WASTE MILK REMEDIATION AND HYDROGEN GAS PRODUCTION USING A HIGH-TEMPERATURE ANAEROBIC DIGESTOR

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1007967
• Project Start Date: Oct 1, 2015
• Project End Date: Sep 30, 2019
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MASSACHUSETTS
(N/A)
AMHERST,MA 01003

Performing Department
Microbiology

Non Technical Summary
Nearly all food and agricultural waste in the U.S. enters landfills, making it the largest contributor of material entering these sites. Biological pre-treatment of large organic molecules by fermentative organisms lowers the high organic carbon load in waste, lowers wastewater treatment costs, and can produce bioenergy to partially offset costs. Conceivably, microbes that grow best above 80°C, or so-called 'hyperthermophiles', could be used to consolidate wastewater heat treatment and organic remediation in a single step to decrease costs while producing H2 as an energy product. My laboratory has adapted the hyperthermophilic archaeon Thermococcus paralvinellae to grow at 82°C on waste milk, including milk containing the bacterial antibiotic Ceftiofur that currently lacks a disposal route. T. paralvinellae produced up to 5 mmol of H2 per liter in bottles in less than 100 hours in a growth medium containing up to 70% milk (v/v). The process killed all of the mesophilic bacteria, including pathogens, present in the milk that otherwise grew on Luria Bertani, Sheep's Blood, and MacConkey agar plates at 37°C. It also removed the Ceftiofur, which is heat labile, from the waste. The next phase of this research is to optimize the growth of this organism for maximal organic compound removal and H2 production from waste milk in a high-temperature, bench-scale anaerobic digestor.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
403	3450	1100	100%

Knowledge Area
403 - Waste Disposal, Recycling, and Reuse;

Subject Of Investigation
3450 - Milk;

Field Of Science
1100 - Bacteriology;

Keywords

waste milk

remediation

hydrogen production

bioenergy

Goals / Objectives
This proposal seeks to make fundamental advances in waste milk remediation and bioenergy generation using a hyperthermophilic microbe. We will study the ability of Thermococcus paralvinellae to degrade waste milk at 80°C, including milk from cows treated for mastitis and containing the bacterial antibiotic Ceftiofur, kill the bacteria present in the waste, remove the Ceftiofur, and produce hydrogen gas as an end product in an anaerobic digestor. Previous research by the PI showed that T. paralvinellae can perform all of these tasks when grown on a small scale in sealed bottles. The next phase of the project is to determine the conditions necessary for maximum organic compound removal and hydrogen gas production in a bench-scale continuous flow anaerobic digestor. This research has three objectives:Determine the maximum concentration of waste milk that can be treated at 80°C by T. paralvinellae in the anaerobic digestor.Determine the optimal solids retention time (SRT) for removal of organic compounds in waste milk and production of H2 in the digestor with concomitant measurements of secondary metabolites, soluble protein, soluble sugars, BOD5, COD, VSS, TSS, contaminant bacteria, and Ceftiofur from treated cows.Adapt T. paralvinellae to degrade lactose through serial transfers and weaning from a similar compound.This research will provide insight into ways to rapidly treat waste milk specifically and food and agricultural wastes in general, the largest contributor to municipal solid waste in the U.S., with concomitant decontamination and bioenergy production.

Project Methods
Cell culturing and analytical techniques. The organism used for this study is Thermococcus paralvinellae ES1 (DSM 27261) from the PI's hyperthermophile culture collection. The base salts solution for the growth media is modified from a medium used previously (Adams et al., 2001; Hensley et al., in prep.). Bottle experiments will contain 50 ml of medium in a 120 ml serum bottle with 1 atm of N2 in the headspace and will be incubated at 80°C in a forced-air incubator. Cells will be counted using a Petroff-Hausser counting chamber and a phase-contrast light microscope. H2 will be measured using a gas chromatograph (Shimadzu GC-8A) equipped with a thermal conductivity detector, a 60/80 Carboxen 100 column (Supelco), and Ar as the carrier gas. Soluble metabolites (formate, acetate, butyrate, isovalerate, succinate) will be measured using a high-performance liquid chromatograph equipped with a photodiode-array detector (Waters) and an Aminex HPX-87H ion exclusion column. Chemical oxygen demand (COD), biological oxygen demand (BOD5), and Ceftiofur will be measured using a COD HR test kit (Hach Co.), a BOD5 Cuvette Test kit (Hach Lange), and a Ceftiofur ELISA test kit (Bioo Scientific) as described by the manufacturers. Abundances of mesophilic bacteria in the waste milk, especially those related to mastitis, will be measured by counting colony forming units on Luria Bertani (general), Sheep's Blood (Gram-positive bacteria), and MacConkey's (Gram-negative enteric bacteria) agar plates. Total suspended solids (TSS) and volatile suspended solids (VSS) will be measured according to the Standard Methods for the Examination of Water and Wastewater.Adaptation to growth on lactose. T. paralvinellae has the gene that encodes for β-galactosidase, the protein that cleaves lactose into glucose and galactose, but a previous attempt at growing the organism on 0.5% lactose was unsuccessful. However, it may be possible to adapt T. paralvinellae to growth on lactose by growing it on cellobiose and lactose. T. paralvinellae grows on cellobiose (Oslowski et al., 2011), which is chemically similar to lactose. The concentration of cellobiose would gradually be reduced until the organism was weaned to grow on lactose only.Adaptation to high milk concentrations. T. paralvinellae grew on waste milk up to 10% (v/v), but growth on higher milk concentrations was not tested. T. paralvinellae would be grown on increasing concentrations of waste milk in bottles and H2 production would be measured to adapt the organism to higher milk concentrations and to determine the upper milk threshold for H2 production.Operation of the bench-scale anaerobic digestor. The PI has grown T. paralvinellae at 82°C in a 2-liter chemostat with stirring, gas-flow control, pH control, temperature control, and a 20-liter reservoir of feed medium that is heated and flushed with N2 (Figure 4). The growth medium contained 0.5% tryptone, and the concentrations of cells and H2 were measured throughout growth (Figure 4). For this project, T. paralvinellae will be grown in the chemostat (i.e., a bench-scale anaerobic digestor) on increasing concentrations of waste milk until the concentration becomes inhibitory to growth and H2 production. Growth on waste milk in the chemostat will also be optimized and modeled at varying dilution rates to determine the optimum solids retention time, waste degradation, and H2 production rate. For each set of conditions, amount of H2, secondary metabolites (e.g., formate, acetate, butyrate, isovalerate), mesophilic bacterial contaminants, TSS, VSS, and Ceftiofur will be measured as described above.

Progress 10/01/15 to 09/30/19

Outputs
Target Audience:Our target audience was the broad scientific community that is interested in high-temperature microbes that can break down organic material at 80ºC and produce H2 as an end product. Changes/Problems:We are testing additional waste streams to make a high-temperature waste-to-hydrogen energy bioreactor more feasible and are analyzing ways to ameliorate hydrogen inhibition within the reactor. What opportunities for training and professional development has the project provided?The project trained a female graduate student on the cultivation of high-temperature anaerobes in bioreactors, RNA isolation, transcriptional differential expression analyses, and flux balance metabolic computational modeling. The project also trained an undergraduate student on the methods for growth of a high-temperature anaerobe. How have the results been disseminated to communities of interest?The results of our H2 inhibition study, including growth and H2 production kinetics, differential gene expression analysis, and flux balance modeling, were published in 2018 in the journal Environmental Microbiology. Metabolite production kinetic analyses for our model organism when grown with and without a H2 syntrophic partner, which demonstrated that H2 inhibition is relieved when there is efficient and effective H2 removal, was published in 2019 in the journal Applied and Environmental Microbiology. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? We found that H2 buildup in the reactor caused the growth of T. paralvinellae to be inhibited, which led to the production of formate in lieu of H2 in the reactor. H2 inhibition led to the synthesis of formate hydrogenlyase, which we believe is used toconvert H2 in the cell to formate during inhibition. We then grew T. paralvinellae with a hyperthermophilic methanogen, Methanocaldococcus jannaschii, that would consume the H2 produced by T. paralvinellae before it led to H2 inhibition. When grown with M. jannaschii, no formate was secreted by T. paralvinellae suggesting that H2 inhibition was relieved. These data demonstrate the need for efficient and effective H2 removal from the reactor in order to obtain the desired waste product.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Topcuoglu BD, Meydan C, Orellana R, Holden JF (2018) Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae. Environmental Microbiology 20:949-957.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Topcuoglu BD, Meydan C, Nguyen TB, Lang SQ, Holden JF (2019) Growth kinetics, carbon isotope fractionation, and gene expression in the hyperthermophile Methanocaldococcus jannaschii during hydrogen-limited growth and interspecies hydrogen transfer. Applied and Environmental Microbiology 85:e00180-19.

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Our target audience was the broad scientific community that is interested in high-temperature microbes that can break down organic material at 80ºC and produce H2 as an end product. Changes/Problems:As described previously, we are testing additional waste streams to make a high-temperature waste-to-hydrogen energy bioreactor more feasible and are analyzing ways to ameliorate hydrogen inhibition within the reactor. What opportunities for training and professional development has the project provided?As reported last year, the project trained a female graduate student on the cultivation of high-temperature anaerobes in bioreactors, RNA isolation, transcriptional differential expression analyses, and flux balance metabolic computational modeling. This past year, the project also trained an undergraduate student on the methods for growth of a high-temperature anaerobe. How have the results been disseminated to communities of interest?The results of our H2 inhibition study, including growth and H2 production kinetics, differrential gene expression analysis, and flux balance modeling, were published last January in the journal Environmental Microbiology. Metabolite production kinetic analyses for our model organism when grown with and without a H2 syntrophic partner, which demonstrated that H2 inhibition is relieved when there is efficient and effective H2 removal, was submitted for publication in the journal Applied and Environmental Microbiology. What do you plan to do during the next reporting period to accomplish the goals?The graduate student working on this project graduated with her Ph.D. last May. The undergraduate working on this project is examining the biochemical mechanisms for formate secretion during H2 inhibition in T. paralvinellae.

Impacts
What was accomplished under these goals? We found that H2 buildup in the reactor caused the growth of T. paralvinellae to be inhibited, which led to the production of formate in lieu of H2 in the reactor. H2 inhibition led to the synthesis of formate hydrogenlyase, which we believe is used to convert H2 in the cell to formate during inhibition. We then grew T. paralvinellae with a hyperthermophilic methanogen, Methanocaldococcus jannaschii, that would consume the H2 produced by T. paralvinellae before it led to H2 inhibition. When grown with M. jannaschii, no formate was secreted by T. paralvinellae suggesting that H2 inhibition was relieved. These data demonstrate the need for efficient and effective H2 removal from the reactor in order to obtain the desired waste product.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Topcuoglu BD, Meydan C, Orellana R, Holden JF (2018) Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae. Environmental Microbiology 20:949-957.
• Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Topcuoglu BD, Meydan C, Nguyen TB, Lang SQ, Holden JF (in review) Growth kinetics, carbon isotope fractionation, and gene expression in the hyperthermophile Methanocaldococcus jannaschii during hydrogen-limited growth and interspecies hydrogen transfer. Applied and Environmental Microbiology.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Nearly all food and agricultural waste in the U.S. enters landfills, making it the largest contributor of material entering these sites. Biological pre-treatment of large organic molecules by fermentative organisms lowers the high organic carbon load in waste, lowers wastewater treatment costs, and can produce bioenergy to partially offset costs. Conceivably, microbes that grow best above 80°C, or so-called 'hyperthermophiles', could be used to consolidate wastewater heat treatment and organic remediation in a single step to decrease costs while producing H2 as an energy product. My laboratory has adapted the hyperthermophilic archaeon Thermococcus paralvinellae to grow at 82°C on waste milk, including milk containing the bacterial antibiotic Ceftiofur that currently lacks a disposal route. T. paralvinellae produced up to 5 mmol of H2 per liter in bottles in less than 100 hours in a growth medium containing up to 70% milk (v/v). The process killed all of the mesophilic bacteria, including pathogens, present in the milk that otherwise grew on Luria Bertani, Sheep's Blood, and MacConkey agar plates at 37°C. It also removed the Ceftiofur, which is heat labile, from the waste. The next phase of this research is to optimize the growth of this organism for maximal organic compound removal and H2 production from waste milk in a high-temperature, bench-scale anaerobic digestor. Changes/Problems:As described above, we are testing additional waste streams to make a high-temprature waste-to-hydrogen energy bioreactor more feasible, and are analyzing ways to ameliorate hydrogen inhibition within the reactor. What opportunities for training and professional development has the project provided?As reported last year, the project trained a female graduate student on the cultivation of a high-temperature anaerobe in a chemostat, RNA isolation and transcriptional differential expression analysis, and flux balance analysis metabolic computational modeling. How have the results been disseminated to communities of interest?The results of our H2 inhibition study, including growth and H2 production kinetics, differential gene expression analysis, and flux balance modeling, were accepted for publication in the journal Environmental Microbiology. What do you plan to do during the next reporting period to accomplish the goals?Because the milk wastestream in Western Massachusetts is too small to justify continuous operation of a waste-to-H2 bioreactor, a new undergraduate in the lab is testing other waste streams that could be used more regularly, that would include periodically added waste milk and other agricultural wastes.

Impacts
What was accomplished under these goals? We found that H2 buildup in the reactor caused the growth of T. paralvinellae to be inhibited, which led to the production of formate in lieu of H2 in the reactor. H2 inhibition led to the synthesis of formate hydrogenlyase, which we believe is used to convert H2 in the cell to formate during inhibition. These data demonstrate the need for efficient and effective H2 removal from the reactor in order to obtain the desired waste product and to stimulate growth.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Topcuoglu BD, Meydan C, Orellana R, Holden JF (2018) Formate hydrogenlyase and formate secretion ameliorate H2 inhibition in the hyperthermophilic archaeon Thermococcus paralvinellae. Environmental Microbiology (in press).

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:The general scientific community through a poster presentation at an undergraduate research symposium at the University of Massachusetts Amherst. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?1. The project trained an undergraduate minority female from the Environmental Sciences program on how to cultivate high-temperature anaerobes and how to conduct a public policy survey to determine the feasibility of the project. 2. The project trained a female graduate student on the cultivation of a high-temperature anaerobe in a chemostat, RNA isolation and transcriptional differential expression analysis, and flux balance analysis metabolic computational modeling. How have the results been disseminated to communities of interest?1. The undergraduate student wrote an honors thesis on her research and presented it as a poster at a statewide undergraduate research symposium at the University of Massachusetts Amherst. What do you plan to do during the next reporting period to accomplish the goals?1. The chemostat, gene transcription, and metabolic modeling results will be submitted for publication in a peer-reviewed journal. 2. A new undergraduate student will be recruited to continue the project and meet the stated goals of the grant.

Impacts
What was accomplished under these goals? 1. An undergraduate student conducted a feasibility study for her honors thesis to determine the need and likelihood that thermophilic milk waste remediation could be implemented in western Massachusetts. She found that within a 30 mile radius of the UMass campus that there are 12 dairy farms. She distributed a questionaire survey to each of the farms and then conducted follow-up interviews with each of the farmers. She found a general unwillingness on the part of the farmers to collect their waste milk, deliver it to a treatment facility, and payment of a fee for its treatment. She also concluded that the volume of waste milk generated for this region alone would not justify implementation of a bioreactor unless it could be combined with other local waste streams. 2. A graduate student conducted bioreactor experiments to determine the effect of H2 buildup and inhibition on the growth and H2 production of the model organism. She found that H2 inhibition caused the organism to switch its metabolite production from H2 to formate. She created a metabolic network model to predict how growth of the model organism is affected by increasing production of formate in lieu of H2 production.

Publications

• Type: Theses/Dissertations Status: Published Year Published: 2016 Citation: Teixeira, M. (2016) Feasibility of using hyperthermophiles to remediate waste milk and produce H2. Commonwealth Honors College Undergraduate Thesis, University of Massachusetts Amherst. 53 pages.