Thanks Yinjie.
What then would an optimal strategy be for using organic wastes as a source of various carbon fuels that are profitable? The various physical fractions of fermented sludge, manure, etc. can be used for different products depending on what is easily produced and refined. Obviously gaseous products like methane are relatively easy to refine by removing contaminants like hydrogen sulfide and water. But the fatty acids you could be producing from the supernatant would still require refining. And what sorts of product concentrations are feasible for these biochemical reactions. Finally engineered E coli is not something that should be let loose into the environment. So what are the licensing restrictions here and at what sort of scale can this be done if the fermentors need to be isolated from the open environment?
--Arno

Answers to Arno:

This project emphasizes on microbial production of biodiesel, an excellent drop-in replacement for diesel and jet fuels. Currently, anaerobic digestion has been proven effective at converting organic wastes into methane. However, methane is a cheap biogas and its primary revenue is for electricity generation. According to Dr. Mark Holtzapple (Texas A&M), methane is currently valued at approximately $4 per million Btu, while drop-in biofuels that can be manufactured from carboxylates produced by anaerobic digestions are worth $20 per million Btu (biomassmagazine.com/articles/7060/an-anaerobic-alternative). His group has built a pilot-scale facility to produce acetate by digestion biomass wastes. The acetate can be chemically converted into ketones and alcohols. However, such conversion is expensive because acetate has to be separated from many other coexisting chemicals. Purification increases the cost significantly. Furthermore, acetate concentrations from these wastes are usually low (<50g/L), making the chemical conversion inefficient.

Based on Dr. Holtzapple’s previous work, We have proposed a modified two-stage conversion process. The first stage is anaerobic digestion to convert waste into acetic acid (the methanogesis can be chemically inhibited by iodoform). In the second stage, acetate can be converted into fatty acids using engineered E.coli strains. The engineered strain can generate medium-chain fatty acids (C12:0–C14:0), which improve biodiesel quality comparing to the natural fat in bio-wastes. This method can be economically feasible because of several reasons, 1) Sources for acetate from anaerobic digestion of wastes are diverse and plentiful. 2) Engineered E. coil could easily grow at various acetate wastes and use other nutrient sources (N and P) in waste streams, preventing the purification of acetic acid from other waste chemicals. 3) The whole process does not require high temperature and pressure, and organic solvent. 4) Acetate is miscible with water, allowing easy mass transfer during fermentation. 5) Acetate has energy content comparable to glucose under aerobic conditions which can be readily converted into diverse products.

As for free lipid extraction method, it requires large amounts of organic solvents and energy consumed by chemical conversion may lead to a significantly environmental concern.

In current stage, I cannot answer the ranges of operating conditions and production scales needed to deploy a profitable and environmental friendly waste-to-biofuel process without a rigorous analysis. In my opinion, the technologies for acetate production from anaerobic digestion of biomass wastes and genetic engineering of E.coli are well-established. Also, there are approximate 180 billion metric tons of agricultural residues (animal manure and crop residues) and 2 billion metric tons of dry municipal solid wastes produced annually in the world. The plentiful resource gives great potential for scaling up our process for advanced fuel production.

Hi,

My research is not on biogas production. We want to make high-value products using wastes. Our suggestion is to use AD for acid accumulation by inhibiting biogas production. Then use acids as the substrate for fermentation. In the past, anaerobic digestion for acetate production has been developed by Mark Holtzapple (Texas A&M). The waste acetate can be purified and chemically converted to biofuel. However, such method is costly since acetate concentration from AD is below 50g/L. There are other complex N, P, C substrates in AD solution. Thereby, we use the engineered strain to convert low concentration waste acetate into high quality fatty acids (in the future, we hope to make FAEE biodiesel directly). FAEE can be easily secreted by cell and recovered as a biofuel.


The project was funded by Gates Foundation (2011~2012). However, we (WUSTL and my collaborators at MSU) did not get the Phase II funding from Gates Foundation to continue this work. I hope people can follow this direction. This technology can be applied in developing countries where the weather is hot and biomass is abundant (e.g., Africa). Such bio-process is particular suitable for development of localized and small operation facilities to fulfill both waste management and bioenergy generations, and thus creating “green” jobs for people in the world.

Dear Yinjie,

I hope you still take the time to respond to Arno's questions.

If I understood right, then Yingjie's research is mainly about fatty acid production (i.e. microbial conversion of one substance into another) and not focussed on biogas production. In fact, quite the contrary, in his research he would aim to inhibit biogas production (and stop the process after the first step of fatty acid production). Correct me if I am wrong.

My questions to Yinjie:

• is this research work ongoing, i.e. do you have more funding for it?
• in which developing country do you see an application for this?

Regards,
Elisabeth

Thanks Yinjie Tang for this interesting report. I have a few simple questions that I am sure can respond to.

1. Have you made any calculations as to the practical scaled up capacity of this sort of acetate transformation process could be?
2. What is the potential lipid content of sewage sludge?
3. What about the sludge from septic tanks and pit latrines?
4. What other sources of acetate are there that can be combined?
5. What class of biofuel are we talking about here and what would be the theoretical tonnage that could be produced say per ton of dry sludge?
6. Any idea about possible relative costs?
7. Have you seen the work from South Korea on the topic? pubs.acs.org/doi/pdf/10.1021/es3019435 Comments on that free lipid extraction method?

Most sincerely
--Arno Rosemarin/Stockholm Environment Institute

The following is an introduction to a research grant under the Grand Challenge Exploration (GCE) Round 7, entitled: "Using fecal sludge for butanol fermentation"
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Title: Integration of sewage sludge digestion with advanced biofuel synthesis

Abstract:
Sewage sludge rich in carbohydrates and other nutrients could be a good feedstock for fuel/chemical production. In this study, fungal and engineered bacterial cultivations were integrated with a modified anaerobic digestion to accumulate fatty acids on sewage sludge. The anaerobic digestion was first adjusted to enable acetogenic bacteria to accumulate acetate. A fungus (Mortierella isabellina) and an engineered bacterium (Escherichia coli created by optimizing acetate utilization and fatty acid biosynthesis as well as overexpressing a regulatory transcription factor fadR) were then cultured on the acetate solution to accumulate fatty acids. The engineered bacterium had higher fatty acid yield and titer than the fungus. Both medium- and long-chain fatty acids (C12:0–C18:0) were produced by the engineered bacterium, while the fungus mainly synthesized long-chain fatty acids (C16:0–C18:3). This study demonstrated a potential path that combines fungus or engineered bacterium with anaerobic digestion to achieve simultaneous organic waste treatment and advanced biofuel production.


Publication:
Liu, Z., Ruan, Z., Xiao, Y., Yu, Y., Tang Y.J., Liao, W., Liu, Y. Integration of anaerobic digestion of sewage sludge with advanced biofuel synthesis. Bioresource Technology. 2013. 132:166-170

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Edit by moderator (EvM):

By the way, the presentation about your grant which you gave last October in Durban is available on the SuSanA website here: www.susana.org/en/resources/library/details/1710


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Edit by EvM: further information by Yinjie:
The grant started November 2011. The project should be ended by April of 2013, but I have accomplished and published the work ahead of time. We have another paper under review now. We will update the final report by the summer.

The research was carried out at Washington University, St. Louis, Missouri, USA

Tang Lab studies microbial cells as factories for production of bio-products and remediation of toxic compounds. Our research involves interdisciplinary fields ranging from microbiology/molecular biology to chemical and bioprocess engineering. On the biological side: we develop and studied the functional genomics tools to provide quantitative readout of metabolic status; (2) apply computer models to describe the cellular metabolism; (3) build microbial strains to produce biofuel or detoxify contaminants. On the engineering side, we will use natural environmental microorganisms or genetically modified species in large scale processes such as in situ bioremediation or reactor engineering process. Our final goal is to bridge the gap between advanced biological researches and their applications in bioprocess/environmental engineering .

Link to Durban presentation