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厌氧发酵体系内小分子底物调控的研究
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摘要
鉴于化石能源日益枯竭,而人类的活动对于能源的消耗与日俱增,能源危机问题逐渐显露。在这一过程中,以生物质能源为代表的绿色能源强势兴起。其中,厌氧消化工艺由于底物选择广泛,且可以在产能过程中对有机质垃圾进行有效的处理而引起越来越多的关注。另一方面,作为一个餐饮大国,中国的餐厨垃圾总量与日俱增又极大的刺激了城市垃圾的排放量,随之而来的后果不容小觑。而将厌氧消化工艺与餐厨垃圾处理相结合,可以有效的解决上述问题。本文针对餐厨垃圾厌氧消化过程中氮抑制现象进行了深入的研究,主要考察了厌氧消化体系内氮抑制现象出现的机制、方式等,并在半连续厌氧消化过程中发现了底物缓冲体系(buffer体系)对于厌氧消化的促进作用;同时对该buffer的功能进行了全面的考察,以期向体系内投放buffer后,改善厌氧消化体系的状态;考察了其最佳的添加浓度,添加方式,接种浓度,及其对厌氧消化体系菌群的影响;并以餐厨垃圾为底物,进行半连续厌氧消化操作。主要结论如下:
     1.厌氧消化体系内,不同无机含氮离子会随浓度的不断升高而抑制体系的产气能力,其中NO_2~--N产生的抑制最剧烈。同时,高浓度的NH_4~+-N可促进体系的甲烷菌活性,当C/N=0.5时,体系的甲烷浓度为高于空白组10%;低浓度的NO_3~--N也可促进体系的产气效率,该效果在NO_3--N浓度上升至48.8ppm时消失,且在84.8ppm时出现显著的抑制。同时,NO_3~--N批次C/N=28组的TOC利用率比NH_4~+-N批次C/N=1组高9.2%,且不易产生抑制。因此,其促进效果比高浓度组的NH_4~+-N的效果更好。厌氧消化体系中的脱氮路径主要为硝化→反硝化路径,其他氮代谢路径活性较低。当氮源以NO_3~--N或NO_2~--N进入底物的时候,体系的脱氮率相对NH_4~+-N组有一定的提高。由于NO_2~--N离子不稳定,在消化后期,NO2—N被氧化为NO_3~--N存在于体系中。
     2.对不同碳氮比厨余/动物粪便废水进行90d的半连续厌氧消化。在整个消化过程中,体系维持了对NH_4~+-N较高的耐受性。在消化最初60d里,较低的碳氮比会显著增加体系微生物的活性及体系的TOC利用率,虽然此时NH_4~+-N浓度已经达到5.65g/L,VFAs浓度已经达到12g/L。但是,C/N=6.5组的TOC利用率仍然比C/N=26组高11.1%,结果表明,在该过程中NH_4~+-N同VFAs结合形成了一个活跃的buffer体系,从而增加了体系的甲烷产量及TOC利用率,也增加了体系对于NH_4~+-N同VFAs的耐受性。在第三阶段(61~90d),由于体系的NH_4~+-N显著积累,体系的buffer降解,最终产生了明显的氮抑制。因此,通过调整半连续厌氧消化体系内的底物C/N可以有效的增加体系的产气效率,提高体系的甲烷产率及TOC利用率。
     3.以1d为HRT进行外源性buffer的添加,由于底物简单,代谢过快,会使体系出现明显的抑制。分别为以4g/L,2g/L及1g/L乙酸铵为底物进行消化时,体系的总产气量同空白组相比分别下降了66.1%,31.2%及17.5%;同时,外源性buffer可有效的提高体系的产甲烷浓度,4g/L组的平均甲烷浓度可达73.66%,远高于空白组的42.72%。同时,外源性buffer的添加可以明显维持体系的pH稳定性;相比于其他产气菌,产甲烷菌需要更长的时间由“不可逆抑制状态”恢复。以半连续法向体系内添加buffer可以有效的促进内源性buffer的形成,以2d为时间间隔,向体系内添加1d的乙酸铵buffer,最有益于体系的消化。最佳的TOC利用率可达到843.31mL CH_4/g TOC。buffer存在的AD体系内,消化的总产气量与种子污泥所稀释的倍数成反比,而产甲烷浓度与所稀释的倍数呈正比。当污泥VS为3.5%时,体系的产气量仅为原污泥(Vs=17.0%)组的49.77%,而平均甲烷产率却高于该组13%;VS=8.1%组的产气情况也相应下降,但是当污泥VS为10.2%时,其平均产气效率仅比原污泥组低3.2%。乙酸铵buffer可以有效的促进体系生物质的形成,并促进体系的产甲烷菌活性。
     4.以HRT=2ds,添加时间为1d,体系的产气效果最好,可达到901.11mLCH_4/g TOC;以HRT=2ds,添加时间为3ds时,即使组内的NH_4~+-N和VFAs浓度虽然分别达到3.27g/L及12.46g/L,体系的TOC仍然可达802.65mL CH_4/g TOC。随buffer添加时间的增加,体系的产甲烷菌活性可以在短时期内被促进,TOC利用率也会相应提高,但随着消化的进行,AD体系会逐渐产生抑制。外源性buffer的添加会对AD体系微生物产生明显的影响,随着抑制的发生,部分厌氧细菌DGGE条带的亮度会明显降低甚至消失;同时,外源性buffer的添加对体系古菌的影响并不明显,在整个反应过程中,体系的古菌条带(以Methanobacterium aarhusense strain H_2-LR为主)基本并无变化。证明以该种方式添加buffer可以促进体系内源性buffer的形成,且不会明显抑制体系的产甲烷菌。
     5.以外源性buffer向餐厨垃圾厌氧消化体系内添加,可以有效的增加体系的稳定性,促进体系的产气及产甲烷状态。当添加buffer总量相同时, HRT=3ds组获得了最佳的甲烷浓度,该组的平均甲烷浓度为55.22%,比空白组高28.0%;而以HRT=1d乙酸铵向体系内添加buffer时,AD体系的平均甲烷浓度为54.87%,且有抑制现象发生;以HRT=5ds向体系内添加时,促进效果并不明显,平均甲烷浓度仅为52.61%。与简单底物相比,以餐厨垃圾为底物,可有明显的降低buffer对体系带来的冲击。在该过程中,体系的NH4+-N并未产生明显的积累。在进行的30ds消化过程可以确定,以3ds HRT进行乙酸铵buffer的添加,添加浓度为1g/L,TOC=1.20g/L,最适宜体系的消化。
In consideration of the increasing depletion of the fossil energy, and theaccelerated energy consumption result from human activities, the energy crisisis gradually emerged. In this process, as a representative of green energy, theutilization of biomass energy arouse passions and concerns of the world.Among the biomass energy productive processes, anaerobic digestion can beapplied to a wide range of substrates, where obtained an effective treatmentability of organic waste. On the other side, as a big catering country, the foodwaste emission of China greatly increases the volume of municipal waste, andthe ensuing consequences could not be underestimated. The combine ofanaerobic digestion technology and food wastes disposal can solve theproblem efficiently. The present study investigated the phenomenon ofN-inhibition (nitrogen inhibition) during the anaerobic digestion processdeeply,especially the mechanism and the performance of the N-inhibition; andduring the study, the promotive ability of buffer system was discovered in thesemi-continuous anaerobic digestion operation. In order to maintained a stableand efficient condition of anaerobic digestion with a buffer supply-operation,the buffer function and characteric was comprehensively investigated also.The optimum buffer addition concentration, addition way, inoculumconcentration were investigated, and also the anaerobic fermentation floravariation during the buffer addition digestion process. At the end of theresearch, the food wastes were regarded as a substrate for the semi-continuousanaerobic digestion, the main results are as follow:
     1. In the AD system, different inorganic nitrogen ions caused a severe drop ofthe biogas productive capacity with the increasing concentration, and theNO_2~--N exhibited the most drastic inhibition. At the same time, the highNH_4~+-N concentration improved the activity of the methanogenic, when the C/N=0.5, the group obtained a10%higher CH_4concentration than theblank. The lower NO_3~--N concentration improved the biogas production ofthe system also, but when the NO_2~--Nconcentration reached48.8ppm, theimprovement disappeared, and inhibited the AD at NO_2~--N concentrationof84.8ppm. Compared with C/N=1group of NH_4~+-N batch, the C/N=28group of NO_3~--Nbatch exhibited a9.2%higher TOC utilization rate, whichmeans, lower NO_2~--N concentration group boosted the AD activity moreefficient than higher NH_4~+-N concentration group. The main N-removalroute of anaerobic digestion was nitrification→denitrification, theactivity of other nitrogen metabolic pathway was lower, Compare withNH_4~+-N, NO_3~--N and NO_2~--N maintained a higher N-removal efficiency inthe AD system. In addition, plenty of NO_2~--N was oxidated to NO_3~--N dueto the instability of NO_2~--N at the end of the digestion.
     2. The characteristics of the long term (90d) anaerobic semi-continuousdigestion of FW/AS wastewater with different C/N ratio were investigated.During the whole process, the system obtained a higher tolerance ofNH_4~+-N accumulation. The lower C/N substrate provides promotiveconditions for microbial activity during the first two periods of digestion(0~60d), increase the utilization of TOC, although the concentration ofNH_4~+reached5.65g/L and the concentration of VAFs around12g/L. Andthe TOC utilization of C/N=6.5case was11.1%higher than C/N=26case,which means the long time AD result in the extra NH_4~+and VFAs(by-products of TOC) form an active buffer system, thus increasing boththe CH_4production and TOC utilization; and also maintained ahigher tolerance to NH_4~+and VAFs concentration, which defusing theimpact of NH_4~+and VAFs to the substrate. In the the third period ofdigestion (61d~90d), with the increasingly accumulated of NH_4~+, thebuffer system was broken up, and then, caused N-inhibited. The resultsindicated that the management of C/N of the the semi-continuous digestionprocess boost the stability because of the formation of buffer system, andin a certain extent, promoting the CH_4production and TOC utilization.
     3. When the HRT=1d, the system exhibited a significant inhibition with thebuffer addition, which result form the simpler substrate and fastermetabolism velocity. When the ammonium acetate concentration were4g/L,2g/L and1g/L, the total biogas production decreased66.1%,31.2% and17.5%, respectively, compare with the blank. In addition, theexogenous buffer improved the CH_4concentration efficiently, the CH_4concentration of4g/L group reached73.66%, however, the the CH_4concentration of blank group was just42.72%at the same time. With theaddition of exogenous ammonium acetate buffer, the system obtained asignificantly safeguard of the pH value, which defused the impact of highNH_4~+-N or VFAsconcentration, kept a constant pH in the digestor. On theother side, compared with the other aerogens, Methanogen need a longerrecovery period formed the N-inhibition for the biogas productive ability.During the semi-continuous anaerobic digestion, when the additioninterval was2ds, the1.5g/L of glucose and2g/L of ammonium acetatewas regard as exogenous buffer, which could promote the endogenousformation, and also improve the efficient of the digestion. The optimumTOC utilization was843.31mL CH_4/g TOC in the process. With theexogenous buffer, the dilution ratio of the seed sludge exhibited a negativeeffect to the biogas production, and a positive effect to the CH_4concentration. When the VS of sludge was3.5%, the biogas production ofthe system was just49.77%of the origin sludge (VS=17.0%), however,the CH_4concentration was13%higher than the VS=17.0%group. TheVS=8.1%group obtained the similar condition but when the VS rose to10.2%, the biogas production was just3.2%lower than the origin sludgegroup. The result means that, the exogenous ammonium acetate buffercause the generation of the biomass, and improved the activity of theMethanogen.
     4. The AD system maintained the optimum condition with the HRT=2ds, and1d added length for the ammonium acetate buffer, the system obtained thebest TOC utilization rate:901.11mL CH_4/g TOC. When the HRT=2ds,and the added length was3ds, although the NH_4~+-N and VFAsconcentration was3.27g/L and12.46g/L, respectively, the systemmaintained the TOC utilization of802.65mL CH_4/g TOC. The CH_4concentration was promoted result from the shorter added cycle, however,caused a lower biogas production becauese of the high bufferconcentration, even the inhibitory phenomenon. During the inhibitionperiod, the amount of bacteria PCR-DGGE bands obtained a significant downward tendency, even disappeared; on the contrary, the methanearchaea PCR-DGGE bands (Methanobacterium aarhusense strain H_2-LRmainly) weren`t significant effected result from the buffer addition, whichmeans, the methanogens exhibited a higher tolerance of exogenous buffer,and the characteric of buffer could improved the formation of endogenousbuffer in the system, without the significant inhibition of the Methanogen.
     5. The addition of exogenous buffer in the food waste AD system maintaineda stable fermentative condition, boost the biogas and CH4production ofthe system. With the same additive buffer concentration, HRT=3dsobtained the optimum average CH4concentration,5522%, which was28.0%higher than the blank group; when the HRT was1d, the systemexhibited the average CH4concentration of54.87%, and a inhibitionphenomenon emerged during the period; when HRT=5ds, the average CH4concentration was just52.61%. Compare with the simple substrate, thefood waste defused the impact of the buffer addition, and during the period,there was no significant accumulation of the NH4+-N. In the30ds foodwastes digest operation, the AD system obtained the optimum condition inHRT=3ds, and the additive cycle=1d.
引文
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