INACTIVATION OF DAIRY MANUREBORNE PATHOGENS BY ANAEROBIC DIGESTION Mark Borchardt, Susan Spencer, and Spencer Borchardt USDA –Agricultural Research Service USGS Wisconsin Water Science Center Becky Larson and Asli Ozkaynak UW-Madison Biological Systems Engineering Travelling Gun Irrigation with Liquid Dairy Manure, September 26, 2012 Manure’s Double-Edged Sword Manure as Asset Manure fieldapplication is a costeffective and sustainable approach for optimal soil tilth and fertility Manure as Liability Manure may contain pathogens harmful to both humans and livestock Societal goal: Maximize the beneficial uses of manure while minimizing environmental pathogen transmission Pathogens in Cattle Manure Bacteria (e.g., Campylobacter, Salmonella, E. coli O157:H7) Protozoa, (e.g., Cryptosporidium, Giardia, Eimeria) Viruses (e.g., adenovirus, enterovirus, rotavirus) Cryptosporidium parvum • Scours in calves • Responsible (with C. hominis) for largest waterborne disease outbreak in US history • Severe diarrhea 21 days median duration • 7-22% of patients hospitalized • Deadly infection in AIDS patients and immunocompromised • Infected children have reduced growth Toxin producing E. coli • E. coli O157:H7, the Jack-in-the-Box bug • No disease in cattle • Severe diarrhea; 4% of cases develop kidney failure • 73,000 cases, 60 deaths/year in US • Walkerton, Ontario outbreak Salmonella enterica • In cattle, diarrhea, milk drop, abortion, rapid death in calves • In people, diarrhea, cramps, fever • Can move from the intestine to bloodstream, bone, and urinary tract • 1.4 million cases, 600 deaths/year in US • Growing antibiotic resistance • Drinking raw milk presents high risk of infection Campylobacter jejuni and C. coli • Most common cause of bacterial gastrointestinal illness in the US • Debatable among veterinarians whether a cattle pathogen • Severe diarrhea, potential complications with liver, heart, other organs • Causes Guillain-Barré syndrome, acute paralysis Other zoonotic pathogens in cattle manure - Infrequent human health effects Microsporidia Brucella species Bacillus anthracis Clostridium perfringens Leptospira species Listeria monocytogenes Mycobacterium bovis Apthovirus (foot and mouth disease) Coxiella burneti Sources Dungan, RS. 2010. J. Anim. Sci. 88:3693-3706 Atwill, ER. Et al. 2012 . NRCS Technical Note No. 9 Pathogens in manure from a single farm by year and season Autumn, 2007 Autumn, 2008 Spring, 2009 Pathogens in manure from a single farm by year and season Autumn, 2009 Spring, 2010 Autumn, 2010 Spring, 2011 Anaerobic Digesters … • Produce methane for generating electricity and heat • And… • Might, as an added side-benefit, inactivate/destroy pathogens in manure that can potentially pose a risk to the health of humans and livestock Operational principle of sanitation since Louis Pasteur •Reducing the number of pathogens reduces the level of exposure which reduces the risk of infection •Principle works at small scale (hand sanitizers) to large scale wastewater and drinking water treatment plants Use in the USA • EPA/USDA AgSTAR program estimates 157 operational digesters as of July 2010 • This is a 5-fold increase from 2002 • Most are located on dairy farms • Midwest, West, and Northeast Current Knowledge • Mesophilic anaerobic digestion can reduce by 99% to 99.9% the levels of several microbes: E. coli, fecal coliform bacteria, fecal streptococci, Listeria monocytogenes, Salmonella senttenberg. • Thermophilic anaerobic digestion is more effective than mesophillic digestion in removing pathogens • Pathogen removal depends on temperature, pH, and retention period Key Knowledge Gaps • Pathogen inactivation in full-scale operational digesters. • Pathogen-specific inactivation rates for the complete array of pathogens potentially present in dairy manure. • Operational parameters of full-scale digesters most important for pathogen removal • Pathogen levels in final solids component used for cow bedding Study Objectives • Measure pathogen inactivation by farm-scale anaerobic digestion of dairy manure • Evaluate several factors for their effect on pathogen inactivation (pathogen type, farm, time of year) • Determine proportion of surviving pathogens in digestate liquid and solid fractions after separation • Determine effect of bedding recovery units (no digestion) on fractionation of pathogens into liquids and solids Study Overview • Nine farms in study, seven farms with anaerobic digesters • • • • and two that have only screw press bedding recovery units. Anaerobic digesters all mesophilic, two complete mix digesters and five plug flow Samples collected approximately every two weeks for eight months, January – August 2012. Four sampling points: 1) Pre-digest (i.e., manure); 2) Post-digest; 3) Solids after separation; 4) Liquid after separation All pathogens and indicators measured by qPCR (i.e., measuring genomes) and reported as genomic copies per gram. Study Sampling Locations Source: Washington Dairies and Digesters, Washington State Dept of Agriculture, AGR PUB 602-343, October 2011 Pathogens and Indicators Analyzed Protozoa Bovine Viruses Cryptosporidium parvum Rotavirus groups A and C Giardia lamblia Enterovirus Adenovirus Polyomavirus Bacteria Bovine viral diarrhea virus types 1 and 2 Enterohemorrhagic E. coli Coronavirus Campylobacter jejuni Salmonella enterica Indicators Clostridium perfringens Bovine bacteroides Mycobacterium avium paratuberculosis Bacteroides-like M3 Bacteroides-like M2 Pathogens Detected during Study Protozoa Bovine Viruses Cryptosporidium parvum Rotavirus group A Giardia lamblia Rotavirus group C Enterovirus Adenovirus Polyomavirus Bovine viral diarrhea virus type 1 Bacteria Bovine viral diarrhea virus type 2 Enterohemorrhagic E. coli Coronavirus Campylobacter jejuni Salmonella enterica Indicators Clostridium perfringens Bovine bacteroides Mycobacterium avium paratuberculosis Bacteroides-like M3 Bacteroides-like M2 Logarithm (Log) Removal • The logarithm of the pathogen concentration in the manure minus the logarithm of the pathogen concentration in the digestate • 1 log removal = 90% reduction • 2 log removal = 99% reduction • 3 log removal = 99.9% reduction, etc. For plug flow digesters the digestate concentration used in the log removal calculation was offset by two sampling periods from the manure concentration to account for retention time of approximately 21 days. Digester Removal by Pathogen Type 10 8- f. 3 4. ll- Log Removal l- oo ? 0+ Pathogen or Microbial Indicator Digester Removal of Bacteroides by Farm Farm Number 2Log Removal ?0.5 - -1.0 1 3* Continuous mix digester Bovine Bacteroides Mean Pathogen Concentration (gc/gm) 100000000 10000000 1000000 100000 Farm #1 10000 Farm #3 1000 Farm #4 100 Farm #5 Farm #6 10 Farm #7 1 Farm #8 Digester Removal of Polyomavirus by Farm Log Removal Farm Number Continuous mix digester Mean Pathogen Concentration (gc/gm) Bovine Polyomavirus 1E+09 100000000 10000000 1000000 100000 Farm #1 10000 Farm #3 1000 Farm #4 100 Farm #5 10 Farm #6 Farm #7 1 Farm #8 Digester Removal of Bacteroides over Time 2.5 I aft-b- - Farml I - .- Farm3 I I Farm-=1 1 Farms 1* C) -2 Farm6 3 5 Farm? - Farms 12/15f2011 1f15/2012 2/15/2012 3/15/2012 -D.5 Digester Removal of Polyomavirus over Time . A 8? 7 _l l" A 12315 A -1 .. Digester Removal of Coronavirus over Time ?O?Seriesl -I- Serie52 I 4" +Serie53 5 .l - (D i +Series4 4 +Serie55 8) +Serie56 ?l 3 Series? 2 1 0 12/15/2011 1/15/2012 2/15/2012 3/15/2012 4/15/2012 5/15/2012 6/15/2012 7/15/2012 Digester Reduction in Percentage Detections Campylobacter 100 - Post I Liquid Percent Detects I Solid Digester Fieduction in Percentage Detections Rntauirus Pest I Liquid Percent Detects I Solid Liquid/Solid Fractionation Bovine Bacteroides 120 100 80 Liquid Percent 60 Solid 40 20 0 Farm1 Farm3 Farm4 Farm5 Farm6 Farm7 Farm8 Liquid/Solid Fractionation Bovine Polyomavirus 120 100 80 Liquid 60 Percent Solid 40 20 0 Farm1 Farm3 Farm4 Farm5 Farm6 Farm7 Farm8 Separated Bedding: Bovine Bacteroides Mean Pathogen concentration (gc/gm) 100000000 10000000 1000000 100000 10000 Farm #2 Farm #9 1000 100 10 1 Manure Separated Liquid Separated Solid 1 Separated Solid 2 Separated Bedding: Bovine Polyomavirus Mean Pathogen concentration (gc/gm) 10000000 1000000 100000 10000 Farm #2 1000 Farm #9 100 10 1 Manure Separated Liquid Separated Solid 1 Separated Solid 2 Study Limitations • Sampling frequency was not based on digester retention time therefore the manure and digestate samples are not truly coupled, particularly for the plug-flow digesters. • Measured inactivation of pathogen/indicator genomes; this is not a measure of infectivity or viability. • Pathogen concentrations in many samples were near the assay limit of detection, which reduces accuracy of the log removal estimate Preliminary Conclusions • Full-scale anaerobic digesters reduced pathogen levels by 90% • • • • • to 99.9% Removal efficiency varied by pathogen type, farm, and time After digestion and separation of the digestate, the liquid fraction contained the majority of pathogens. Separation of undigested manure by screw press bedding recovery units resulted in the liquid fraction containing the majority of pathogens. Although the solids fraction contained fewer pathogens, the concentration could still be above the infectious dose, particularly for calves Heating the solids should result in complete pathogen inactivation but this was not always the case Does Anaerobic Digested Manure have Reduced Health Risks? Findings and perspectives to keep in mind… • Pathogen types and concentrations in manure (i.e., the herd) are highly variable over time • Pathogen inactivation by anaerobic digestion is highly variable • Because pathogen concentrations in manure can be very high, a 99% reduction (i.e., 2-log removal) does not mean pathogen levels become low • 99% of the pathogens in the digestate after separation partition into the liquid fraction • Digesters are designed to produce methane, not inactivate pathogens. Questions? Comments?