig, poultry). It is also
recommended that other environmental and seasonal variables such as wind
76
velocity and trajectory be measured in order to estimate their effects on compost
mass losses, oxygen profiles, temperatures and particle size.
3.6 SUMMARY AND CONCLUSION
The different turning frequencies and pile sizes used in this study did not
appear to have a great impact on compost properties, temperatures or oxygen
concentrations during composting (p > 0.05). Neither moisture content, bulk
density nor volatile solids losses were significantly affected by turning frequency
or pile size (p> 0.05). Similar oxygen concentrations and temperatures were
observed in all windrow treatments and although oxygen concentrations rose
transiently after turning, they returned to preturn levels after two hours indicating
that this is not an important mechanism of aeration. The seasonal effects on
composting were primarily related to moisture content mostly due to ambient
temperatures which affect water holding capacity by air. pH was also affected by
composting season possibly as a result of ammonia volatilization during summer
and condensation during winter. The bulking agent was the main factor affecting
total operational costs. But when amendment costs are low, windrow size and
turning frequency can also considerably affect those costs. Results of this study
indicate that composting is possible in any season and infrequent turning (every
10 days) with larger windrow sizes could potentially be used to reduce the
operating costs associated with unseparated dairy manure composting.
77
CHAPTER 4
BIOLOGICAL AND MOLECULAR PARAMETERS DURING COMPOSTING OF
DAIRY MANURE/SAWDUST (DM+S) IN FREQUENT AND INFREQUENT
TURNED WINDROWS
4.1 ABSTRACT
Composting is a biological process which contains diverse microbial communities
due to the wide range of conditions prevalent during the process. These
microbial communities mediate some of the most valuable properties of
composts including plant disease suppression and nutrient availability. However
the effects of dairy manure compost production practices on community structure
an on compost maturity have not yet been previously studied. The objectives of
this study were to determine the effects of turning frequency (frequently-every 3
days, infrequently-every 10 days), size (windrow and pile) and season (winter
and summer) on the microbial community structure in DM+S composts of
different ages and their impacts on compost maturity determined by plant growth
bioassays. A mixture of dairy manure and sawdust (3:1 w/w) was composted in
windrows/piles and samples were collected on days 0, 30, 60, 90, and 120.
78
Bacterial populations were characterized using T-RFLP analysis of amplified 16S
rDNA sequences. PCR products were digested with HhaI and the Terminal
Restriction Fragment (TRF) sizes were compared to fragment sizes predicted by
in silico amplification and digestion (RDP v.9.0). TRF fragments sizes were also
compared to a clone library of 263 sequences from composted dairy manure.
Clustering, pairwise comparison, principal component analysis (PCA) and kruskal
Wallis tests were used to determine the similarities and differences between
microbial communities in the different treatments. Plant growth bioassays
showed a high emergence percentage (≥ 80%) and shoot dry weight for all
compost treatments that were correlated with carbon and nitrogen content of the
compost and to fertilizer application. Pairwise comparision on day 30, showed
that piles of different sizes and turning frequencies have very similar microbial
communities (>60%) but that composts of different seasons and ages were less
similar (~30%). Principal component analysis revealed variations in the
communities in response to age, size and season. In each treatment a different
subset of TRFs contributed considerably to the variation along the first three
* A (Every three days), B (every 10 days), C (every ten days) ** Winter Day zero =Julian day 008, day 120= Julian date 125; Summer day zero = Julian date 220, day 120 = Julian date 340 (2007) a Analysis of total C and N were performed in the StarLab at the OARDC Wooster campus. b Percentage compared to the shoot dry weigh of plants grown in a peat control. N- means without fertilizer Table 4.18Biochemical changes of composite samples in frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C) for the full scale study
Figure 4.17Effects of compost age on Total N supplied by compost and shoot dry weight of cucumber plants (C.sativus. L.cv) produced in the three different compost amended potting mixes treatments. Compost physical and chemical conditions are shown in previous results.
92
4.4.2 Quantitative Assessment of Microbial Community
The amount of extractable DNA is strongly correlated with the total
microbial biomass and reflects the size of genetic material pools of
microorganisms (Kelly, 2003; Howeler et al., 2003; Yang et al., 2007). However,
the DNA content does not reflect the ability of these microorganisms to be
activated physiologically and metabolically. Blagodatskaya et al., (2003)
characterized microbial communities by the amounts of extractable DNA
quantified by PicoGreen. He found a strong correlation between microbial
biomass and DNA contents in environmental samples of different types (r = 0.8);
thus, the DNA content of the compost samples analyzed in this study can be
used to characterize the compost microbial community (Blagodatskaya et al.,
2003; Yang et al., 2007).
The genomic DNA for the composite compost samples of every treatment
after turning was quantified using PicoGreen ® dsDNA Quantitation reagent
(Molecular probes). Initial DNA concentrations were 0.36 μg DNA/g of compost
and 4.57 μg DNA/g of compost for winter and summer respectively. The
concentration of genomic DNA at the end of the composting phase did not vary
greatly (p > 0.05) between seasons and treatments (10.36 ± 2.54 μg DNA/g wet
compost) (Table 4.2). According to Howeler et al., (2003) an average extraction
and purification of wet compost is 18.2 ± 3.8 μg DNA/g; the amount of DNA
recovered with the kit used in this study depends greatly on the sample; on the
other hand the binding capacity of the spin filter is 20 μg of DNA. Even though
93
DNA concentrations were not as high as those values reported in the literature
(Howeler et al., 2003; Yang et al., 2007), results showed similar patterns in the
concentration of genomic DNA in all composts.
During the first 30 days, DNA concentration for the infrequently turned
piles for both seasons, showed the highest increase. There was a high
correlation of the concentration of genomic DNA with turning frequency and pile
size (r > 0.7). A high positive correlation with temperature (r > 0.6), in all
treatments and seasons, and oxygen (r > 0.6) in the summer replicates was
observed (Figure 3.3), verifying that the microbial community is highly dependent
on its surroundings (i.e. temperature, oxygen, moisture).
The DNA purification methods produced DNA sufficiently pure to allow
restriction enzymes and DNA polymerase enzymes to function; and although
PCR amplification is extremely sensitive to humic acid contamination (Howeler et
al., 2003; LaMontagne et al., 2002), humic acid concentration was reduced
sufficiently and the PCR products of the expected size were present in the
majority of the samples (25/30). Molecular analyses were limited to those
samples that had sufficient digestion product for T-RFLP analysis. Samples from
day 90 (frequently turned windrow, Aw), day 120 (infrequently turned windrow
Bw) of the winter study; and samples from day 90 and 120 of the infrequently
turned windrow and day 90 of the infrequently turn pile of the summer study (Bs,
Cs) were not considered due to poor DNA recovery.
94
4.4.3 Analysis of bacterial community structure
Terminal Restriction Fragment Length polymorphism was used to asses
the microbial community structure. The profiles of the samples were evaluated,
using UPGMA clustering based on pearson correlation coefficients. The resulting
dendogram generated four large clusters, assigned 1 to 4 (Figure 4.2). Cluster 1
contained samples from day zero (fresh dairy manure sawdust compost from a
composite mix of winter and summer replicate) and showed low similarity with
the rest of the treatment samples (r = 1.75%). The great majority of the young
samples (day 30 and 60) for winter and summer grouped in the same cluster (4)
with ≥ 41.49% similarity in almost all the treatments. Older samples (> 60 days)
also clustered together (3), but similarity among these samples was less (>
13.61%). There was no clustering of samples based on season, turning
frequency or pile size.
TRF profiles from the clone sequences, representing active (50 days-
A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days) * Temperature and oxygen gradients are averages of all depths, before and after turning **Composite samples. DNA concentration was based on Picogreen results The number in parenthesis is the age of compost
Table 4.29Concentration of genomic DNA and conditions for the frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C) compost treatments.
96
100
95908580757065605550454035302520151050
As(90)
Aw(60)
Cs2(30)
Cw(60)
Aw(120)
Cw(90)
AS(60)
As(120)
Bw(90)
Cw(120)
I(50)
III(330)
II(155)
Cs(120)
Aw2(30)
Bs(30)
Bw(60)
Cs(30)
Bw(30)
Cw(30)
Cw2(30)
As(30)
As2(30)
Bs2(30)
Bw2(30)
Aw(30)
Bs(60)
Cs(60)
Zero
3
4
2
1
A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days)*. w=winter, s=summer. I(50) The number in parenthesis is the age of compost. Samples day 30, Samples day 60
Samples day 90, Samples day 120 . Figure 4.28Dendogram-Relatedness of T-RFs profiles of HhaI-digested of 16S rDNA from frequently turned windrows (A), infrequently turned windrows (B), infrequently turned piles (C) and clone compost samples (I, II, II). (The UPGMA, single linkage, was used to performed the cluster patterns and obtain the similarity dendogram)
97
Pairwise comparisons of the TRFLP profiles between the seasons for the
frequently turned windrow (A) showed a similarity from 30 to 70% during the
composting process (day 30-120). In winter, a high similarity (70%) between
infrequently turned (B-C) composts made in piles of different sizes suggested no
effect of pile size on the microbial community. In Table 4.3 similarities between
the middle depths in day 30 of compost for all seasons, pile size and turning
frequency are shown; a low similarity between summer pile compost and the
other treatments was observed.
Aw Bw Cw As Bs Cs Aw X 61% 59% 45% 61% 22% Bw X X 43% 61% 64% 0% Cw X X X 30% 40% 0% As X X X X 85% 6% Bs X X X X X 0% Cs X X X X X X
A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days)*. W=winter, S=summer. * Pairwise comparisions were performed using Bionumerics (Apllied Math v.3.5) Table 4.310Similarity coefficients between the TRFs from the middle of the pile (120cm) on day 30 of frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C) compost
During composting (day zero through day 120) there was low similarity
between various treatments, therefore further statistical analyses were performed
to determine where the variation occurred. Principal component analyses were
98
performed to determine the overall effect of each treatment (turning frequency,
pile size, age and season) on the observed populations of 16S rDNA TRFs. For
this part of the study each TRF was considered as a different variable.
Samples from different seasons, turning frequencies, pile sizes and ages
were analyzed separately to identify were variation. Ordination plots, generated
from the mean principal components scores, were used to interpret the observed
treatment separations. Variation explained by the first two principal components
ranged from 40% to 95% among season and age. There was an apparent effect
of season on bacterial community structure (Figure 4.3). The variation between
winter and summer explained by the first two principal components ranged from
46% to 70% among all turning frequencies and sizes samples (data not shown).
T-RFLP profiles from day 30 and 60 appeared to be influenced by turning
frequency and pile size (Fig. 4.4).. For day 30 in the summer replicate,
separation between winter treatments was observed in the infrequently turned
pile and windrow, but not the frequently turned ones, suggesting no significant
effect of turning in the microbial communities but some effect of season. Overall
winter replicates separated from the summer replicate along the second principal
component (17%) and only the infrequently turned pile (C) for winter and summer
was separated along the first component (45%) (Figure 4.3).
The observed TRFs from the clone sequence (I,II,II) were compared
according to age, (samples were collected from the compost pad and did not
have any treatment- turning frequency or pile size). These TRFs showed high
99
similarity with the rest of the samples but separated from the other treatments
along the second PC (17%) with day 30 of the infrequently turned windrow
(summer).
A response to pile size was also observed in the PCAs of the T-RFLP
profiles in both seasons for each date evaluated. During day 30, after turning, the
frequently turned windrow in summer (As) showed 10% of variation along the
second PC between the replicate from winter (Aw). The communities also depict
differences in day 30 among windrow/pile size. The windrow (As) is separated
from the pile (Cs) along the second PC (10%). For 120cm depth on day 30,
before turning, the pile in summer (Cs) was separated from the rest of the
replicates along the first component (44%). However the pile in winter (Cw) did
not separate from the other treatments (Figure 4.4).
For day 60 the highest variation among communities was observed for the
infrequently turned windrow during winter but not for summer (PC2 10%). For
day 90 and 120 the frequently turned windrow (A) is separated from the
infrequently turned pile (C) along the second PC suggesting an effect of pile size.
The pair wise comparison and the cluster analysis showed differences and
effects in the microbial communities with compost age but not season, pile size
A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days)*. w=winter, s=summer. The number in parenthesis is the age of compost. Figure 4.39Effects of composting age, turning frequency and pile size during winter and summer in bacterial community structure for the frequently turned windrows (A), infrequently turned windrows (B), infrequently turned piles (C) and clone compost samples (I, II, II). Ordination plots from the first two principal components (PC) are shown with the corresponding standard error bars. The PCA was performed using the 16S rDNA terminal restriction fragment (HhaI) relative abundance data obtained from composts collected on day zero, 30, 60, 90 and 120 exposed to different management practices.
Plots show the mean principal component (PC) scores for each treatment with the corresponding error bars. The PC analyses of TRf’s were performed using 16S rDNA terminal restriction from Hha. A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days)*. w=winter, s=summer. The number in parenthesis is the age of compost. Figure 4.410Effects of season variability, turning frequency, depth and pile size in day 30 on bacterial community structure for the frequently turned windrows (A), infrequently turned windrows (B) and infrequently turned piles (C).
C 93 227 371 * TRFsize was predicted by a computer-simulated amplification and digestion of complete 16S gene sequences obtained from the Ribosomal Database using TAP TRFLP software (RDP v9.0) A frequently turned windrow (Every three days), B infrequently turned windrow (every 10 days), C infrequently turned piled (every ten days)*. W=winter, S=summer. ** Winter Day zero =Julian day 008, day 120= Julian date 125; Summer day zero = Julian date 220, day 120 = Julian date 340 (2007) Table 4.41116S rDNA terminal restriction fragment with factor loadings |x|>0.60 on the four principal components (PC) for each experimental treatment
103
Only a small subset of TRFs contributed strongly to the variation observed
in the PCA. TRFs with a factor loading |x| ≥0.60 for each of the first four principal
components are summarized in Table 4.4. The first four principal components
explained from 40% to 96% of the variation among the different experimental
treatments. In all treatments, A TRF with a size of 371 bp (H371) contributed
more than 40% of the variation. H371 had factor loading values of |x| ≥ 0.8 in all
treatments except in the older composts (day 90-120). Other TRFs that largely
contributed to the variation to the first four principal components in more than one
scenario were H93, H215 and H481. TRFs with high factor loadings in day 30
from 120cm depth for summer did not show any similarities with those found in
winter, in addition the factor loading for M379 did not meet (x≤0.4) our selection
criteria.
In order to determine if the relative abundance of individual TRFs was
influenced by treatment and if there was a significant difference in bacterial
community structure, the nonparametric Kruskall-Wallis test was also used. Even
though uncommon TRFs were found in the different treatments (season, age,
turning frequency and depth); p (α =0.1) values revealed that differences in
abundance of TRFs were directly affected by age but not season, turning
frequency or pile/windrow size. Only TRF M371 was directly associated with age
(p < 0.1). Nevertheless the loading factor for H371 for summer (0.673) was more
highly associated to the variation than in winter (0.605).
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The clone library of active (I), stable (II) and mature (III) composts
generated a total of 87, 85 and 91 16S rRNA gene clones respectively that were
sequenced. Phylogenetic analysis of the sequences using RDP Naïve Bayesian
classification indicated that 9, 11 and 12 different Phyla were found in composts
I, II and III, respectively (data not shown). In all three composts, Proteobacteria
ribotypes predominated. Many sequences from the phyla Actinobacteria,
Bacteroidetes, Firmicutes, and Chloroflexi were also found (Table 4.5). The
numbers of different Classes of bacteria observed among the cloned sequences
from the three composts was 14, 18 and 17 for composts I, II and III,
respectively. The class Gamma Proteobacteria predominated in composts I and
II while Actinobacter was the most prevalent class in compost III. Clostridia,
Chloroflexi and Sphingobacteria were observed in all three composts. The
BLAST nearest relatives of the clones from all three composts did not include
any pathogenic bacteria (data not shown). Sequences related to classes of
bacteria not previously described in composts, such as Chlorofexi, Anaerolineae,
Thermomicrobia, Gemmatimonadetes and Acidobacteria, were found.
Conversely, entire phyla such as the Acidobacteria or the Chloroflexi are poorly
represented among the sequence databases but are widely abundant in natural
environments (Mering C.von., 2007). Only two sequences corresponding to
Bacillus, the predominant culturable genus found in composts (Strom, 1985),
were observed.
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Phylogenetic identity of the terminal restriction fragments (± 2bp) for each
experimental treatment with factor loadings |x| ≥ 0.60 on the PCA (Table 4.4)
were performed using TAP TRFLP software (RDP v9.0) and by comparisons to
the clone database (Table 4.5). Phylogenetic assignments for H371 ± 5bp with
the clone database suggested the presence of Cytophaga sp (Bacterioidetes),
E.coli and Ewingella (Gammaproteobacteria), Rothia and/or Bifidobacterium
cuniculi ATCC 27916 (Actinobacteria) in young compost samples.
Comparisons of the representative TRFs in the PCA for each treatment
(Table 4.4) with the clone database was consistent with the presence of 4
different Phyla. Among these TRFs, Proteobacteria ribotypes were the most
prevalent, followed by Firmicutes, Bacterioidetes and Actinobacteria (Table 4.5).
The number of different Classes of bacteria consistent with the TRFs from
all the treatments was 14. The class Gamma proteobacteria was consistent with
the largest number of TRFs (H61, H205, H215, H371) while Actinobacteria and
Alpha proteobacteria were consistent with TRFs in all the samples regardless of
turning frequency, pile size, season or age.
In this study the TRFs found indicated that the microbial community varies
with time during composting and that there is a succession of bacterial lineages
during composting. Microbial communities are very diverse within dairy manure
composts (Wang et al., 2007; Bolta et al., 2003; Morales et al 2005; Guo et al.,
2007) throughout the composting process but do not differ significantly with
* TRFsize and identity was predicted by a computer-simulated amplification and digestion of complete 16S rDNA gene sequences obtained from the Ribosomal Database using TAP TRFLP software (RDP v9.0) and comparing fragment sizes to a clone sequence database (HhaI disgested) matching (fragment size ≥ 50bp, ±1bp for TRFs < 100, ±2bp for TRFs between 100 and 200bp, and ±5bp for TRFs > 200). Table 4.512Predicted bacterial genera to generate a terminal restriction fragments (TRFs) with factor loadings |x| ≥ 0.60 on the PCA for each experimental treatment
107
4.5 RECOMMENDATIONS FOR FUTURE RESEARCH AND APPLIED
AGRICULTURE
Even though several studies have described the composition and
dynamics of microbial communities during composting (Wang et al., 2007), none
have examined the impacts of turning frequency, season or pile size on microbial
community structure on dairy manure composts or the effect of these process
parameters on compost maturity.
This study is the first, to our knowledge, to determine these effects.
Although the dairy manure sawdust compost supported growth of cucumber
without added fertilizer, the fertilized pots had higher shoot plant weight. It is
recommended to conduct some further studies to control, quantify and optimize
the concentration of nutrients in potting media to reduce nutrient leaching and,
therefore, improve the sensitivity of this test to compost maturity.
This work can be used as the first step of a step-wise approach for
identifying and confirming the predominant bacterial populations in composts. It
is also recommended to evaluate different microbial communities based on
different ribosomal subunits (i.e, 18S rDNA T-RFLP profiles) with different
restriction enzymes (i.e, RsaI, MspI, TaqI, Mbo, etc), and to use functional genes
to identify, if possible, the metabolic activities of the microorganisms present .
108
4.5 SUMMARY AND CONCLUSIONS
The different turning frequencies and pile sizes did not appear to have a
great impact on plant growth (dry and wet weight) and bacterial community
structure (p > 0.05). Cluster analysis on TRFLP profiles of the different
treatments revealed low similarities between composts of different age and
season but high similarities between composts of different turning frequencies
and pile sizes. Ppairwise comparison showed low similarities between small
windrows and larger piles (≤ 6%), but high similarities between microbial
communities from composts of different seasons and ages (≥ 70%). Principal
Component Analysis revealed changes in the bacterial communities in response
to age, size and season. In each treatment (turning frequency, pile size and age)
a different subset of TRFs contributed considerably to the variation along the first
three principal components. However, terminal restriction fragment M371
contributed significantly (p<0.1) to the observed variation with compost age.
According to RDP and a clone database, , fragment M371 is consistent with
Cytophaga sp, E.coli, Ewingella, Rothia and Bifidobacterium cuniculi. The effects
of management differences (turning frequency, pile size and season) did not
appear to affect significantly (p > 0.05) microbial communities; but different
classes of organisms predominated during different stages of composting.
109
APPENDIX A
PHYSICAL. CHEMICAL, BIOLOGICAL AND MOLECULAR PARAMETERS
ANALYZED DURING THE COMPOSTING PROCESS
WINTER
110
Physical. chemical, biological and molecular parameters analyzed during the composting process---WINTER
Variables: - Hc Hauling cost ($/mile/Mg) - Lr rate of labor ($/h) - S speed (mph) - Rt Rent truck ($/h) - Am Additional mileage ($/mile) - Fe fuel efficiency ($/h) - Ml machine-truck load - Cm Cost of manure ($/Mg) - Cs Cost of sawdust ($/Mg) - Dh Distance hauled (miles) - Ir Initial bulking ratio - Mtype:I, ii, iii, iv Machinery type used (fuel efficiency) ($/h) - Ө Time (minutes) - P personnel
Assumptions:
- No costs of manure - Cost of Sawdust $35.00/Mg (Dalton Wood Products) - Initial ratio 3:1 (hardwood sawdust/manure) - Initial Moisture content 60% (This study) - Final Moisture content 40% (Market moisture) - Weight wet losses 70% (This study) - Constant speed 30mph - Medium Dump truck load: 7Mg - Fuel efficiency of Medium Dump Truck: 13.91 gal/h (Grisso et al., 2007) - Average fuel price $4.15/gal - Labor Rate: $ 15.00/h - Rent costs $60/h plus additional mileage $0.50/mile
121
122
APPENDIX D
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