Ciliate protozoa affect rumen microbiome composition and metabolism and act as ecosystem engineers

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Before I started my Ph.D., I never thought that a cow could be such a fascinating animal. I imagined them grazing in the field or chewing straw and hay  in the confined dairy farm while supplying milk and meat for human nutrition but not more than that. I recognized their agricultural importance but also was aware of their infamous methane emission, which is a potent greenhouse gas that highly contributes to global climate change [1]. Three years later, from the beginning of my deep-dive research on the rumen microbiome, I realized I was completely wrong.  Cows belong to Ruminantia, a Latin word describing their repeated chewing behavior, and consist of many other  herbivores mammals that have specialized stomachs enabling plant material degradation at the beginning of their digestive system, the rumen [2]. This simplified name led me to the realization that I am not the only one who oversimplifies cows, but the taxonomy itself underestimates ruminants. Not only that the rumen function and biology are much more complex than chewing, but the credits for the plants' degradation wrongly attributed solely to the cow, rather than to the microbial community, which are the 'hard workers' that successfully exploit the unreachable plant's energy by performing fermentation[3]. We can imagine the rumen cow as the skeleton of this biological machinery and the microbes as the functioning interior organs.

Gladly, with recent technological advances including the emergence of high throughput sequencing methods, cutting-edge bioinformatics tools, and the expansion of the common biological concept, including a comprehensive grasp of the high microbial diversity, the scientific spotlight begins to move slowly towards the microbial world [4]. Microbes were found to be omnipresent with diverse functions.  It is true also for the rumen environment, as the effects of the prokaryotic cells on cow productivity, health and disease states, and the effects on the environment were revealed [5]. Unfortunately, a lot of work has to be done on changing common old-school conceptions including the one that considers microbial communities exclusively as prokaryotes [6][7]. In the rumen, the eukaryotic population, which is mainly composed of ciliate protozoa, can contribute up to 50% of the microbial biomass [8]. Although most of the time the ruminal protozoa were neglected, few studies showed high effects on the consumption and production of metabolites in the rumen, mainly the effect on volatile fatty acid production (the energetic currency of the cow) and methane emission. The methane results were striking because even though the only known methane producers in the rumen are methanogenic archaea, elimination of ciliate protozoa led to a decrease in methane emission while methanogens abundance was not affected at all [9]. Other results showed that ciliate protozoa affect the abundances of some prokaryotic taxa, which is not surprising as it is well known from other environments that protozoa can serve as predators in ecosystems, and predation is a controlling force in ecological systems enabling species coexistence [9][10].

In my research, I'm trying to elucidate the neglected microbial component- ciliate protozoa and to uncover their effect on the microbial community and metabolism while dealing with microbe-microbe interactions. I am sure that there is much more than meets the eye and I would like to reveal protozoa's true nature.   

So in order to answer our intriguing questions regarding protozoa, we designed a microcosm experiment that will illustrate the effect of protozoa populations on the overall natural prokaryotic community in the rumen. To this end, we separated protozoa into populations by their size, and exposed the same ruminal prokaryotic environment to these various protozoa size populations. I was enthusiastic to ask my questions on the real natural community and not on a narrow subset of microbes like was done before.  

One of the striking results we have got is that prokaryotic communities that were exposed to protozoa showed an increase in their diversity. It is an available possibility that protozoa, as the ruminal predators, increase diversity, based on  theoretical models and previous studies that were done on simplified communities. But surprisingly the highest diversity was obtained in the intermediate size population. Strikingly, the diversity within  genus populations was highly increased suggesting that protozoa allow for the co- existence of phylogenetically and possibly metabolically, similar species.  Also, we obtained enrich in some specific taxa with the presence of protozoa.  

Then we asked ourselves how protozoa affect methane emission and surprisingly methane emission patterns across the protozoa population resembled the diversity. Protozoa presence increases methane emission and the peak was observed in intermediate populations where prokaryotic diversity was also at its peak. 

Based on our results, and in line with previous studies, we assume that there is a kind of tradeoff between predation and competition, and in a given ecological system each taxon uses a different strategy to survive and multiply. The predator, ciliate protozoa in our case, affects the competition dynamics while reducing competition pressure in the system by introducing predation force. Taking all this into account, we think that in systems with no protozoa biomass, like our free-protozoa microcosms, there is no predation pressure at all so the very high competition level in the system leads to species exclusion. On the other hand, with very high protozoa biomass, such as microcosms containing large populations, the increase in predation pressure reaches the point where diversity can not be fully maintained and diversity is only slightly higher than free- protozoa communities. In the microcosms that contain intermediate populations, the balance between predation and competition is optimal for species coexistence and as a result the highest increase in diversity.  Our left open questions are how increase in microbial diversity is related to the increase in methane emission? Do specific protozoa populations directly interact with methanogens and does this interaction result in higher methane emission? Or maybe the increase in the overall microbial diversity lead to these methane changes? 

As it often does, our result raised many more questions than it answered, making this aspect of rumen microbiome a fresh avenue for many new and exciting questions waiting to be resolved. The effect of protists in microbial communities should not be underestimate, and a true understanding can only arise by studying all their components.

Link to our paper:


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Ronnie Solomon

Student, Ben Gurion university