Single-Layer Metasurface with Controllable Multiwavelength Functions.

In this paper, we report dispersion-engineered metasurfaces with distinct functionalities controlled by wavelength. Unlike previous approaches based on spatial multiplexing or vertical stacking of metasurfaces, we utilize a single phase profile with wavelength dependence encoded in the phase shifters' dispersion. We designed and fabricated a multiwavelength achromatic metalens (MAM) with achromatic focusing for blue (B), green (G), yellow (Y), and red (R) light and two wavelength-controlled beam generators (WCBG): one focuses light with orbital angular momentum (OAM) states ( l = 0,1,2) corresponding to three primary colors; the other produces ordinary focal spots ( l = 0) for red and green light, while generating a vortex beam ( l = 1) in the blue. A full color (RGB) hologram is also demonstrated in simulation. Our approach opens a path to applications ranging from near-eye displays and holography to compact multiwavelength beam generation.

Nitrogen source effects on the denitrifying anaerobic methane oxidation culture and anaerobic ammonium oxidation bacteria enrichment process.

The co-culture system of denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (Anammox) has a potential application in wastewater treatment plant. This study explored the effects of permutation and combination of nitrate, nitrite, and ammonium on the culture enrichment from freshwater sediments. The co-existence of NO3-, NO2-, and NH4+ shortened the enrichment time from 75 to 30 days and achieved a total nitrogen removal rate of 106.5 mg/L/day on day 132. Even though ammonium addition led to Anammox bacteria increase and a higher nitrogen removal rate, DAMO bacteria still dominated in different reactors with the highest proportion of 64.7% and the maximum abundance was 3.07 ± 0.25 × 108 copies/L (increased by five orders of magnitude) in the nitrite reactor. DAMO bacteria showed greater diversity in the nitrate reactor, and one was similar to M. oxyfera; DAMO bacteria in the nitrite reactor were relatively unified and similar to M. sinica. Interestingly, no DAMO archaea were found in the nitrate reactor. This study will improve the understanding of the impact of nitrogen source on DAMO and Anammox co-culture enrichment.

Simultaneous enrichment of denitrifying anaerobic methane-oxidizing microorganisms and anammox bacteria in a hollow-fiber membrane biofilm reactor.

In this study, the coculture system of denitrifying anaerobic methane oxidation (DAMO) microbes and anaerobic ammonium oxidation (anammox) bacteria was successfully enriched in a hollow-fiber membrane biofilm reactor (HfMBR) using freshwater sediment as the inoculum. The maximal removal rates of nitrate and ammonium were 78 mg N/L/day (131 mg N/m2/day) and 26 mg N/L/day (43 mg N/m2/day), respectively. Due to the high rate of methane mass transfer in HfMBR, the activity of DAMO archaea continued to increase during the enrichment period, indicating that HfMBR could be a powerful tool to enrich DAMO microorganisms. Effects of partial methane pressure, temperature, and pH on the cocultures were obvious. However, the microbial activity in HfMBR could be recovered quickly after the shock change of environmental factors. Furthermore, the result also found that DAMO bacteria likely had a stronger competitive advantage than anammox bacteria under the operating conditions in this study. High-throughput sequencing 16S rRNA genes illustrated that the dominant microbes were NC10, Euryarchaeota, Proteobacteria, Planctomycetes, and Chlorobi with relative abundance of 38.8, 26.2, 13.78, 6.2, and 3.6 %, respectively.

Experimental evaluation of the metabolic reversibility of ANME-2d between anaerobic methane oxidation and methanogenesis.

The "reverse methanogenesis" hypothesis as the metabolic pathway of AOM has recently been supported in the novel ANME lineage ANME-2d in denitrifying anaerobic methane oxidation (DAMO). However, no previous studies have experimentally evaluated the reversal of methane oxidation and methane production in this archaea. In the present study, the metabolic reversibility of ANME-2d from AOM to methanogenesis was evaluated using H2/CO2 and acetate as substrates. The results showed that the system produced methane from H2/CO2 but not from acetate. However, the clone library and real-time PCR analysis of the culture showed that both the percentage and quantity of ANME-2d decreased significantly under this condition, while methanogen abundance increased. Further high-throughput sequencing results showed that the archaea community did not change at the fourth day after H2/CO2 was supplied, but changed profoundly after methanogenesis took place for 3 days. The percentage of DAMO archaea in the total archaea decreased obviously, while more methanogens grew up during this period. Comparatively, the bacteria community changed profoundly at the fourth day. These results indicated that ANME-2d might not reverse its metabolism to produce methane from H2/CO2 or acetate. After archaea were returned to DAMO conditions, DAMO activity decreased and the amount of ANME-2d continued to fall, implying that the lineage had suffered from severe injury and required a long recovery time.

Environmental evaluation of coexistence of denitrifying anaerobic methane-oxidizing archaea and bacteria in a paddy field.

The nitrate-dependent denitrifying anaerobic methane oxidation (DAMO) process, which is metabolized together by anaerobic methanotrophic archaea and NC10 phylum bacteria, is expected to be important for the global carbon and nitrogen cycles. However, there are little studies about the existence of this process and the functional microbes in environments. Therefore, the coexistence of DAMO archaea and bacteria in a paddy field was evaluated in this study. Next-generation sequencing showed that the two orders, Methanosarcinales and Nitrospirales, to which DAMO archaea and DAMO bacteria belong, were detected in the four soil samples. Then the in vitro experiments demonstrated both of nitrite- and nitrate-dependent DAMO activities, which confirmed the coexistence of DAMO archaea and DAMO bacteria. It was the first report about the coexistence of DAMO archaea and bacteria in a paddy field. Furthermore, anammox bacteria were detected in two of the four samples. The in vitro experiments did not show anammox activity in the initial period but showed low anammox activity after 20 days' enrichment. These results implicated that anammox bacteria may coexist with DAMO microorganisms in this field, but at a very low percentage.

The role of paraffin oil on the interaction between denitrifying anaerobic methane oxidation and Anammox processes.

Methane is sparingly soluble in water, resulting in a slow reaction rate in the denitrifying anaerobic methane oxidation (DAMO) process. The slow rate limits the feasibility of research to examine the interaction between the DAMO and the anaerobic ammonium oxidation (Anammox) process. In this study, optimized 5 % (v/v) paraffin oil was added as a second liquid phase to improve methane solubility in a reactor containing DAMO and Anammox microbes. After just addition, methane solubility was found to increase by 25 % and DAMO activity was enhanced. After a 100-day cultivation, the paraffin reactor showed almost two times higher consumption rates of NO3 (-) (0.2268 mmol/day) and NH4 (+) (0.1403 mmol/day), compared to the control reactor without paraffin oil. The microbes tended to distribute in the oil-water interface. The quantitative (q) PCR result showed the abundance of gene copies of DAMO archaea, DAMO bacteria, and Anammox bacteria in the paraffin reactor were higher than those in the control reactor after 1 month. Fluorescence in situ hybridization revealed that the percentages of the three microbes were 55.5 and 77.6 % in the control and paraffin reactors after 100 days, respectively. A simple model of mass balance was developed to describe the interactions between DAMO and Anammox microbes and validate the activity results. A mechanism was proposed to describe the possible way that paraffin oil enhanced DAMO activity. It is quite clear that paraffin oil enhances not only DAMO activity but also Anammox activity via the interaction between them; both NO3 (-) and NH4 (+) consumption rates were about two times those of the control.

New primers for detecting and quantifying denitrifying anaerobic methane oxidation archaea in different ecological niches.

The significance of ANME-2d in methane sink in the environment has been overlooked, and there was no any study evaluating the distribution of ANME-2d in the environment. New primers were thus needed to be designed for following research. In this paper, a pair of primers (DP397F and DP569R) was designed to quantify ANME-2d. The specificity and amplification efficiency of this primer pair were acceptable. PCR amplification of another pair of primers (DP142F and DP779R) generated a single, bright targeted band from the enrichment sample, but yielded faint, multiple bands from the environmental samples. Nested PCR was conducted using the primers DP142F/DP779R in the first round and DP142F/DP569R in the second round, which generated a bright targeted band. Further phylogenetic analysis showed that these targeted bands were ANME-2d-related sequences. Real-time PCR showed that the copies of the 16s ribosomal RNA gene of ANME-2d in these samples ranged from 3.72 × 10(4) to 2.30 × 10(5) copies µg(-1) DNA, indicating that the percentage of ANME-2d was greatest in a polluted river sample and least in a rice paddy sample. These results demonstrate that the newly developed real-time PCR primers could sufficiently quantify ANME-2d and that nested PCR with an appropriate combination of the new primers could successfully detect ANME-2d in environmental samples; the latter finding suggests that ANME-2d may spread in environments.

Simultaneous enrichment of denitrifying methanotrophs and anammox bacteria.

Interaction between denitrifying anaerobic methane oxidation (DAMO) and anaerobic ammonium oxidation (anammox) processes may play an important role in global carbon and nitrogen cycles. In this study, a coculture of denitrifying methanotrophs (DAMO archaea and DAMO bacteria) and anammox bacteria, initially sourced from the environment, was enriched with a supply of methane, nitrate, and ammonium. After a 4.5-month enrichment, simultaneous oxidation of methane and ammonium and reduction of nitrate were observed. The highest rate of nitrate reduction in the suspended DAMO culture was 4.84 mmol/L/day, and simultaneously, the highest ammonium removal rate was 4.07 mmol/L/day. Fluorescence in situ hybridization and analysis of 16S rRNA gene clone libraries revealed the coexistence of DAMO archaea, DAMO bacteria, and anammox bacteria. The development of anammox bacteria might reduce the enrichment time of DAMO microorganisms and promote the activity of DAMO archaea. The activity of the reactor fluctuated during the long-term operation, which might be caused by the formation of microbial clusters whereby DAMO archaea grew in aggregates that were surrounded by anammox and DAMO bacteria. This study is the first to demonstrate that it is feasible to establish a coculture of DAMO archaea, DAMO bacteria, and anammox bacteria from environmental inocula.

Hollow fiber membrane bioreactor affects microbial community and morphology of the DAMO and Anammox co-culture system.

Denitrifying anaerobic methane oxidation (DAMO) and Anammox co-culture system was investigated in hollow fiber membrane bioreactor (HfMBR) for the change of microbial community morphology and proportion. NO3--N and NH4+-N removal rates reached 85.33 and 37.95mg/L/d on 193d. The inoculum microorganisms were flocs and the proportion of DAMO archaea, DAMO bacteria and Anammox bacteria was 11.0, 24.2 and 0.4%, respectively, but it changed to 74.3, 11.8, 5.6% in HfMBR, respectively. Interestingly, microorganisms formed biofilms on fibers surface and the biofilms included two layers: inner layer was thin and dominated by DAMO bacteria and Anammox bacteria; while the outer layer was thick made up of granules with 100-200µm diameter and dominated by DAMO archaea. The spatial distribution of microorganisms in HfMBR was different from simulation results in the literature. Likely, HfMBR changed the interaction between DAMO and Anammox microorganisms, and the reactor configuration was beneficial for DAMO archaea growth.

Decoupling of DAMO archaea from DAMO bacteria in a methane-driven microbial fuel cell.

Anaerobic oxidation of methane (AOM) contributes significantly to the global methane sink. Previously, studies of anaerobic methanotrophic (ANME) archaea have been limited as they have not been separable from their bacterial partners during the AOM process because of their dependence on the bacteria. A microbial fuel cell (MFC) is a device capable of directly transforming chemical energy to electrical energy via electrochemical reactions involving biochemical pathways. In this study, decoupling of denitrifying anaerobic methane oxidation (DAMO) archaea and DAMO bacteria was investigated in an microbial fuel cell (MFC) using methane as the fuel. The DAMO fuel cell worked successfully but demonstrated weak electrogenic capability with around 25 mV production. After 45 days' enrichment, the sequencing and fluorescence in situ hybridization results showed the DAMO archaea percentage had increased from 26.96% (inoculum) to 65.77% (electrode biofilm), while the DAMO bacteria percentage decreased from 24.39% to 2.07%. Moreover, the amount of ANME-2d had doubled in the electrode biofilm compared with the inoculum. The sequencing results also showed substantial enrichment of the Ignavibacterium and Geobacter genera. The roles of Ignavibacterium and Geobacter in the MFC system need to be further investigated. Nevertheless, these results illustrate that an MFC device may provide a possible approach to separate DAMO archaea from DAMO bacteria.

Tracking the activity of the Anammox-DAMO process using excitation-emission matrix (EEM) fluorescence spectroscopy.

Coupling of anaerobic ammonium oxidation (anammox) and denitrifying anaerobic methane oxidation (DAMO) microorganisms in a hollow-fiber membrane biofilm reactor (HfMBR) is a potential strategy for simultaneous anaerobic removal of nitrogen and methane in wastewater streams. In these systems, effluents contain dissolved organic substances from anammox and DAMO microorganisms, but their characteristics and relationships have not been investigated. In the present study, excitation-emission matrix (EEM) fluorescence spectroscopy was used to characterize effluent dissolved organic matter (EfDOM) from an Anammox-DAMO HfMBR. Four component types (Component 1-4) were identified by parallel factor analysis (PARAFAC) of EEM data. Component 1 was produced when anammox and DAMO microorganisms simultaneously starved, whereas Component 4 was only generated through the starving period of DAMO microorganisms, and the longer the starving period, the higher the fluorescence intensity of the components. Components 2 and 3 were generated via active and starving periods of co-cultures. More efficient nitrogen removal was accompanied by a higher fluorescence intensity and microbial activity. Compared to measuring both influent and effluent nitrogen concentrations, monitoring EfDOM can obtain other information about the reactor, such as nitrogen removal activity of the reactor, status of the microbes and the duration of starving period the reactor suffered, which therefore offers a complementary but direct tool for assessing reactor performance in complex co-culture systems.

Cr(VI) reduction coupled with anaerobic oxidation of methane in a laboratory reactor.

The process of anaerobic oxidation of methane (AOM) is globally important because of its contribution to the carbon cycle in the environment. Besides, microorganisms play important roles in the environmental fate of chromium. However, there have been no studies to date on the interaction between methane and chromium in batch reactor systems. In this study, biological Cr(VI) reduction was investigated using methane as the sole electron donor. Isotopic (13)CH4 in the batch experiments and long-term performance in the reactor demonstrated that Cr(VI) reduction is coupled with methane oxidation. High-throughput sequencing of the 16S rRNA genes demonstrated that the microbial community had changed substantially after Cr(VI) reduction. The populations of ANME-2d archaea were enhanced, and they became the only predominant AOM-related microbe. Interestingly, other bacteria with significant increases in abundance were not reported as having the ability to reduce Cr(VI). According to these results, two mechanisms were proposed: 1) Cr(VI) is reduced by ANME-2d alone; 2) Cr(VI) is reduced by unknown Cr(VI)-reducing microbes coupled with ANME-2d. This study revealed the potential relationship between Cr(VI) reduction and CH4 oxidation, and extended our knowledge of the relationship between the AOM process and biogeochemical cycles.

Iron reduction in the DAMO/Shewanella oneidensis MR-1 coculture system and the fate of Fe(II).

Dissimilatory iron reduction and anaerobic methane oxidation processes play important roles in the global iron and carbon cycle, respectively. This study explored the ferrihydrite reduction process with methane as a carbon source in a coculture system of denitrifying anaerobic methane oxidation (DAMO) microbes enriched in laboratory and Shewanella oneidensis MR-1, and then characterized the reduced products. Ferrihydrite reduction was also studied in the DAMO and Shewanella systems alone. The ferrihydrite was reduced slightly (<13.3%) in the separate systems, but greatly (42.0-88.3%) in the coculture system. Isotope experiment of (13)CH4 addition revealed that DAMO microbes coupled to S. oneidensis MR-1 in a ferric iron reduction process with (13)CH4 consumption and (13)CO2 production. Compared with ferrihydrite, the reduced products showed increased crystallinity (from amorphous state to crystallinity 77.1%) and magnetism (from paramagnetic to ferromagnetic). The produced ferrous iron was formed into minerals primarily composed of siderite with a small amount vivianite and magnetite. A portion of products covered the cell surface and hindered further reactions. The results presented herein widen the current understanding of iron metabolism and mineralization in the ocean, and show that the coculture systems of DAMO microbes and Shewanella have the potential to be globally important to iron reduction and methane oxidation.

Design and evaluation of universal 16S rRNA gene primers for high-throughput sequencing to simultaneously detect DAMO microbes and anammox bacteria.

To develop universal 16S rRNA gene primers for high-throughput sequencing for the simultaneous detection of denitrifying anaerobic methane oxidation (DAMO) archaea, DAMO bacteria, and anaerobic ammonium oxidation (anammox) bacteria, four published primer sets (PS2-PS5) were modified. The overall coverage of the four primer pairs was evaluated in silico with the Silva SSU r119 dataset. Based on the virtual evaluation, the two best primer pairs (PS4 and PS5) were selected for further verification. Illumina MiSeq sequencing of a freshwater sediment and a culture from a DAMO-anammox reactor using these two primer pairs revealed that PS5 (341b4F-806R) was the most promising universal primer pair. This pair of primers detected both archaea and bacteria with less bias than PS4. Furthermore, an anaerobic fermentation culture and a wastewater treatment plant culture were used to verify the accuracy of PS5. More importantly, it detected DAMO archaea, DAMO bacteria, and anammox bacteria simultaneously with no false positives appeared. This universal 16S rRNA gene primer pair extends the existing molecular tools for studying the community structures and distributions of DAMO microbes and their potential interactions with anammox bacteria in different environments.

Fatty acids production from hydrogen and carbon dioxide by mixed culture in the membrane biofilm reactor.

Gasification of waste to syngas (H2/CO2) is seen as a promising route to a circular economy. Biological conversion of the gaseous compounds into a liquid fuel or chemical, preferably medium chain fatty acids (caproate and caprylate) is an attractive concept. This study for the first time demonstrated in-situ production of medium chain fatty acids from H2 and CO2 in a hollow-fiber membrane biofilm reactor by mixed microbial culture. The hydrogen was for 100% utilized within the biofilms attached on the outer surface of the hollow-fiber membrane. The obtained concentrations of acetate, butyrate, caproate and caprylate were 7.4, 1.8, 0.98 and 0.42 g/L, respectively. The biomass specific production rate of caproate (31.4 mmol-C/(L day g-biomass)) was similar to literature reports for suspended cell cultures while for caprylate the rate (19.1 mmol-C/(L day g-biomass)) was more than 6 times higher. Microbial community analysis showed the biofilms were dominated by Clostridium spp., such as Clostridium ljungdahlii and Clostridium kluyveri. This study demonstrates a potential technology for syngas fermentation in the hollow-fiber membrane biofilm reactors.