Recovery of cyanophycin granule polypeptide from activated sludge: carbon source dependence and aggregation-induced luminescence characteristics

Abstract

● Sodium acetate significantly enriched the CGP synthetase-encoding gene.

● The highest CGP yield was obtained from activated sludge fed with sodium acetate.

● Biofilm is more conducive to CGP accumulation compared with floc sludge.

● Aggregation-induced luminescence of CGP was first reported.

In the sewage treatment process, facilitating the conversion of pollutants into value-added resources holds great potential for reducing the amount of greenhouse gas emissions and promoting economic circulation. Cyanophycin granule polypeptide (CGP), a recently discovered high value-added biopolymer present in activated sludge, has provided new avenues for the recovery of resources. However, the mechanisms that regulate CGP synthesis and the characteristics of this biopolymer in activated sludge remain unclear thus far. This study investigated the synthesis of CGP, polyhydroxyalkanoates (PHA), and alginate-like exopolysaccharides (ALE) in various microbial aggregates under different carbon sources feeding conditions. Our results showed that the CGP yields was superior that of PHA and ALE when subjected to identical carbon source feeding conditions. Furthermore, biofilm was more conducive to CGP accumulation than floc sludge. Compared with glucose and methanol, sodium acetate significantly enriched the CGP synthetase-encoding gene (cphA_abundance = ~17419), resulting in the highest CGP yield (average 107.1 mg/g MLSS) in both biofilm and floc sludge. This study is the first to reported the characteristic fluorescence of CGP (Ex/Em = ~360/450 nm) caused by the aggregated luminescence of arginine on the side chains. Overall, this study highlights the potential application of CGP as a fluorescent material and offers insights into CGP recovery from activated sludge in wastewater treatment plants.

Graphical abstract

Keywords

Cyanophycin / Polyhydroxyalkanoate / Alginate-like exopolysaccharides / Bioresource recovery / Aggregation-induced luminescence

References

[1] Adames K , Euting K , Broker A , Steinbuchel A . (2013). Investigations on three genes in Ralstonia eutropha H16 encoding putative cyanophycin metabolizing enzymes. Applied Microbiology and Biotechnology, 97(8): 3579–3591

[2] Amorim de Carvalho C, Ferreira Dos Santos A, Tavares Ferreira T J, Sousa Aguiar Lira V N, Mendes Barros A R, Bezerra Dos Santos A (2021). Resource recovery in aerobic granular sludge systems: Is it feasible or still a long way to go? Chemosphere, 274: 129881

[3] Chen H , Chen Z , Chu X , Deng Y , Qing S , Sun C , Wang Q , Zhou H , Cheng H , Zhan W , Wang Y . (2022a). Temperature mediated the balance between stochastic and deterministic processes and reoccurrence of microbial community during treating aniline wastewater. Water Research, 221: 118741

[4] Chen X , Lee Y J , Yuan T , Lei Z , Adachi Y , Zhang Z , Lin Y , Van Loosdrecht M C M . (2022b). A review on recovery of extracellular biopolymers from flocculent and granular activated sludges: cognition, key influencing factors, applications, and challenges. Bioresource Technology, 363: 127854

[5] Chen Y D , Yang Z , Ren N Q , Ho S H . (2020). Optimizing the production of short and medium chain fatty acids (SCFAs and MCFAs) from waste activated sludge using different alkyl polyglucose surfactants, through bacterial metabolic analysis. Journal of Hazardous Materials, 384: 121384

[6] Dueholm M K D , Besteman M , Zeuner E J , Riisgaard-Jensen M , Nielsen M E , Vestergaard S Z , Heidelbach S , Bekker N S , Nielsen P H . (2023). Genetic potential for exopolysaccharide synthesis in activated sludge bacteria uncovered by genome-resolved metagenomics. Water Research, 229: 119485

[7] Frommeyer M , Wiefel L , Steinbuchel A . (2016). Features of the biotechnologically relevant polyamide family “cyanophycins” and their biosynthesis in prokaryotes and eukaryotes. Critical Reviews in Biotechnology, 36(1): 153–164

[8] González-Martínez A , Calderon K , Gonzalez-Lopez J . (2016). New concepts of microbial treatment processes for the nitrogen removal: effect of protein and amino acids degradation. Amino Acids, 48(5): 1123–1130

[9] Hao X , Wu D , Li J , Liu R , Van Loosdrecht M . (2022). Making waves: a sea change in treating wastewater―Why thermodynamics supports resource recovery and recycling. Water Research, 218: 118516

[10] Huang R , Xu J , Xie L , Wang H , Ni X . (2022). Energy neutrality potential of wastewater treatment plants: a novel evaluation framework integrating energy efficiency and recovery. Frontiers of Environmental Science & Engineering, 16(9): 117

[11] Khomlaem C , Aloui H , Oh W G , Kim B S . (2021). High cell density culture of Paracoccus sp. LL1 in membrane bioreactor for enhanced co-production of polyhydroxyalkanoates and astaxanthin. International Journal of Biological Macromolecules, 192: 289–297

[12] Kim N K , Mao N , Lin R , Bhattacharyya D , Van Loosdrecht M C M , Lin Y . (2020). Flame retardant property of flax fabrics coated by extracellular polymeric substances recovered from both activated sludge and aerobic granular sludge. Water Research, 170: 115344

[13] Li H , Cheng J , Deng H , Zhao E , Shen B , Lam J W Y , Wong K S , Wu H , Li B S , Tang B Z . (2015). Aggregation-induced chirality, circularly polarized luminescence, and helical self-assembly of a leucine-containing AIE luminogen. Journal of Materials Chemistry. C, Materials for Optical and Electronic Devices, 3(10): 2399–2404

[14] Li H , Zhang J , Shen L , Chen Z , Zhang Y , Zhang C , Li Q , Wang Y . (2019). Production of polyhydroxyalkanoates by activated sludge: correlation with extracellular polymeric substances and characteristics of activated sludge. Chemical Engineering Journal, 361: 219–226

[15] Li J , Hao X , Gan W , Van Loosdrecht M C M , Wu Y . (2021a). Recovery of extracellular biopolymers from conventional activated sludge: potential, characteristics and limitation. Water Research, 205: 117706

[16] Li J , Hao X , Gan W C M , Van Loosdrecht M , Wu Y . (2022). Controlling factors and involved mechanisms on forming alginate like extracellular polymers in flocculent sludge. Chemical Engineering Journal, 439: 135792

[17] Li Y Y , Huang X W , Li X Y . (2021b). Use of a packed-bed biofilm reactor to achieve rapid formation of anammox biofilms for high-rate nitrogen removal. Journal of Cleaner Production, 321: 128999

[18] Lu L , Guest J S , Peters C A , Zhu X , Rau G H , Ren Z J . (2018). Wastewater treatment for carbon capture and utilization. Nature Sustainability, 1(12): 750–758

[19] Lu Z , Ye J , Chen Z , Xiao L , Lei L , Han B P , Paerl H W . (2022). Cyanophycin accumulated under nitrogen-fluctuating and high-nitrogen conditions facilitates the persistent dominance and blooms of Raphidiopsis raciborskii in tropical waters. Water Research, 214: 118215

[20] Miyakawa T , Yang J , Kawasaki M , Adachi N , Fujii A , Miyauchi Y , Muramatsu T , Moriya T , Senda T , Tanokura M . (2022). Structural bases for aspartate recognition and polymerization efficiency of cyanobacterial cyanophycin synthetase. Nature Communications, 13(1): 5097

[21] Qin P , Cui H , Li P , Wang S , Fan S , Lu J , Sun M , Zhang H , Wang S , Su X . . (2023). Early stage of biofilm assembly on microplastics is structured by substrate size and bacterial motility. iMeta, 2023: e121

[22] Remminghorst U , Rehm B H . (2006). In vitro alginate polymerization and the functional role of alg8 in alginate production by Pseudomonas aeruginosa. Applied and Environmental Microbiology, 72(1): 298–305

[23] Sharon I , Haque A S , Grogg M , Lahiri I , Seebach D , Leschziner A E , Hilvert D , Schmeing T M . (2021). Structures and function of the amino acid polymerase cyanophycin synthetase. Nature Chemical Biology, 17(10): 1101–1110

[24] Sharon I , Pinus S , Grogg M , Moitessier N , Hilvert D , Schmeing T M . (2022). A cryptic third active site in cyanophycin synthetase creates primers for polymerization. Nature Communications, 13(1): 3923

[25] Simon R D , Lawry N H , Mclendon G L . (1980). Structural characterization of the cyanophycin granule polypeptide of Anabaena cylindrica by circular dichroism and raman spectroscopy. Biochimica et Biophysica Acta. Protein Structure, 626(2): 277–281

[26] Tuck C O , Perez E , Horvath I T , Sheldon R A , Poliakoff M . (2012). Valorization of biomass: deriving more value from waste. Science, 337(6095): 695–699

[27] van Loosdrecht M C , Brdjanovic D . (2014). Anticipating the next century of wastewater treatment. Science, 344(6191): 1452–1453

[28] Wang L , Wang D H , Zhang G H , Xu D , Deng W X . (2016). Crystal structure, spectroscopic investigation and thermal properties of l-lysine p-toluenesulfonate. Journal of Molecular Structure, 1108: 179–186

[29] Wu W , Liu B . (2021). Aggregation-induced emission: challenges and opportunities. National Science Review, 8(6): nwaa222

[30] Zhang C, Hu P, Liu Q, Lu Z, Cao B, Tang Y, Hao T (2022). Biopolymer recovery from waste activated sludge toward self-healing mortar crack. Science of the Total Environment, 858(Pt 3): 160107

[31] Zhang X , Lin Y , Wu Q , Wang Y , Chen G Q . (2020). Synthetic biology and genome-editing tools for improving PHA metabolic engineering. Trends in Biotechnology, 38(7): 689–700

[32] Zhang Z , Zhou Y , Zhang J , Xia S , Hermanowicz S W . (2016). Effects of short-time aerobic digestion on extracellular polymeric substances and sludge features of waste activated sludge. Chemical Engineering Journal, 299: 177–183

[33] Zhu J , Wang J , Chen Y P , Qing T P , Zhang P , Feng B . (2022). Quantitative proteomics and phosphoproteomics elucidate the molecular mechanism of nanostructured TiO₂-stimulated biofilm formation. Journal of Hazardous Materials, 432: 128709

[34] Ziegler K , Deutzmann R , Lockau W . (2002). Cyanophycin synthetase-like enzymes of non-cyanobacterial eubacteria: characterization of the polymer produced by a recombinant synthetase of Desulfitobacterium hafniense. Journal of Biosciences, 57(5–6): 522–529

[35] Zou K , Huang Y , Feng B , Qing T P , Zhang P , Chen Y P . (2022). Cyanophycin granule polypeptide: a neglected high value-added biopolymer, synthesized in activated sludge on a large scale. Applied and Environmental Microbiology, 88(14): e0074222

RIGHTS & PERMISSIONS

Higher Education Press