Abstract
Microorganisms are key components for plant biomass breakdown within rumen environments. Fibrobacter succinogenes have been identified as being active and dominant cellulolytic members of the rumen. In this study, F. succinogenes type strain S85 was adapted for steady state growth in continuous culture at pH 5.75 and confirmed to grow in the range of pH 5.60–5.65, which is lower than has been reported previously. Wild type and acid tolerant strains digested corn stover with equal efficiency in batch culture at low pH. RNA-seq analysis revealed 268 and 829 genes were differentially expressed at pH 6.10 and 5.65 compared to pH 6.70, respectively. Resequencing analysis identified seven single nucleotide polymorphisms (SNPs) in the sufD, yidE, xylE, rlmM, mscL and dosC genes of acid tolerant strains. Due to the absence of a F. succinogenes genetic system, homologues in Escherichia coli were mutated and complemented and the resulting strains were assayed for acid survival. Complementation with wild-type or acid tolerant F. succinogenes sufD restored E. coli wild-type levels of acid tolerance, suggesting a possible role in acid homeostasis. Recent genetic engineering developments need to be adapted and applied in F. succinogenes to further our understanding of this bacterium.
Highlights
Understanding and overcoming plant biomass recalcitrance is a crucial step to enable industrial biofuels and biomaterials production[1, 2]
Ml scale in a bioreactor, wild-type F. succinogenes S85 was first grown overnight in a serum bottle with 50 ml of medium supplemented with 3 g/L cellobiose, which was inoculated into a bioreactor that contained 500 ml of fully reduced complete medium at pH 6.70
One of the mutations we identified in the acid tolerant F. succinogenes genome sequence is in the sufD gene, encoding a structural protein that may be involved in transfer of sulfur groups to form Fe-S clusters in the SufBCD complex[51, 52]
Summary
Understanding and overcoming plant biomass recalcitrance is a crucial step to enable industrial biofuels and biomaterials production[1, 2]. Anaerobic cellulolytic bacteria are not known to grow (i.e., increase microbial cell mass) below pH 6.0 and the majority of fermentative microorganisms grow within a relatively narrow pH range near circumneutral pH5. Efficient biomass breakdown in the rumen is facilitated by a complex consortium of anaerobic microorganisms and host actions[8,9,10], and building an expanded gene catalogue for biomass deconstruction is one area of applied interest[11]. Modern cattle diets contain rapidly fermentable feedstuffs, which results in low rumen pH for much of the day, reducing the efficiency of forage utilization and can lead to a condition known as sub-acute ruminal acidosis or chronic acidosis[16]. Amongst other challenges, obtaining ruminal pH samples and analyses can be complicated as ruminal pH is not homogenous and different analytical techniques can produce different results[26]
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