Direct Effects Of Biochar On Microbial Denitrification And The Mechanisms

To mitigate climate change caused by anthropogenic greenhouse gas emissions,there is an increasing demand to reduce carbon emissions and increase carbon sinks,and biochar,which has the effect of sequestering carbon and increasing sinks,is gaining more and more attention.Microbial denitrification is an important source of N₂O emissions,and biochar has been widely reported to affect denitrification rates and N₂O emissions.However,previous studies have mainly focused on the indirect effects of biochar on microbial denitrification,such as biochar affecting p H and microbial nitrification processes,etc.In this paper,the direct effects of biochar on model denitrifying bacteria（Paracoccus denitrificans,P.denitrificans）were investigated firstly,and the contributions of the water-soluble organic compounds（WSOCs）and granular components（GCs）of biochar to denitrification were elucidated.Then,the possible structures of the active molecules in WSOCs with promoting effects were explored,and the molecular mechanisms of the active molecules in WSOCs regulating denitrification electron transfer and enzyme activity were revealed by combined transcriptome-proteome analysis and other methods.Finally,the way in which GCs directly influences the carbon and nitrogen metabolism of P.denitrificans was investigated,and the mechanism was elucidated in terms of microbial key metabolic pathway activity,carbon flux distribution,reducing power level,and electron distribution,and energy distribution.Firstly,the direct effects of different components of biochar on the metabolism and cell growth of P.denitrificans were investigated.The effects of biochar prepared from corn straw and wheat straw at low（300°C）and high（500°C）temperatures on denitrification efficiency,cell growth and individual metabolic activity of P.denitrificans were investigated under the conditions that the initial environmental parameters（p H,dissolved oxygen,substrate levels,etc.）were maintained.It was found that the addition of 0.1%-1%（w/w）of biochar prepared at low and high temperatures significantly improved the denitrification efficiency and significantly reduced the accumulation of N₂O,and the effect of biochar was related to the pyrolysis temperature but not to the source of straw;The low-temperature biochar increased the individual metabolic activity of P.denitrificans but did not affect their growth rate,which was mainly related to the composition and content of the WSOCs;The high-temperature biochar not only increased the growth rate of P.denitrificans but also enhanced the individual metabolic activity,which was mainly related to the composition and content of the surface functional groups of GCs.Secondly,the characteristics and composition of active molecules in WSOCs of low-temperature biochar were investigated.The low molecular weight（<500 Da）,medium molecular weight（500-10000 Da）and high molecular weight（>10000 Da）groups accounted for 77.8%,12.4%and 9.8%of WSOCs,respectively.Of this,the LMW WSOCs played a key role in enhancing denitrification.Further analysis by excitation emission matrix fluorescence-parallel factor analysis（EEM-PARAFAC）,Fourier transform ion cyclotron resonance mass spectrometry（FT-ICR-MS）and ultra performance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry（UPLC-QTOF-MS）revealed that LMW fulvic acids were the active components in WSOCs,which mainly included some highly unsaturated phenolic acids represented by 3,5-Diacetoxybenzoic acid,Fraxetin,and 7-（Carboxymethoxy）-4-methylcoumarin.Furthermore,the mechanism of active molecules in WSOCs affecting denitrifying nitrogen metabolism was investigated.The results based on denitrification electron transfer,enzyme activity,transcriptomics and proteomics analysis showed that the active molecules not only significantly increased the electron transfer rates in the denitrification membrane electron transfer system and the periplasmic electron transfer chain,but also enhanced the activities of the complexes（Complex I and Complex III）in the membrane electron transfer system,the membrane-bound nitrate reductase（NAR）,and nitrite reductase（NIR）,nitric oxide reductase（NOR）,nitrous oxide reductase（NOS）in the periplasmic electron transfer chain.This was mainly due to the up-regulation of the transcription and translation of genes encoding denitrification enzymes（nar G,nar I,nir S,nor B,nos Z）and the expression of genes encoding DNA repair,transcriptional regulators,protein translation and other related genes.Next,the mode of action and the site of action of GCs of high-temperature biochar to promote denitrification were investigated.Although extracellular electron transfer is widely considered as an important reason for biochar to promote microbial metabolism,the results of a series of experiments including direct contact between GCs and P.denitrificans,interaction between GCs and extracellular metabolites,metabolomic analysis,and molecular simulation studies showed that it is not the main reason for enhanced denitrification.GCs might affect the microbial metabolism through the chemisorption of surface oxygen-containing functional groups represented by carboxyl groups.The surface oxygen-containing functional groups of GCs could spontaneously dock with amino acid-like metabolites such asγ-glutamylcysteine,nicotinic acid,methionine,glutamate,L-dopa,etc.,and selectively adsorb these substances through hydrogen bonding,van der Waals forces andπ-πstacking.The higher the amount of metabolites adsorbed by GCs with higher carboxyl group content,the greater the cell growth rate and the faster the denitrification rate of P.denitrificans.Finally,the mechanism of GCs affecting denitrification carbon metabolism and nitrogen metabolism was revealed.The results of intracellular metabolome,carbon flux allocation,reducing power level,electron allocation,energy allocation,and proteome studies showed that GCs increased the activity of intracellular carbohydrate metabolism,energy metabolism,and amino acid metabolism pathways,and decreased the activity of lipid metabolism pathway,which led to more allocation of carbon flux to oxidative catabolism rather than being fixed as an internal carbon source.This resulted in a higher rate of denitrification and more electrons available for N₂O reduction.Although the expression and activity of NAR did not change significantly,the expression and activity of NIR,NOR,and NOS were upregulated,which optimized the distribution of electrons among denitrifying enzymes and decreased the accumulation of N₂O.The reduced accumulation of internal carbon sources allowed more energy generated by oxidative phosphorylation to be used for cell growth,thus increasing cell yield.The above research contributes to an in-depth understanding of the mechanism of biochar affecting the metabolism of denitrifying bacteria,and also provides important theoretical guidance for the use of biochar to promote denitrification and reduce N₂O emissions.The research results are of high academic value and certain application value.