As a product of phosphorous recovery from anaerobic digestion (AD) of waste activated sludge (WAS), vivianite has received increasing attention. However, key factors controlling vivianite formation have not yet been fully addressed. Thus, this study was initiated to ascertain key factors controlling vivianite formation. A simulation of chemical equilibriums indicates that interfering ions such as metallic ions and inorganic compounds may affect vivianite formation, especially at a PO₄^3-concentration lower than 3 mM. The experiments demonstrated that the rate of ferric bio-reduction conducted by dissimilatory metal-reducing bacteria (DMRB) and the competition of methane-producing bacteria (MPB) with DMRB for VFAs (acetate) were not the key factors controlling vivianite formation, and that ferric bio-reduction of DMRB can proceed when a sufficient amount of Fe³⁺ exists in WAS. The determined affinity constants (K_s) of both DMRB and MPB on acetate revealed that the K_HAc constant (4.2 mmol/g VSS) of DMRB was almost 4 times lower than that of MPB (15.67 mmol/g VSS) and thus MPB could not seriously compete for VFAs (acetate) with DMRB. As a result, vivianite formation was controlled mainly by the amount of Fe³⁺ in WAS. In practice, a Fe/P molar ratio of 2:1 should be enough for vivianite formation in AD of WAS. Otherwise, exogenously dosing Fe³⁺ or Fe²⁺ into AD must be applied in AD.

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Alginate like extracellular polymers (ALE) recovered from flocculent sludge has been identified as a kind of highly valuable biomaterials. However, the extraction protocols limit the production of biopolymers as ALE extracted from flocculent sludge is at a lower level, around 90–190 mg/g VSS. Under this circumstance, the eco-friendly and effective optimizations for the ALE extraction protocols are expected, and thus surfactants have gained an attention to enhancing the ALE extraction. With this study, different surfactants with different structures and chemical characteristics, such as sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (CTAB) and octyl phenyl polyoxyethylene ether (Triton X-100), were experimented to improve the ALE extraction, and in turn the optimal conditions and the associated mechanisms were evaluated and figured out. The experimental results indicated that surfactants could enhance the ALE extraction but also improve the alginate purification of ALE. With the optimal dosage of surfactants, the ALE extraction increased from 124.1 mg/g VSS to about 222.8–281.9 mg/g VSS, and the alginate purify was at around 54%–70%, in which the efficiency of the ALE extraction was improved by 79.5%–127.2%. Among others, Triton X-100 had the best performance on improving the ALE extraction, followed by CTAB and SDS. The mechanisms of surfactants on enhancing the ALE extraction and improving the alginate purify can be attributed to: i) surfactants micelles, which can solubilize flocs and extracellular biopolymers; ii) similar structures of surfactants and ALE, which follows the rule of “like dissolves like”; iii) functional groups adsorption, which facilitates the ALE release from matrixes. In a word, the optimized extraction protocol by using surfactants can be effectively applied to extract ALE from flocculent sludge.

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Sludge incineration and seawater desalination are two approaches that can be used in the disposal of waste activated sludge (WAS) and for obtaining fresh water. As resource recovery from wastewater treatment and water purification is a topic of particular interest in these times, “water mining” has become a focus of research, with phosphate/P-recovery from WAS incineration ash, and extraction of useful elements from the brine of desalination being important steps in the pursuit of a circular/blue economy. However, P-recovery from ash involves removing metals, which need to be disposed of carefully, as does the brine collected. If cations in the ash and anions in the brine could be combined in order to produce coagulants/flocculants, a new circular model would be established. A preliminary experiment for this purpose has demonstrated that a liquid poly‑aluminum chloride (PAC) could be synthesized from the aluminum ion/Al³⁺ removed from the ash and the original brine. With this work, we synthesized the liquid PAC by a hydrothermal method, and the results from infrared spectrometer demonstrated that the synthesized PAC was similar to a commercial PAC. Moreover, the synthesized PAC was able to efficiently reduce the effluent turbidity of wastewater treatment plants (WWTPs), especially when compared with the commercial PAC. It is therefore important that research in this area be continued in order to improve the quality of synthesized coagulants and to produce different coagulants based on cations and anions in ash and brine.

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Entropy is a concept defined by the second law of thermodynamics. Applying this concept to the world we live in, entropy production must be minimized and negentropy (negative entropy production) should be accelerated, in order to produce a healthy and stable ecological system. The present wastewater treatment, however, contributes to entropy production. This means that conventional wastewater treatment, without recovery of resource and energy, will gradually but inevitably contribute to a deteriorating ecological balance. When the self-cleaning ability of the natural ecological system is limited, the need to develop sustainable wastewater treatment in order to delay entropy production and accelerate negentropy becomes urgent. Resource and energy recovery from wastewater should be the first priority, as they can contribute significantly towards minimizing entropy production and accelerating negentropy. Sustainable wastewater treatment must focus on recovering recyclable high value-added organic chemicals from wastewater and/or excess sludge to minimize entropy production caused by methane (CH₄, once combusted, is converted into CO₂ - an even higher substance in entropy) via anaerobic digestion. Instead of CH₄, thermal energy present in the effluent can be utilized for heating/cooling buildings and also for drying excess sludge towards incineration to recover more energy. Overall, this can lead to a carbon-neutral operation and even creating a “carbon sink” could be possible for wastewater treatment.

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地下式污水处理厂建设近年在我国兴起，且有扩展之势。地下污水厂地表可形成的景观环境直观、易觉，这就使污水处理厂可与景观环境建设合二为一的说法盛行。反观更加缺地的欧洲、日本等地，地下式污水处理厂其实并不多见，让人不免心生疑窦。因此，有必要通过一些方法对地下式污水处理厂进行综合影响评价，以揭示其综合效益之优劣。以国内某全地下式污水处理厂为研究实例，并以相同工艺的地上式污水处理厂作为对比参照物，通过全生命周期环境影响评价（LCIA）、全生命周期成本（LCC）评价及全生命周期生态效益（LCEE）评价3种方法分别对地下式污水处理厂进行全方位影响评价，最后将3种评价结果归一化为全生命周期综合影响（LCCI）评价。综合影响评价结果显示，地下式污水处理厂在环境影响、基建投资、生态效益三方面的综合负面影响较地上式要高出约20%。虽然地下式污水处理厂地表园林景观会产生一定生态效益，但这并不能“中和”其环境影响以及基建投资所产生的负面效益。因此，地下式污水处理厂建设并非优选方式，需要因地制宜，选址要特别慎重。 Abstract: Constructing underground wastewater treatment plants (UWWTPs) prevails recently and tends to extend largely in China. Ecological landscape saved by UWWTPs is easy to be seen, which makes an opinion popular that wastewater treatment could combined with ecological landscape. In Europe and Japan much shorter of land, however, it is rare to see UWWTPs, which makes us a query. Therefore, it is necessary to comprehensively assess UWWTPs in some ways and to reveal their pluses and minuses. Based on life cycle impact assessment (LCIA), life cycle cost (LCC) assessment and life cycle ecological efficiency (LCEE) assessment, the study assessed omnibearingly the impacts and efficiencies of UWWTPs, and finally the assessed results from each assessment were normalized into life cycle comprehensive impact (LCCI) assessment. The assessment was based on a real domestic UWWTP, and a proposed surface WWTP was used as a counterpart for comparison. The LCCI results revealed that the comprehensively negative efficiency of UWWTP on the environment, infrastructure investment and ecological efficiency was about 20% higher than that of its counterpart. Although the ecological landscape restored by the UWWTP could form a certain ecological efficiency, it could not neutralize the negative efficiency of the UWWTP on the environment and the infrastructure investment. As a result, UWWTPs are not a preference approach to municipal wastewater treatment. So constructing UWWTP should act according to circumstances and selecting sites of UWWTP have to be prudent.@en

Extracellular polymeric substances (EPS) are biopolymers that can be recovered from excess sludge, which could contribute to a more sustainable wastewater treatment plant (WWTP) operation. An example is alginate like extracellular polymers (ALE) contained in the biopolymers could be a potential resource with a highly-added value. EPS extraction for ALE from aerobic granules sludge (AGS) has already been well studied and applied in the Netherlands. On the other hand, there is little attention to the recovery of biopolymers from conventional activated sludge (CAS). In this study, flocculent sludge from eight CAS-WWTPs in China was collected and their EPS/biopolymers were extracted to investigate their recovery potential, chemical & physical properties and limiting factors. The results revealed that the biopolymers extracted and purified from CAS ranged from 90 to 190 mg/g VSS. The compositional characteristics of the biopolymers were observed by FT-IR, 3D-EEM and UV-Visible spectra, demonstrating some differences in the composition and property of the biopolymers from the different WWTPs. The biopolymers had a similarity of about 60% to a commercial alginate with respect to chemical functional groups and the alginate equivalent was >400 mg/g biopolymers. Moreover, the biopolymers consisted of poly (guluronic acid) blocks (20%-30%) and poly (guluronic acid-mannuronic acid) blocks (8%-28%), and the ionic hydrogel formation tests indicated that condensed beads were immediately formed once the drops of the biopolymers came in contact with CaCl₂ solution. These results demonstrated that the biopolymers extracted had a relatively high gel-forming capacity and might also have a potential application as commercial biopolymers. Furthermore, the factors influencing the biopolymers’ formation such as influent substrate, nutrient content and microbial community and the related mechanisms were investigated. Among them, increasing soluble organics (SCOD) content and low nutrient content (C/N/P) in the influent could promote the biopolymers’ formation. Also, different bacteria in BNR processes might have positive or negative effects on the biopolymers’ formation. In conclusion, the diversity and abundance of bacteria were identified to be a crucial and decisive factor controlling biopolymers’ extraction and composition.

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Anaerobic digestion (AD) is an effective approach to recovering chemical (organic) energy from excess sludge, but the conversion efficiency for energy is usually not very high. One of the obstacles comes from the severe inhibition of humic acid (HA) on both hydrolytic and methanogenic process on the AD. Therefore, it is necessary to ascertain some effective approaches to relieving the inhibition of HA for obtaining a high methane (CH₄) yield. With the “clean” sludge (cultured by synthetic wastewater) containing almost no HA and metal ions, the inhibition of HA on the AD process was designed by dosing HA at 15% VSS, and relieving the inhibition by metal ions was also designed by dosing the different amounts of Ca²⁺ and Al³⁺. Based on the batch AD experiments, solo Ca²⁺=100 mg /L or Al³⁺=70 mg/L added realized the highest relieved efficiency of 65%, respectively. Interestingly, dual metal ions added at the low concentrations (Ca²⁺=50 mg/L and Al³⁺=10 mg/L) could reach up to 80 % of the relieved efficiency, which was attributed to the synergistic effect of 1+1>2. The mechanisms behind the phenomena could be that metal ions might interact with HA via electrostatic force, cation exchange and sweep flocculation. Thus, some key hydrolytic and methanogenic enzymes could indirectly be reactivated and degradation of organic substances could be enhanced in the AD process. In wastewater treatment plants, metal ions contained in excess sludge would “inherently” relieve the inhibition of HA to an extent, which depends on the effective and/or optimal concentration of metal ions at a free (unabsorbed and/or unwrapped) state.

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突发新型冠状病毒肺炎（COVID-19）已蔓延至全球几乎所有国家，累计确诊病例已超过1亿例。这一全球大流行病尽管现阶段在我国多点散发，但已得到基本控制，由于不容乐观的全球疫情扩散，依然存在疫情反弹之巨大风险。在新冠疫苗实现大规模接种之前，COVID-19暴发有效预警机制依然是最需要的技术手段。鉴于感染者粪便/尿液中存在病毒粒子或遗传物质核糖核酸（RNA），荷兰学者提出了基于污水流行病学（WBE）监控SARS-CoV-2（诱发COVID-19的病毒）出现并传播的设想。目前，污水中SARS-CoV-2有限RNA检测研究结果已经证明了这一技术作为疫情预警的可行性，亦可能根据WBE检测结果表征病毒传播动态与趋势、估算感染群体比例，甚至在一定范围内进行感染群体追踪。在总结WBE技术在我国发展应用的基础上，对已有污水中SARS-CoV-2的核酸检测研究结果、应用价值和指导意义进行综合分析，总结WBE研究标准化方案，辨析亟需深入研究的方向领域。这对我国下一阶段疫情防控具有相当启发与参考价值。@en

Handling excess sludge produced by wastewater treatment is a common problem worldwide. Due to limited space available in landfills, as well as difficulties involved in using excess sludge in agriculture, there is a need for alternative disposal methods. Although anaerobic digestion (AD) is widely used in processing sludge, only partial energy recovery from methane and sludge volume reduction can be achieved, resulting in a substantial amount of sludge remaining, which needs to be disposed of. Direct incineration after sludge drying is one possible option, a practice that is already in place in some cities in China. A comparison between direct incineration and conventional AD (with or without pretreatment by thermal hydrolysis) has to be made with respect to the energy balance and investment & operational (I & O) costs. This comparison reveals direct incineration to have the lowest energy deficit and I & O costs. Therefore, it is expected that direct incineration without AD will become the preferred sustainable approach to handling sludge.

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In dealing with wastewater, chemical energy has traditionally been perceived as the only source of recoverable energy in moving towards the carbon-neutral operation of wastewater treatment plants. Based on an estimation of practically recoverable energy embedded in municipal wastewater, however, the potential for thermal energy (90% recovery from wastewater) is much higher than for chemical energy (COD, 10% recovery). The carrier of chemical energy (COD) has a high exergy value which should, from a sustainability point of view, be utilized to the greatest extent possible. Rather than being converted into methane (and subsequently into carbon dioxide), carbon (COD) contained in wastewater should be converted into highly valuable organic products. Thermal energy could be utilized for district heating/cooling, agricultural greenhouses, and even for drying dewatered sludge. In this way, thermal energy can indirectly offset the energy demand for wastewater treatment. The limitations in utilizing thermal energy are not generally based on technical difficulties; in fact, they can be mainly attributed to supply distances and governmental policies. It would, therefore, be greatly beneficial if municipal authorities would work together to jointly plan utilization of this thermal energy.

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Inhibition by humic substances (HSs) on anaerobic digestion of excess activated sludge is one of the limitations for converting more organics into biogas. In order to counteract the inhibition from HSs, the present study was initiated to understand the dynamic changes (content and structure) of HSs during the anaerobic digestion of synthetic and real sludge. For the first time, the present work studied the dynamics of HSs separately in the liquid and solid phases. These observations on HSs conversions by potentiometric titration, UV–Vis, FTIR, and two-dimensional infrared spectra, confirmed that the dynamic changes of HA and FA compositions were caused by losing aliphatic moieties and enriching aromatic moieties in the structural compositions. This changes increased the humification degree, aromaticity, and the amounts of oxygen-containing functional groups. Based on the observations, strategies to alleviate the inhibition effect were discussed.

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Conventional wastewater treatment plants (WWTPs) clean wastewater and minimize water pollution; but, while doing so, they also contribute to air pollution and need energy/material input with associated emissions. However, energy recovery (e.g. anaerobic digestion) and resource recovery (e.g. water reuse) allow us to offset the adverse environmental impacts of wastewater treatment. Life cycle assessments (LCA) have been used more and more to evaluate the environmental impacts of WWTPs and to suggest improvement options. There is a need to search for resource recovery applications that genuinely realize a net-zero impact on the total environment of WWTPs. In this work, a scheme with highly efficient energy and resource recovery (especially for thermal energy) is proposed and evaluated. The environmental impact of a conventional WWTP in comparison with the scheme proposed here, with energy/resource recovery included, was calculated, and discussed with reference to LCA methodology. In the process of using LCA, it was necessary to choose a regional situation to focus on. In this case, a Chinese situation was focused as a reference, but the qualitative information gained is of worldwide relevance. The results clearly revealed that conventional WWTP does not benefit the total environment as a whole while the new scheme benefited the total environment via resource/energy recovery-based processes. Among others, thermal energy recovery played a significant role towards a net-zero LCA analysis (contributing around 40%) which suggests that more attention and research should be focused on it.

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Anaerobic digestion (AD) is a technology for recovering chemical energy as methane from excess sludge/waste. Unfortunately, humic acids (HA) contained in excess sludge can have the effects of inhibiting the efficiency of energy conversion. Based on a batch experiment, the impact of HA on a semi-continuous AD process was sequentially investigated, with the impact on the associated enzymes and microorganisms being measured. The results of this semi-continuous experiment indicate that the inhibition of the microbial community increased with an increased HA:VSS ratio. Long-term cultivation did not result in the adaption of methane production to the presence of HA. Moreover, at HA:VSS = 20%, the strongest inhibition (74.3%) on energy conversion efficiency was observed in the semi-continuous experiment, which was two-fold higher than that recorded in the batch experiment. This is attributed to serious and irreversible inhibition of both acidogenic and methanogenic microorganisms, as well as the physical-chemical reactions between HA and the associated enzymes which, it was concluded, were the dominant mechanisms of inhibition in the batch experiment.

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Anaerobic digestion (AD) is a sustainable pathway towards recovering chemical energy from excess sludge, and humic substances (HSs) contained in sludge can inhibit energy (methane/CH ₄ ) conversion efficiency. This study aims to investigate the impact of humic acids (HA) on the various processes in a batch anaerobic digestion process. For this purpose, “clean” sludge was cultivated in a laboratory to avoid HSs presence. The cultivated sludge was used in a series of batch experiments, with humic acids added at different levels. A complete AD test, as well as three sub-phase tests (hydrolytic phase; acidogenic phase; methanogenic phase) was performed and analyzed with and without HA dosing. In the single-phase AD system, dosing with HA inhibited the methanogenic efficiency by 35.1% at HA:VSS = 15%. However, the effects of HA on the three sub-phases revealed something very different. HA inhibited hydrolytic efficiency by 38.2%, promoted acidogenic efficiency by 101.5%, and finally inhibited methanogenic efficiency by 52.2%. The combined efficiency of the three sub-phases without HA dosing is calculated at 15.7%; and with HA dosing (HA:VSS = 15%) at 10.2%. Overall, the combined inhibition efficiency of the three sub-phases is equal to 35.0%, which is almost identical (35.1%) to the result observed in the single-phase AD process. The possible mechanisms behind the phenomena were analyzed and summarized in the context.

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The potential crisis of phosphorus resource is unprecedentedly pushing phosphorus recovery from wastewater and other sources. Although products of phosphorus recovery are variable, economic and high value-added products could promote owners to do so voluntarily. Among others, vivianite (Fe 3 (PO 4 ) 2 •8H 2 O), which has been found to exist largely during biological sludge (biomass), is arousing attention to researchers. In fact, vivianite, a stable (K sp = 10 -36 ) ferrophosphorus compound, widely exists in natural sediments. Besides such inherent factors as rich iron and phosphate, there are also some external factors determining formation of vivianite, like low oxidation-reduction potentials (ORP<-300 mV) and suitable pH conditions (6~9), etc. Fortunately, these inherent and external factors could be satisfied in wastewater and/or processes of wastewater treatment, and some wastewater treatment plants (WWTPs) in the Netherlands declared that they indeed found a large amount of vivianite presence in biological sludge. To promote both fundamental and applied researches on vivianite, the article reviews vivianite in: i) chemical properties, economic value and recovered potential; ii)controlling factors like pH, ORP, microorganisms, sulfide, etc.; iii) pathways of vivianite formation in biological sludge; iv) separation and purification of vivianite. @en

The Paris Climate Treaty implies the coming of a new era towards carbon-neutral operation in wastewater treatment plants (WWTPs), and thus energy self-sufficiency from biosolids and/or heat in the form of wastewater temperature has to be considered. In this regard, anaerobic digestion (AD) of sludge should be paid renewed attention to resolve a low conversion efficiency of biosolids. As a potential way to energy positive operation, exogenous iron has been proposed to enhance methane production in recent years. In this review, the authors provided a deep insight into the feasibility of iron-enhanced AD system. Hydrogen evolution from iron corrosion and its effects on CH₄ production is firstly reviewed; then the roles of iron in reducing ORP was illustrated with regard to its impact on fermentation type; serving as an essential element and potential electron donor, the stimulating effects of iron on microorganisms and enzyme activities were also elaborated, and thus the technical feasibility of iron-based AD could be evaluated. In regards of the environmental and economic impacts of iron-based AD, life cycle assessment (LCA) was employed to calculate its economic feasibility, and the results revealed that iron-based AD system could reduce both operational costs and carbon emissions. Conclusion was drawn that iron-based anaerobic digestion is promising on technical level as well as economic perspective, and is expected to contribute to carbon-neutral operation of WWTPs. Iron-based anaerobic digestion is such a promising and sustainable strategy towards circular economy that it could be applied to many cross-disciplinary fields.

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The decreased activity (i.e. decay) of anaerobic ammonium oxidation (Anammox) bacteria during starvation can be attributed to death (i.e. decrease in the amount of viable bacteria) and activity decay (i.e. decrease in the specific activity of viable bacteria). Although they are crucial for the operation of the Anammox process, they have never been comprehensively investigated. This study for the first time experimentally assessed death and activity decay of the Anammox bacteria during 84 days’ starvation stress based on ammonium removal rate, Live/Dead staining and fluorescence in-situ hybridization. The anaerobic and aerobic decay rates of Anammox bacteria were determined as 0.015 ± 0.001 d⁻¹ and 0.028 ± 0.001 d⁻¹, respectively, indicating Anammox bacteria would lose their activity more quickly in the aerobic starvation than in the anaerobic starvation. The anaerobic and aerobic death rates of Anammox bacteria were measured at 0.011 ± 0.001 d⁻¹ and 0.025 ± 0.001 d⁻¹, respectively, while their anaerobic and aerobic activity decay rates were determined at 0.004 ± 0.001 d⁻¹ and 0.003 ± 0.001 d⁻¹, respectively. Further analysis revealed that death accounted for 73 ± 4% and 89 ± 5% of the decreased activity of Anammox bacteria during anaerobic and aerobic starvations, and activity decay was only responsible for 27 ± 4% and 11 ± 5% of the decreased Anammox activity, respectively, over the same starvation periods. These deeply shed light on the response of Anammox bacteria to the starvation stress, which would facilitate operation and optimization of the Anammox process.

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Methane (CH4) recovery from excess sludge plays an important role in wastewater treatment aimed at achieving carbon neutrality. Besides pretreatment for excess sludge, exogenous CO2and even H2have already been tested in enhancing CH4production from anaerobic digestion (AD). However, exogenous H2injected into AD might simultaneously influence both upstream acidogenesis and downstream methanogenesis. In this study, a series of batch tests were conducted to ascertain the influences of hydrogen partial pressure (PH2) on these two processes. Interestingly, the batch tests demonstrated that injecting exogenous H2into AD at the beginning of each batch cycle can both stimulate acidogenesis (organic conversion rate) and enhance methanogenesis (both CH4and biogas productions) at an appropriate PH2(=0.33 bar) and an optimal revolution (Rs = 200 rpm) of a shaker.@en

Microalgae cultivation is a promising technology for integrated effluent polishing and biofuel production, but poor separability of microalgal cells hinders its industrial application. This study intended to selectively enrich settleable microalgal consortia in mixed culture by applying “wash-out” pressure, which was realized by controlling settling time (S_T) and volume exchange ratio (VER) in photo-SBRs. The results demonstrated that highly settleable microalgal consortia (settling efficiency>97%; SVI = 17–50 mL/g) could be enriched from indigenous algal cultures developed in WWTP's effluent. High VER was the key factor for the fast development of settleable microalgae. VER was also a controlling factor of the algal community structure. High VERs (0.5 and 0.7) resulted in the dominance of diatom, while low VER (0.2) facilitated the dominance of cyanobacteria. The settleable microalgal consortia were very efficient in phosphorus removal (effluent PO₄ ^3--P<0.1 mg/L; removal efficiency>99%), which was largely attributed to intensive chemical precipitation of phosphate induced by high pH (8.5–10). However, the high pH decreased the bioavailable inorganic carbon, resulting in incomplete nitrate removal (effluent NO₃ ⁻-N = 2.2–4 mg/L; removal efficiency = 61–79%) under high VERs and low lipid content (up to 10%) in the settleable microalgae. This problem could be resolved by sparging CO₂ or controlling pH. Overall, this study demonstrated a simple and effective method to overcome the separation challenge in scale-up of microalgae biotechnology for advanced wastewater purification and biofuel production.

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Carbon-neutral operation of wastewater treatment plants (WWTPs) requires enhancing anaerobic digestion (AD) of excess sludge for a higher energy conversion efficiency. Among others, iron has been identified to function on enhancing methane production in AD. As an industrial residual, waste iron scraps (WISs) have been reported as potentially enhancing CH₄ production in AD. With this study, the mechanisms of AD enhanced by WISs are analysed in a two-phase process: acidogenic phase (AP) and methanogenic phase (MP). Semi-continuous tests substantially excluded ORP reduction and hydrogen-evolution corrosion induced by WISs in enhancing CH₄ production, although WISs (10 g Fe/L) could indeed increase CH₄ production by 10.1% and 21.4% when added in AP and MP respectively. Detection on both FISH and enzymatic activities of involved microorganisms revealed that the stimulating effects of WISs on anaerobes (both catabolism and anabolism) could play an important (96.3%) role in enhancing CH₄ production, which would facilitate hydrolysis of refractory organics and improvement of electron transport rate (ETR).

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