Control of Hydrogen Sulfide-producing Bacteria

By Annel K. Greene, PhD, Professor and Center Director
Clemson University Animal Co-Products Research and Education Center

Hydrogen sulfide-producing bacteria include a wide variety of bacterial species that are ubiquitous in the environment and can grow in high protein products such as chicken feathers. Growth of the organisms can lead to production of hydrogen sulfide, which can be hazardous to worker health. Dr. Xiuping Jiang, professor in the Department of Food, Nutrition, and Packaging Sciences at Clemson University and a member of the Clemson University Animal Co-Products Research and Education Center, has recently concluded a proof-of-concept study developing a safe, biological control method for preventing growth of hydrogen sulfide-producing bacteria.

Raw animal by-products can contain many species of bacteria, including those organisms that can produce hydrogen sulfide gas under anaerobic conditions. The most common species of these hydrogen sulfide-producing bacteria are Pseudomonas, Citrobacter, Aeromonas, Salmonella, and Escherichia coli, but numerous other species are also recognized as being capable of generating hydrogen sulfide. These microorganisms can cause rapid spoilage of the materials at ambient temperatures. Hydrogen sulfide production not only decreases the quality of raw materials, but the gas is also extremely toxic for humans and animals. During a 12-hour transportation and storage time, the concentration of hydrogen sulfide in raw animal materials has been measured to easily increase to 700 parts per million, which is a level that can cause immediate death in humans (Beauchamp et al. 1984).

Bacteriophages are small viruses that act on specific bacteria. Discovered more than a century ago, these minute viruses have been used for bacterial control for over 60 years. Bacteriophages must find a precisely matching receptor on a bacterial cell wall of a particular species of bacteria. Upon finding a match, the bacteriophage will inject its nuclear material into the bacteria where the virus then commandeers the controls of the bacteria, produces multiple copies of itself, and then kills the cell and releases an “epidemic” of new, safe, bacteriophage particles to seek additional target bacteria – in this case, the hydrogen sulfide-producing bacteria. Bacteriophages are the most plentiful microbial entity on earth and are completely incapable of infecting anything except its target bacteria.

Prior to the discovery of antibiotics, bacteriophages were utilized as antimicrobials in medicine. Currently, several bacteriophages have been accepted by the United States Food and Drug Administration as “generally recognized as safe” for use in packaged luncheon meats to prevent pathogen growth. In recent years, interest in bacteriophages has grown as scientists seek alternatives to antibiotics in light of the rapid development of antibiotic resistance by bacteria along with the concurrent need for bacterial control methods. Bacteriophages were used in a study by Greer in 1986 to control growth of Pseudomonas spp. on steaks; phage treatment increased shelf life of the beef from 1.6 days to 2.9 days. Greer and Dilts (2002) also used bacteriophages in improving shelf life of pork products. Using a bacteriophage against the common pork spoilage organism Brochothrix thermosphacta, the study indicated that the shelf life of pork treated with phage was doubled from four to eight days. Bacteriophages have also been used successfully for reducing pathogens in other studies in live animals (Smith and Huggins 1983; Sheng et. al. 2006; Atterbury et. al. 2007).

Chao “James” Gong, a PhD student in microbiology, along with fellow doctoral students Spencer Heringa and Randhir Singh and post-doctoral Dr. Jinkyung Kim, first isolated several bacteriophages specific to hydrogen sulfide-producing bacteria from raw animal rendering materials. The team collected meat, chicken offal, and feather samples from local grocery stores and rendering processing plants and isolated 142 strains of hydrogen sulfide-producing bacteria from these materials, including Escherichia coli, Citrobacter freundii, and Hafnia alvei. They then isolated 52 bacteriophages specific to these hydrogen sulfide-producing bacteria. Using electron microscopy, the team identified the nine bacteriophages selected for use in a cocktail belonging to the families of Siphoviridae and Myoviridae.

Using a method of analysis involving restriction enzyme digestion with the endonuclease DraI, six different patterns were distinguished among the nine phages. The team developed methodology for quantitating the impact of the bacteriophages on hydrogen sulfide production. When these phages were used experimentally, it was determined that the treatment could delay the growth of hydrogen sulfide-producing bacteria for 10 hours at 86 degrees Fahrenheit (F) (30 degrees Celsius) and for two hours at 72 degrees F (22 degrees C). This delay in growth of hydrogen sulfide-producing bacteria means a delay in production of hazardous hydrogen sulfide as well as improved freshness of the raw materials. Additional time would allow transport of materials to rendering facilities before onset of microbial degradation. The team also studied methods to maximize bacteriophage production and submitted a manuscript on this portion of the study to the Canadian Journal of Microbiology, which has been accepted and is currently being published.

In the second portion of the study, graduate student Chao Gong, post-doctoral Dr. Xiaohua Liu, and Jiang applied the isolated bacteriophages to raw animal materials. Using laboratory and greenhouse conditions, the team simulated transportation and rendering facility conditions to measure bacteriophage impact in a controlled experiment. The team prepared test strips impregnated with lead acetate to measure hydrogen sulfide production and used an electronic hydrogen sulfide monitor. Using laboratory conditions, application of phage to spoiled chicken, chicken offal, chicken feathers, and fresh chicken meat inoculated with hydrogen sulfide-producing bacteria resulted in 25 to 69 percent reduction in hydrogen sulfide production at temperatures between 68 and 98.6 degrees F (20 to 37 degrees C). Under greenhouse conditions, use of the phage resulted in 30 to 85 percent reduction of hydrogen sulfide in chicken offal and feathers. The team is currently working on a manuscript of these findings for submission to a refereed journal.

The significance and impact of this study is proof that bacteriophage control could limit growth of hydrogen sulfide-producing bacteria in raw rendering materials, thereby leading to less spoilage and providing increased safety for workers.

Atterbury, R.J., M.A.P. Van Bergen, F. Ortiz, M.A. Lovell, J.A. Harris, A. De Boer, J.A. Wagenaar, V.M. Allen, and P.A. Barrow. 2007. “Bacteriophage therapy to reduce Salmonella colonization of broiler chickens.” Applied and Environmental Microbiology 73 (14): 4543-4549.

Beauchamp, R.O., J.S. Bus, J.A. Popp, C.J. Boreiko, D.A. Andjelkovich, and P. Leber. 1984. “A critical review of the literature on hydrogen sulfide toxicity.” Critical Reviews in Toxicology 13 (1): 25-97.
Greer, G.G. 1986. “Homologous bacteriophage control of Pseudomonas growth and beef spoilage.” Journal of Food Protection 49: 104-109.

Greer, G.G. and B.D. Dilts. 2002. “Control of Brochothrix thermosphacta spoilage of pork adipose tissue using bacteriophages.” Journal of Food Protection 65 (5): 861-3.

Sheng, H., H.J. Knecht, I.T. Kudva, and C.J. Hovde. 2006. “Application of bacteriophages to control intestinal Escherichia coli O157: H7 levels in ruminants.” Applied and Environmental Microbiology 72 (8): 5359-5366.

Smith, H.W., M.B. Huggins, and K.M. Shaw. 1987. “Factors influencing the survival and multiplication of bacteriophages in calves and in their environment.” Microbiology 133 (5): 1127-1135.

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