Caffeine-degrading microbes could offer vital bioremediation services as well as upcycling coffee waste into valuable pharmaceutical compounds, a new review suggests.

The review by researchers at the University of Alabama is published in a paper, ‘Microbial Metabolism of Caffeine and Potential Applications in Bioremediation’, which appears in the Journal of Applied Microbiology, an Applied Microbiology International publication.

Caffeinesnip

The paper is a compilation and in-depth analysis of the current knowledge pertaining to the microbial degradation of caffeine which will facilitate the engineering of bacterial strains designed to both reduce environmental contamination from waste caffeine and generate high-value pharmaceuticals from a renewable source, says corresponding author Dr Meredith Mock.

Caffeine waste

“With increasing global consumption of caffeine-rich products, like coffee and tea, there is also an increase in urban and processing waste full of residual caffeine with limited disposal options. This waste caffeine has been found to leach into the surrounding environment where it poses a threat to microorganisms, insects, small animals, and entire ecosystems,” she says. 

“A select number of microorganisms have been found to be capable of harnessing caffeine as a carbon and nitrogen source, opening up the possibility of harnessing this environmental contaminant. 

“Recently observed promiscuity of caffeine-degrading enzymes in vivo has further expanded the potential applications to include the engineering of bacterial strains capable of producing a wide variety of valuable caffeine derivatives from a renewable resource. These engineered strains can be used to reduce the negative environmental impact of leached caffeine-rich waste through bioremediation efforts.

Caffeine-degrading microbes

“To supplement these efforts, it is imperative we compile a comprehensive list of what is currently known about caffeine degrading microorganisms, as well as increase our understanding of external factors, applications, and new techniques, such as the impact of heavy metals and cell immobilisation on caffeine degradation efficiency.

“In this work, we have compiled information pertaining to all identified caffeine-degrading microbial strains available in literature with particular focus on bacterial strains due to the large number identified, 79 in total. We have discussed their metabolism and related enzymology, and investigated their potential application in bioremediation. We have identified 79 bacterial strains, 8 yeast strains, and 32 fungal strains capable of metabolizing caffeine by N-demethylation and/or C-8 oxidation.”

The review reveals that as much as 80% of the world’s population consumes at least one caffeinated product daily, primarily in the form of coffee, and as consumption of caffeine-based products increases, the resulting environmental contamination also increases.

Useful derivatives

In spite of the well-known hazards of caffeine, the derivatives of caffeine may actually provide the benefits frequently attributed to caffeine, such as anti-inflammatory and stimulatory properties, but with fewer side effects.

Coffee plant-based waste is often burned or abandoned and left untreated to decompose naturally. These processes are ineffective at removing residual caffeine and have resulted in leaching of caffeine into the environment, which can lead to potential human health risks and ecotoxic effects.

Despite the antimicrobial effects of caffeine, a variety of microorganisms have developed the ability to metabolize caffeine. There are two primary routes of caffeine degradation: N-demethylation and C-8 oxidation. Both of these routes allow the microorganisms to harness the carbon and nitrogen found in caffeine.

 Coffee-loving Pseudomonas

Out of the 119 microorganisms identified in this paper with a natural ability to degrade caffeine, 79 are bacteria, and 37 of the bacterial strains have been reported to belong to the genus Pseudomonas. 18 of the Pseudomonads are reported to be of the species Putida.

“The largest challenge in this work lies in the fact that there is no standardized method for reporting the efficiency of bacterial caffeine degradation. The mechanism of degradation and generation of the resulting products are not always recorded in studies,” Dr Mock says.

“Similarly, there is a large variation in culture conditions and a general lack of description as to cell growth phase, which makes direct comparisons of strains difficult.

“The C-8 oxidative pathway has only been partially characterized; however, the overall pathway and enzymes involved have been proposed based on identified intermediates.“

”The N-demethylation pathway appears to be the more common caffeine catabolic pathway in bacteria. This pathway involves the sequential removal of methyl groups from the nitrogen atoms of the xanthine structure and has been identified in 23 strains of bacteria, one strain of yeast, and fourteen strains of fungi.”

Fungal pathways

All of the caffeine metabolic pathways that have been characterized in fungi have been found to be N-demethylation pathways. In contrast to bacteria, theophylline is the primary metabolite produced from caffeine by fungi, Dr Mock says.

“The ability of microorganisms to metabolize caffeine can have a variety of applications, from pharmaceutical production to the removal and repurposing of waste caffeine. Significant work has been performed on the optimization of caffeine degradation in controlled conditions with some emphasis specifically on pharmaceutical production,” she says.

“Controlled, laboratory conditions would vary significantly from waste-based media with complex compositions.When considering bioremediation applications, variation in substrate composition becomes substantially more problematic.

“The variability and potential toxicity of process waste samples can create environments even more extreme and toxic towards bacterial cells than media supplemented with caffeine alone, making treatment more challenging without a specialized and optimized system.

Waste and soil samples

“Current bacterial studies primarily focus on the degradation of caffeine added to complex or defined media and have rarely been tested on caffeine-rich waste or soil samples. Commonly used methods of caffeine detection are discussed here as well.

“The primary applications for biological caffeine degradation include bioremediation, biopharmaceutical production, and decaffeination. We described the strains engineered to date and the potential medical applications of caffeine derivatives. While these processes are independent, there is room for overlap between them or a synergism where one process may benefit or directly lead into another.”

Dr Mock says she found it surprising that out of all of the strains currently identified and characterized, only two caffeine degradation pathways have been found, even among yeast and fungi.

“About half of the caffeine-degrading organisms identified are pseudomonads, and the primary catabolic pathway found in these microorganisms is N-demethylation.

Widespread contamination

“I was also surprised at how widespread caffeine contamination is, and the lack of uniformity in the reporting of information, including failure to identify the primary compounds generated from caffeine /route of degradation in many of these organisms.”

Dr Mock says increasing global consumption of caffeine has resulted in an increase in global caffeine contamination from residual and waste caffeine that is not fully removed in some wastewater treatment plants or is leached into the soil and groundwater from sources like coffee plant waste. Caffeine has been detected in locations around the world at concentrations high enough to pose a hazard to the environment, and the paper explores the possibility of reducing caffeine contamination by taking advantage of microorganisms already resistant to caffeine and even able to break down and consume caffeine as a source of carbon and nitrogen.

“To facilitate the goal of successful caffeine bioremediation, this article was assembled as a comprehensive review of caffeine-degrading microorganisms, collecting all of the relevant information into one accessible source. It is intended for this paper to be a pool of information for researchers to use to quickly reference previous work,” she says.

Gaps in knowledge

“This article is also intended to facilitate the identification of the gaps in our knowledge that need to be filled in order to move this research forward. This includes information such as the prevalence of heavy metals in real world samples and what impact they may have on caffeine-degrading microorganisms. 

“A lot of research has been done investigating caffeine degradation in controlled, laboratory settings, but little work has been done using more complex media representative of potential real-world samples. This article has emphasized the lack of uniformity in reporting key information regarding these organisms and will hopefully encourage researchers to be more thorough and consistent in how they present their data pertaining to caffeine-degrading microorganisms.

“It is also our hope that researchers will see the potential for both bioremediation as well as biopharmaceutical production by harnessing caffeine-degrading microorganisms. Residual and waste caffeine represents a non-competitive renewable source that can be up-cycled into valuable derivatives of caffeine with promising medical applications.” 

Biosynthetic production

The article was designed to facilitate work investigating the biosynthetic production of high-value caffeine derivatives, as well as the bioremediation of residual waste caffeine by compiling and analyzing the available, relevant information in a way that is accessible. 

“Researchers will be able to use the information compiled in this article to enhance the quality of their work. This article will facilitate more informed strain selection for experiments, targeted engineering of microbial pathways, comparisons between different wild-type strains as well as engineered strains, improved consistency in reporting and characterization strategies, and experimental design,” Dr Mock says.

“More work is necessary to fully explore and characterize the caffeine metabolizing abilities of many of these organisms, and those gaps are highlighted in this work. More investigation is also needed into the effects that complex samples, such as samples taken from an industrial site or wastewater samples, may have on activity. Specifically, how does such variation of substrate affect the optimization of bacterial strain(s), and can a strain be engineered and/or a biodegradation process be established to be successful in both environments as an effective caffeine bioremediate?”

This study was led by Dr. Meredith Mock and Dr. Ryan M. Summers. The work was supported by University of Alabama research funds.

‘Microbial Metabolism of Caffeine and Potential Applications in Bioremediation’ appears in the Journal of Applied Microbiology‘.

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