Over 90% of municipal sewage and more than 50% of industrial wastewater are treated by using activated sludge (AS) process in China. The annual amount of sewage and wastewater, 76.5 billion metric tons a year, accounts for 1/36 of the accessible water resource of China. However, the 4000-plus AS sewage treatment plants of China are facing great challenges such as high energy-consumption, frequent sludge bulking and foaming, and particularly the huge amount of excessive sludge (400 billion metric tons a year) without proper disposal, posing a risk of secondary contamination.    

To develop novel approaches to address these issues, a research team led by Prof. QIU Dongru from Institute of Hydrobiology (IHB) of Chinese Academy of Sciences, studied the microbiome of activated sludge and particularly the floc-forming bacteria. Their findings provide the first direct experimental evidence that PEP-CTERM proteins are required for bacterial floc formation and shed light on the role of PEP-CTERM protein sorting systems in general. Their study may also provide important insights into the innovation of novel activated sludge-based technology and the efficient disposal and utilization of excessive sludge.   

Bacterial floc formation plays a central role in the AS process, allowing for gravitational sludge-and-effluent separation and recycling of AS, two key characteristics of this technology for enriching microbes. The formation of AS flocs has long been known to require bacterial biosynthesis of the extracellular polymeric substance (EPS) including exopolysaccharides. However, EPS is responsible for the high water-absorbing capacity and high water content (>95%) of activated sludge, a trait making it very difficult to dispose and utilize the excessive sludge.   

By using molecular genetics and comparative genomics approaches, the IHB team previously identified a large gene cluster involved in the extracellular polysaccharide (EPS) biosynthesis and floc formation by the predominant AS bacterium Zoogloea resiniphila and interestingly two asparagine synthetase paralogues encoded in this gene cluster are also involved in floc formation (An et al., 2016. Water Research 102: 494-504).    

Subsequently the same team had demonstrated that an RpoN sigma factor was required for floc formation but not exopolysaccharide biosynthesis of another floc-forming bacterium, Aquincola tertiaricarbonis (Yu et al., 2017. Applied and Environmental Microbiology, 83(14). pii: e00709-17).    

Most of the bacterial exopolysaccharides were secreted and released rather than bound to the cells of the rpoN mutant, indicating that an RpoN-regulated component may be required for floc fomration.    

Recently, the IHB team has further demonstrated an additional requirement for the so-called PEP-CTERM protein in Zoogloea resiniphila. Members of a wide-spread family of high copy number-per-genome PEP-CTERM genes, transcriptionally regulated by RpoN sigma factor and PrsK-PrsR two-component system, must be expressed for Zoogloea to build the floc structures that allow gravitational sludge settling and recycling.    

Without PrsK or PrsR, Zoogloea cells were planktonic rather than flocculated, and exopolysaccharides were released into the growth broth in soluble form, which is consistent with their previous findings in Aquincola.    

Moreover, similar extracelluar polysaccharide biosynthesis genes, asparagine synthetase genes, prsK-prsR and PEP-CTERM genes are present in other important activated sludge bacteria such as Thauera, Dechloromonas, Azoarcus, Candidatus Accumulibacter phosphatis, ammonia-oxidizing and comammox bacteria, which may be the reason why these crucial microbes could be enriched via the sludge settling and recycling for water purification.    

These novel findings have just been written into a paper, entitled “Both widespread PEP-CTERM proteins and exopolysaccharides are required for floc formation of Zoogloea resiniphila and other activated sludge bacteria”, and published on-line ahead of print by the international academic journal Environmental Microbiology.