Potential And Mechanisms Of Plant-growth Biostimulants Production From High-solid Sewage Sludge Stabiliation Treatments

As the urbanization is quickly developing in China,the treatment capacity of wastewater exceeded 200 million m³/d.Sewage sludge,the inevitable by-product from the wastewater biological treatment process,is generated over 60 million ton annually（total solid content≈20%）.Sewage sludge is considered to have high fertilizing potential with nitrogen,phosphorus,potassium,and micronutrients.The land application of sewage sludge is an environmentally friend way of waste-to-fertilizer,which is in line with the goals of and carbon neutral by 2050.Stabilization treatment of sludge is the premise of sewage sludge land application.In recent years,to improve the fertilizing effectiveness of stabilized sewage sludge,the identification and production potential of micromolecular plant-growth biostimulants（Micro-PBs）are attracting increasing interest.The known species of Micro-PBs can be categorized as phytohormones and allelochemicals,which are in the forms of trace organic acids with well-leveraged effects on promoting plant growth.As for the well-known macromolecular PB（Macro-PB）,i.e.,humic acids（HA）,their formation requires the assembly of amino acids and carboxyl acids,most of which can be classified into Micro-PBs.In other words,the successful construction of the Macro-PB also depends on Micro-PBs production.Thus,it is of primary importance to develop methods to maximize the Micro-PBs production from sewage sludge before assembling them into Macro-PB.This study attempted to promote effective Micro-PBs production from sewage sludge and Macro-PB formation through advanced anaerobic digestion,composting and abiotic treatments and explore the underpinning regulation mechanisms.The main research contents and results are as follows:1.Establishing the characterization system of sludge HA according to the changes of their core structures consisting of O-Aryl and quinones.In the stabilized high-solid sewage sludge after biotreatment,this study found the sludge HA formation mainly rely on proteins（PN）transformation and sludge HA are easily cross-linked with PN and PN derives through via various forces of metal bridges,humic self-assembly and hydrophobic interaction.Moreover,the HA-PN structures tend to aggregate in extracellular polymeric substances（EPS）,which dominates the extracellular organic stabilization in sludge.Notably,HA and PN can be distinguished by their aromatic conjugated origins（O-aryl and quinones as the core structures in HA while aryl as the aromatic residues in aromatic amino acids）.Besides,the O-aryl and quinones are also the bioactive center of groups to make the HA act as plant-growth biostimulants.Thus,this study proposed a novel characterization strategy of sludge supramolecular HA that monitoring aromaticity via spectroscopy methods,measuring the total changes of aromatic carbon of O-aryl and aryl via ¹³C NMR spectra and determining the content and occurrence state of quinones via electrochemical tests.2.Revealing the sludge humification mechanisms during high-solid AD.High-solid anaerobic digestion（HAD）can directly treat dewatered sewage sludge（total solid content≥10%）with superior volume efficiency.Sludge stabilization during HAD is expected to achieve by throughout organic degradation and conversion towards methane-rich biogas release and humic formation.The important characteristics and mechanisms of sludge humification in HAD found in this present study are follows.i)The formation of O-Aryl and quinone groups as the HA core structures are restricted during anaerobic digestion,and the process of the sludge humification is essentially the endogenous HA core drives the rearrangement of extracellular organic matter,especially the aromatic amino and carboxyl groups.ii)The preparation of the O-Aryl core and aromatic amino precursor for the formation of HA in anaerobic digestion is almost completed in the early stage of digestion.The preparation of the core refers to the deconstruction of the extracellular protein and the release of endogenous HA at the initiation stage to expose the aromatic core,while the preparation of aromatic aminos refers to the biosynthesis of aromatic amino acids at the early stage of digestion.iii)The aromatization of HA mainly occurred in the middle and late stage of digestion,which showed that the accumulation of Aryl in the extracellular polymer of conventional high solid content anaerobic digestion sludge（SRT=28d）was obvious at12-28d,and the aromatization of HA in delayed high solid content anaerobic digestion sludge（SRT=48d）was higher than that of the original sludge at 24-48d.Therefore,enhancing the generation of aromatic compound precursors and appropriately prolonging the running time of digestion may enhance the generation of HA in sludge.iiii)The microbial driving force of HA aromatization during the mid and late anaerobic digestion of high solid sludge can be attributed to defense mechanisms.Increased HA aromatization sites at this time can solidify the phytotoxicity of small amides,inorganic salts and metal ions,thus significantly reducing free salt content and improving plant germination index.3.Developing a novel method of hyperthermophilic pretreatment composting（HPC）to enhance sludge HA formation and exploring the underlying mechanisms.Composting of sewage sludge is widely used for stabilization treatment to realize the reduction of moisture and pathogen as well as the formation of humic substances.However,the major limitations were protein hydrolysis and functional bacteria activity.This study aimed to verify the potential of hyperthermophilic pretreatment composting（HPC）with HP at 120℃ for 30 min to improve sludge stabilization and to explore the key mechanism for enhancing sludge humification and improving organic nitrogen（N）retention.This study conducted the lab-scale facility of HPC in contrast to the control composting（CC）without thermal pretreatment.Surprisingly,HPC was more efficient at sludge degradation and phytotoxicity removal,further,the end sludge compost of HPC achieved 123.3%increase of the humification degree（HAC/FAC）and 14.2%growth of organic N retention.HPC enhanced protein transformation towards HA’aromatic structural development with greater quinones regeneration and condensation.The essential effects of HP are on（1）activating redox groups e.g.quinones in HA to accelerate proteins degradation as well as to support the energy intensive regeneration of new aromatic core structures and aromatic amino materials that benefited for HA’s formation;（2）selectively enriching functional bacteria that survived in high temperature of HP and could strength protein utilization with less N loss,further stimulating subsequent beneficial bacteria to polymerize proteinaceous intermediates into HA structures.For the perspective of the application,the sludge compost rich with HA products characterizing with high aromaticity and organic N retention is promising for efficient soil amendment and the fixation of sludge N resource.4.Exploring the potential and the underpinning regulation mechanisms of thermal hydrolysis pretreatment-anaerobic digestion to enhance sludge Micro-PBs production.Firstly,all the effective sludge Micro-PBs whose content positively correlated with sludge germination index were found to be originated from aromatic amino acids（AAAs）.Secondary,thermal hydrolysis pretreatment at 120℃ and 160℃ coupling with anaerobic digestion,i.e.THP（120℃）-AD and THP（160℃）-AD,improved the effective Micro-PBs production as compared to control group（N-AD）and reached the highest at day 12.However,this improvement achieved in THP（160℃）-AD was much more greater than that induced by THP（120℃）-AD.The maximum level of effective Micro-PBs in digested sludge from THP（160℃）-AD were 80.73 mg/kg VS of indoleacetic acids（IAA）and hydroxy-phenylacetic acids（HPAs）for phytohormones as well as417.75 mg/kg VS of aromatic carboxylic acids and indolic derivatives for allelochemicals.Thirdly,the metabolomic and metagenomic results revealed that,as compared to THP（120℃）-AD and N-AD,THP（160℃）-AD uniquely upregulated the AAAs biosynthesis and consequently improved the AAAs metabolism towards effective Micro-PBs production.Further analyses for related microbial pathways and metabolites suggested the upregulated AAAs biosynthesis in THP（160℃）-AD at the early stage was partially attributed to the enhanced carbohydrate release.More importantly,the amount of quinones,which could facilitate energy generation via acting as electron transfer mediator,was significantly positive-correlated with the abundances of AAAs biosynthesis genes（R²=0.93）.Hence,the improved initial release and biosynthesis of quinones are critical to stimulate the upregulation of AAAs biosynthesis in THP（160℃）-AD.Moreover,the enhanced quinones supply and the consequent active AAAs transformation in THP（160℃）-AD reinforced the humification process,highly supporting effective Micro-PBs stabilization.The important roles of quinones in effective Micro-PBs production and stabilization in sludge anaerobic digestion should be considered in technology development for Micro-PB recovery.5.Investigating the feasibility to produce high-quality liquid fertilizer with sludge N-PGN and N-PGB recovery via alkaline thermal hydrolysis（ATH）using Ca（OH）2.For the dewatered sludge with a low VS/TS ratio where biological treatment is inapplicable,the abiotic thermal treatment coupling with liquid-solid separation can be promising to stabilize sludge and to produce liquid fertilizer.Moreover,the liquid fertilizer rich with nitrogen-containing plant-growth nutrients（N-PGN）and plant-growth biostimulants（N-PGB）from sewage sludge is attracting increasing interest recently,due to its superior fertilizing effect and the ease of application.As compared to single thermal hydrolysis（TH）treatment,the alkaline thermal hydrolysis（ATH）treatment was superior in N solubilization（TSN/TN>54%）and organic N maintenance in sludge liquor（>80%）when compared to single thermal hydrolysis（TH）.More surprisingly,ATH also promoted the production of N-PGN and N-PGB.As for N-PGN,the maximum free amino acids（FAAs）accumulation in ATH liquor was 56.82 g/L at120℃ while soluble protein（SPN）and soluble humic acid（SHA）reached 8.30-8.88g/L and 1.88-2.05 g/L at 140-160℃.The greatest N-PGB produced by ATH treatment was achieved at 160℃,with the detection of 1.156 mg/L phytohormones（indole-3-acetic acid and hydroxyphenyl acetic acids）and 4.95 mg/L allelochemicals（indolic derives and aromatic carboxylic acids）.The 2D correlation FTIR maps analyses suggested,compared with TH,ATH could achieve protein hydrolysis before polysaccharides solubilization and denaturation with the temperature increased,thus avoiding Maillard reaction and benefiting N-PGB production.Moreover,the laboratory investigation and field study indicated the usage of ATH liquor improved the growth of plants without inducing heavy metal contamination and soil salinization.In general,for the first time,this study systematically explored the potential（concentration level and occurrence state）and the mechanisms of HA and Micro-PBs production from high-solid sewage sludge stabilization treatments（biological ways of anaerobic digestion and aerobic composting as well as the abiotic ways of single thermal hydrolysis and alkaline thermal hydrolysis）.This study provides a novel theoretical method for the subsequent research on sludge land utilization and resource utilization,and has important theoretical and scientific significance.