Philippa Kaur

SettingThis study was conducted in approximately 40-year-old naturally regenerated P. densiflora stands in Wola National Experimental Forest in Gyeongnam province in South Korea (35°12′ N, 128°10′ E; Table 1). The productivity of this forest is low, with a dominant tree height of 10 m at 20 years of age. Over the last 10 years, the mean annual precipitation was 1490 mm, of which one third fell during summer (July–August), and the mean temperature was 13.1 °C. The vegetation growing season generally lasts for approximately 200 days, extending from early April to October. The soil texture is a silt loam originating from sandstone and shale (clay 13.0 ± 0.8%, silt 44.1 ± 1.3%, sand 42.9 ± 1.6%; n = 18). The given texture results in volumetric water contents at 13.4 ± 0.7% (m3 m−3) at permanent wilting point (1500 kPa) and 40.7 ± 1.2% at field capacity (10 kPa)55. The understory is covered with Lespedeza spp., Quercus variabilis, Q. serrata, Smilax china, and Lindera glauca.In 2010, we selected two adjacent P. densiflora stands approximately 100 m apart from each other (180 m and 195 m above sea level, on slopes of 15° and 33°, both stands face south). Following a completely randomized design, we established nine plots (10 × 10 m2 with a 5 m untreated buffer) within each stand, of which three were randomly assigned to annual NPK fertilization, three to PK fertilization, and the rest to a control treatment without fertilization. The fertilizer, composed of urea, fused superphosphate and potassium chloride (N3P4K1) or P4K1 was added manually by deposition on the forest floor for 3 years in April 2011, April 2012, and March 2013. Over these 3 years, the NPK plots received 33.9 g N, 45 g P, and 11.1 g K m−2, while the PK plots received 45 g P and 11.1 g K m−2.Tree and stand measurementsThe standing biomass of trees was estimated using a combination of site-specific allometric equations based on destructive harvesting56 and repeated measurements of the dimensions of all trees in each plot (5–18 trees plot−1). The stem diameter at 1.2 m (D) was measured for all trees in each plot for which D was ≥ 6 cm. Selecting a representative tree in size for each plot within the 4 × 4 m2 center of the plot, we measured the tree height (H) and crown base for the representative trees. Measurements were performed in April and September 2011, September 2012–2014, and October 2021. We observed no effect of fertilization on the relationship between D and H or between D and crown base, so we assumed no effect on the allometric functions for foliage or branch biomass. A dendrometer band (Series 5 Manual Band, Forestry Suppliers Inc., Jackson, MS, USA) was installed on 18 representative trees (one per plot) to monitor radial growth monthly.Three 0.25 m2 circular litter traps were installed 60 cm above the forest floor in each plot in April 2011. Litter was collected at 3-month intervals between June 2011 and March 2015. The litter from each trap was transported to the laboratory and then oven-dried at 65 °C for 48 h. All dried samples were separated into needles, bark, cones, branches, and miscellaneous components, and weighed separately.In September 2014, we estimated the biomass of understory vegetation, separately for woody plants and herbaceous plants. All woody plants  More

Goal and scope of the studyThe goal of this study was to assess the planetary footprints of GHG mitigation strategies for the global production of plastics. To calculate planetary footprints, we apply LCA in combination with the planetary boundaries framework as proposed by ref. 22. As GHG mitigation strategies, we consider recycling, bio-based production and production via CCU, and compare their planetary footprints to the planetary footprints of fossil-based plastics. We use a bottom-up model covering >90% of global plastic production for 2030 (and 2050, Supplementary Information, section 3). The bottom-up model builds on the plastic production system from ref. 10 and includes plastic production, the supply chain and the disposal of plastics at the end of life.Functional unitIn LCA, the functional unit quantifies the functions of the investigated product system. In this study, the function of the product system is the production and disposal of >90% of global plastics. To cover >90% of global plastics, we define the functional unit as the yearly global production and disposal of 14 large-volume plastics (summarized in Supplementary Table 5). We estimated the yearly production volumes for 2030 and 2050 based on the production volumes in 2015 and the annual growth rates shown in Supplementary Table 5.Our assessment includes plastic disposal. However, the production and disposal of plastics do not necessarily occur in the same year. For instance, while polyolefins used for plastic packaging have an average lifetime of 6 months, the average lifetime of polyurethane used in construction is 35 years11. Including the lifetime of plastics, and hence, the temporal difference between production and disposal, would lead to an increasing plastic stock. An increasing stock, in turn, represents a carbon sink during the production year that appears to enable the production of net-negative GHG emission plastics based on biomass or CCU. However, the plastic stock is not a permanent carbon sink, which would be required for producing net-negative GHG emission plastics55. To avoid misleading conclusions about net-negative bio- and CCU-based plastics, we assign the planetary footprints from disposal to the year of plastic production. Thereby, we conservatively assess the planetary footprints of plastics.In addition, we address the challenge highlighted in ref. 56 that the increasing demand for plastics renders determining the absolute sustainability of plastics difficult. We meet this challenge by assuming a steady-state production system with a recurring functional unit in the same amount every year. We thereby analyse discrete scenarios with constant consumption levels for plastics. Therefore, our conclusions depend on the accuracy of the demand forecasts and apply only to the production volumes considered.System boundariesWe use cradle-to-grave system boundaries, including plastic production and supply chain, potential recycling and final disposal at the end of life. Assessing the use phase of plastics is not possible because of a lack of data. The versatile properties of plastics result in a wide range of applications that cannot be represented in a single study. Furthermore, it would be necessary to consider not only the emissions of the use phase (probably relatively small) but also the system-wide environmental consequences of using plastics in each application compared to other materials. Thus, a consequential assessment of the plastic use phase is desirable but beyond the scope of this study.The plastics supply chain includes several intermediate chemicals such as monomers, solvents or other reactants. The bottom-up model covers the production of all intermediate chemicals in the foreground system. As a background system, we use aggregated datasets from the LCA database ecoinvent. A list of all intermediate chemicals and all aggregated datasets can be found in Supplementary Information, section 1. In addition, the foreground system of the bottom-up model does not include environmental impacts from infrastructure and transportation because of a lack of data. However, we consider the environmental impacts of infrastructure and transportation from other industrial sectors by aggregated datasets, for example, from electricity generation and biomass cultivation.The bottom-up model includes the best available fossil-based technologies and the following technologies for plastic disposal and virgin production based on biomass and CCU.Plastic waste disposalThe bottom-up model includes three options for plastic waste disposal: landfilling, incineration with energy recovery and recycling. Plastic waste can occur in several forms: as sorted fraction of municipal solid waste, as mixed plastics and residues from sorting, and as residues from mechanical recycling. For all fractions, we include waste incineration with energy recovery and landfilling.Landfilled plastic waste is assumed to degrade by approximately 1% of the contained carbon, which is in line with the ecoinvent database45. Mechanical recycling is only modelled for sorted fractions of packaging waste owing to impurities of mixed and non-packaging wastes. In contrast, chemical recycling can be applied to all plastic fractions. In this study, we model chemical recycling as pyrolysis to refinery feedstock, that is, naphtha. The pyrolysis has yields of 29 to 69% depending on the type of plastic (details in Supplementary Information, section 1). Furthermore, we include options for chemical recycling of plastic waste to monomers, which are still early-stage technologies. To derive the minimal necessary recycling rate in Fig. 5, we apply an optimistic scenario with a 95% yield of chemical recycling processes following common modelling in life-cycle inventories of chemicals (Supplementary Information, section 3)57. All calculations are constrained to maximum recycling rates of 94% as the remaining 6% are assumed to be the minimal landfilling rate until the middle of the century11. The assumption is based on historical trends in end-of-life treatment of plastics.Bio-based productionBio-based GHG mitigation is frequently discussed in the literature and is often associated with competition with the food industry58. To avoid competition with the food industry, the bottom-up model is restricted to lignocellulosic biomass as feedstock, that is, energy crops, forest residues and by-products from other industrial biomass processes (for example, bagasse). In this study, unless mentioned otherwise, we model biomass as energy crops because of their potential for large-scale application (Supplementary Information, section 3). However, we conduct a sensitivity analysis for other lignocellulosic biomass sources to assess the sustainability of bio-based plastics in more detail.For each biomass type, we account for the carbon uptake during the biomass growth phase by giving a credit corresponding to the biomass carbon content. We do not consider land use change emissions as current literature lacks an assessment of land use change effects on other Earth-system processes besides climate change.For biomass processing, we include the following high-maturity processes: gasification to syngas and fermentation to ethanol, and the subsequent conversion to methanol and ethylene (Supplementary Table 1). Methanol and ethylene can be further converted to propylene and aromatics, which all together represent the building blocks for all plastics in this study.CCU-based productionCCU-based plastic production particularly requires CO2 and hydrogen. For CO2 supply, we consider CO2 capture from highly concentrated point sources within the plastics supply chain. Highly concentrated point sources include the conventional fossil-based processes, ammonia production, steam methane reforming, ethylene oxide production, the bio-based processes for ethanol and syngas, and plastic waste incineration. Capturing from processes within the plastics supply chain is limited by the amount of CO2 emitted by these processes and avoids the corresponding emissions. For these processes, we considered the energy demand for compressing the CO2 with 0.4 MJ of electricity59. For waste incineration, we consider a decrease in energy output when capturing CO2. All further CO2 sources are conservatively approximated by direct air capture. For 1 kg CO2 captured via direct air capture, we include an uptake of 1 kg of CO2 equivalent while considering the energy demand of 1.29 MJ electricity and 4.19 MJ heat60.Hydrogen for CCU is produced by water electrolysis, with an overall efficiency of 67%61. Previous studies have already shown that renewable electricity is required for CCU to be environmentally beneficial13. Thus, we conduct a sensitivity analysis for multiple electricity technologies to assess their influence on the sustainability of CCU-based plastics (Supplementary Information).For CCU-based production, we include high-maturity technologies, such as CO2-based methanol and methane, as well as subsequent production of olefins and aromatics (Supplementary Table 1). We do not consider CCS as an additional scenario, as fossil resources and storage capacities are ultimately limited. Therefore, CCS may serve as an interim solution for GHG mitigation but stands in contrast to long-term sustainability as the goal of this study.Pathway definitionWe assess nine pathways for the plastics industry towards sustainability. Pathway 1 is fossil-based plastic production (current recycling rate of 23%) that serves as a reference. We also include two pathways that combine all circular technologies: Pathway 2, which minimizes the climate change impact (climate-optimal), and pathway 3, which minimizes the maximal transgression of the share of SOS of the plastics industry (balanced) (Fig. 2). To assess the impact of switching from fossil to renewable feedstocks, we introduce pathway 4, which is bio-based, and pathway 5, which is CCU-based (Fig. 3). Pathways 4 and 5 include the current recycling rate of 23%. In addition, we introduce three pathways with the maximum recycling rates of 94%: pathway 6, in which the remaining virgin production is based on fossil resources; pathway 7, in which it is based on biomass; and pathway 8, in which it is based on CO2 (Fig. 3). Pathway 9 combines biomass, CCU and recycling, and additionally includes chemical recycling of polymers to monomers to calculate the minimal recycling rate to achieve sustainable plastics (Fig. 5).The planetary boundaries frameworkWe follow the recommendations for absolute environmental sustainability assessment in ref. 29 and choose the planetary boundaries framework for the assessment. The planetary boundaries framework suits the goal of the study best because of its precautionary principles for the definition of environmental thresholds, the SOS. We assess eight of the nine Earth-system processes suggested in ref. 21, namely, climate change, ocean acidification, changes in biosphere integrity, the biogeochemical flow of nitrogen and phosphorus (referred to as N cycle and P cycle), aerosol loading, freshwater use, stratospheric ozone depletion, and land-system change. We do not assess the Earth-system process of novel entities since neither control variables nor the boundary itself is yet adequately defined22. We consider the global boundaries for the Earth-system processes in line with the scope of this study. These global boundaries and the corresponding calculation of planetary footprints are subject to assumptions and thus incorporate uncertainty (Supplementary Information, section 2).For the two subprocesses for climate change (namely, atmospheric CO2 concentration and energy imbalance at the top-of-atmosphere), we only consider the energy imbalance at the top-of-atmosphere quantified by radiative forcing. We focus on radiative forcing, as the control variable is more inclusive and fundamental, and the global limits are stricter than for atmospheric CO2 concentration21. Thereby, we conservatively assess climate change.Biosphere integrity is divided into functional and genetic diversity of species. Preserving functional diversity ensures a stable ecosystem by maintaining all ecosystem services. We assess the functional diversity of species using the method proposed in ref. 18. The method covers the mean species abundance loss caused by the two main stressors, direct land use and GHG emissions, as a proxy for the biodiversity intactness index. Genetic diversity provides the long-term ability of the biosphere to persist under and adapt to gradual changes of the environment21. Genetic diversity is often approximated by the global extinction rate. However, using the global extinction rate does not fully cover variation of genetic composition, resulting in high uncertainties when quantifying genetic diversity18. Thus, we focus on functional diversity.Downscaling of the safe operating spaceAs the plastics industry accounts for only a fraction of all human activities, we assign a share of the SOS to plastics. The plastics industry should operate within its assigned share to be considered environmentally sustainable. To assign a share of SOS to the plastics industry, we apply utilitarian downscaling principles. Utilitarian downscaling principles are tailored to maximize welfare in society29. We approximate welfare by consumption expenditure on plastics as an economic indicator for consumer preferences and human needs62. An extensive discussion on the other downscaling principles and their implications can be found in Supplementary Information.Although the final consumption expenditures on plastics are negligible, the industry consumes plastics to produce other goods and services. Accordingly, plastics are produced mostly in the upstream supply chain to support the final consumption of other goods and services. Thus, consuming other goods and services induces plastic production. To account for this inducement of plastic production, we used the total global plastic production xplastics to represent the global intermediate and final consumption expenditure on plastics. For this purpose, we use the gross output vector x of the product-by-product input–output table of EXIOBASE for the year 2020 (ref. 63). To calculate the share of SOS of the plastics industry, we divide the total global plastic production xplastics by the gross world product. The gross world product equals the total global final consumption expenditure. Analogously, we also consider the end-of-life treatment of plastics to be consistent with the system boundaries of the environmental assessment.We estimate the share of SOS for the plastics industry for 2030 and 2050 based on data for the year 2020. Accordingly, we assume that the market share of the plastics industry and, therefore, its share of SOS do not change in the coming years despite the increasing production volume of plastics. Thereby, we implicitly assume that all industries grow equally economically. Alternatively, economic forecasting models could estimate future market shares of plastics. However, applying economic forecasting models is complex, and the results would still be highly uncertain, especially if industry pursues low-carbon technology pathways. Therefore, estimating future market shares is beyond the scope of this study.Technology choice modelTo calculate the planetary footprint of plastics, we use a bottom-up model of the plastics industry. The model builds on the technology choice model (TCM) that allows for linear optimization of production systems27. The TCM represents the production system based on the following elements: technologies, intermediate flows, elementary flows and final demands. Ref. 27 describes each element in detail.The TCM is based on the established computational structure of LCA64. This structure arranges the data that represent the physical production system in the technology matrix A and the elementary flow matrix B. In the technology matrix A, columns represent technologies, and rows represent intermediate flows. Therefore, the coefficient aij of the A matrix corresponds to an intermediate flow i that is either produced (aij  > 0) or consumed (aij  More

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Artificial intelligence (AI) and machine learning could revolutionize the search for life on other planets. But before these tools can tackle distant locales such as Mars, they need to be tested here on Earth.A team of researchers have successfully trained an AI to map biosignatures — any feature which provides evidence of past or present life — in a three-square-kilometre area of Chile’s Atacama Desert. The AI substantially reduced the area the team needed to search and boosted the likelihood of finding living organisms in one of the driest places on the planet. The results were reported on 6 March in Nature Astronomy1.Kimberley Warren-Rhodes, a senior research scientist at the SETI Institute in Mountain View, California, and lead author on the paper, has been chasing biosignatures since the early 2000s, when she realized how few tools existed to study the biology of other planets. She wanted to combine her background in statistical ecology with emerging technologies such as AI to help mission scientists, “who are under a lot of pressure to find biosignatures” but tightly constrained in how they do so. Rovers that are controlled remotely from Earth, for example, can travel only limited distances and collect relatively few specimens, placing a premium on sampling locations that are the most likely to yield life. Mission scientists base these predictions in part on Mars analogues on Earth, where scientists scour extreme habitats to determine how and where living organisms thrive.Searching for lifeBeginning in 2016, Warren-Rhodes’ group travelled to the high, parched plateau of the Atacama Desert — a proposed Mars analogue at an elevation of around 3,500 metres in the Chilean Andes — to search for rock-dwelling, photosynthetic organisms called endoliths. To fully characterize the environment, the researchers collected everything from drone footage to geochemical analyses to DNA sequences. Together, this data set mimics the types of information researchers are collecting on Mars with orbital satellites, drones and rovers.Warren-Rhodes’ team fed its data into an AI-based convolutional neural network (CNN) and a machine-learning algorithm that in turn predicted where life was most likely to be found in the Atacama.

Aerial view (left) and ground view from a rover of a biosignature probability map of the same area.Credit: M. Phillips, K. A. Warren-Rhodes & F. Kalaitzis

By targeting their sample collection on the basis of AI feedback, the researchers were able to reduce their search area by up to 97% and increase their likelihood of finding life by up to 88%. “At the end, you could plop us down, and instead of wandering around for a long time, it would take us a minute to find life,” Warren-Rhodes says. Specifically, the team found that endoliths in the Atacama were most often found in a mineral called alabaster — which is porous and retains water — and tended to aggregate in transitional areas between various microhabitats, such as where sand and alabaster crystals abut one another.“I’m very impressed and very happy to see this suite of work,” says Kennda Lynch, an astrobiologist at the Lunar and Planetary Institute in Houston, Texas, who studies biosignatures. “It’s really cool that they can show some success with an AI to help predict where to go and look.”Graham Lau, an astrobiologist at the Blue Marble Space Institute of Science who is based in Boulder, Colorado, worked on another Mars analogue in the Canadian Arctic as a graduate student, to study how biology influences the formation of rare minerals that can serve as biosignatures on other planets. “Ever since I first read Frank Herbert’s Dune as a young child, I was struck by this idea of applying ecology to planets,” he says. But up until the last decade or so, the tools and data weren’t available to address such questions with scientific rigour. “The place where we have almost unlimited data possibilities is through these orbital observations and drone imaging,” he says, “and I do see this paper as being one of many pieces along the pathway to doing these larger analyses.”Deceptively simpleThe new method will need to be verified across multiple ecosystems, Lau and Lynch say, including those with more complex geology and greater biodiversity. The Atacama, Lau notes, is relatively simple in terms of the habitats and the types of life that are likely to be found there. And on Mars, the high level of ultraviolet radiation striking the planet’s surface means that scientists might need to detect clues that hint at life below ground.

NASA’s Perseverance rover collected its first rock sample from an area in Mars’ Jezero Crater.Credit: NASA/JPL-Caltech/ASU/MSSS

Ultimately, Warren-Rhodes says she would like to see a comprehensive database of different Mars analogues that could feed valuable information to mission scientists planning their next sampling run. Her team’s advance, she adds, might appear “deceptively simple” to anyone who grew up watching Star Trek explorers scanning alien worlds with a tricorder. But, it represents an important advance in extraterrestrial research, in which biology has often lagged behind chemistry and geology. Imagine, for instance, virtual-reality headsets that feed mission scientists real-time data as they scan a surface, using a rover’s ‘eyes’ to direct their activities. “To have our team make one of these first steps towards reliably detecting biosignatures using AI is exciting,” she says. “It’s really a momentous time.” More

Preparation of the pao cai soupFirst, 35 kg of white radish (Raphanus sativus), 35 kg of cabbage (Brassica oleracea), 2 kg of chili pepper (Capsicum frutescens), 1 kg of ginger (Zingiber officinale), 1 kg of peppercorns (Zanthoxylum bungeanum), 2.5 kg of rock sugar, and 210 kg of cold boiled water (containing 6% salt) were divided into six ceramic jars. After 7 days of natural fermentation at room temperature, the pao cai was filtered out with sterile gauze to obtain 200 kg of pao cai soup. To ensure an even distribution of microorganisms in the soup, the soup was mixed well and then left to rest for 12 h, the supernatant was taken, and the soup was left to rest for 12 h again.The plants used in this study were cultivated vegetables which purchased from the vegetable market at the study site. All local, national or international guidelines and legislation were adhered to in the production of this study.Establishment of the microcosm systemSeventy-eight for each size of 10 ml, 20 ml, 50 ml, 100 ml, 250 ml, 500 ml, and 1000 ml sterile glass culture flasks were filled with pao cai soup, the bottle mouth was sealed with sterile sealing film, and the bottle was capped without leaving any air (Fig. 1). Each flask became a microcosm and was cultured in a 25 °C incubator.Figure 1Schematic diagram of the establishment of the microcosmic system.Full size imageSample collectionBefore the microcosm system was established, a sample of well-mixed pao cai soup was taken as a reference to establish background biodiversity. The microbial community dynamics should change the fastest at the beginning of the microcosm system establishment and gradually become slower over time. Considering the workload and cost, this study collected samples daily for 1–10 day after the establishment of the microcosm and then collected every 2 days for 10–30 day and every 5 days for 30–60 day. Three different microcosms of the same volume were established. Monitoring was carried out for 60 days, and a total of 546 samples of 7 volumetric gradients were obtained at 26 time points. At the time of sampling, the pao cai soup in the microcosm was mixed, and 50 mL of sample (10 mL of sample was collected for microcosm systems with a volume of less than 50 mL) was collected. The sample was centrifuged at 8000 rpm for 10 min, the supernatant was collected for pH determination, and the pellet was stored in a − 80 °C freezer.Microbial analysesMicrobial DNA was extracted from pao cai samples using the E.Z.N.A.® Soil DNA Kit (Omega Biotek, Norcross, GA, U.S.) according to the manufacturer’s protocols. For bacteria, we targeted the V3-V4 region of the 16S ribosomal RNA (rRNA) gene, using the 338F (5′-ACTCCTACGGGAGGCAGCAG-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) primer pairs. For fungi, we targeted the ITS1-1F region of the nuclear ribosomal internal transcribed spacer region (ITS rDNA) gene, using ITS1-1F-F (5′-CTTGGTCATTTAGAGGAAGTAA-3′) and ITS-1F-R (5′-GCTGCGTTCTTCATCGATGC-3′). PCRs were performed in triplicate in a 20 μL mixture containing 4 μL of 5 × FastPfu Buffer, 2 μL of 2.5 mM dNTPs, 0.8 μL of each primer (5 μM), 0.4 μL of FastPfu Polymerase and 10 ng of template DNA. The PCR program for the 16S rRNA gene was as follows: 3 min of denaturation at 95 °C; 27 cycles of 30 s at 95 °C, 30 s of annealing at 55 °C, and 45 s of elongation at 72 °C; and a final extension at 72 °C for 10 min. For the ITS1-1F region, the PCR program was as follows: samples were initially denatured at 98 °C for 1 min, followed by 30 cycles of denaturation at 98 °C for 10 s, primer annealing at 50 °C for 30 s, and extension at 72 °C for 30 s. A final extension step of 5 min at 72 °C was added to ensure complete amplification of the target region. The resulting PCR products were extracted from a 2% agarose gel, further purified using the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) and quantified using QuantiFluor™-ST (Promega, Madison, WI, USA).Purified amplicons were pooled in equimolar amounts and paired-end sequenced (2 × 300) on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) according to standard protocols. The analysis was conducted by following the “Atacama soil microbiome tutorial” of QIIME2 docs along with customized program scripts (https://docs.qiime2.org/2019.1/). Briefly, raw data FASTQ files were imported in the QIIME2 system using the qiime tools import program. Demultiplexed sequences from each sample were quality filtered, trimmed, denoised, and merged, and then the chimeric sequences were identified and removed using the QIIME2 DADA2 plugin to obtain the feature table of amplicon sequence variants (ASVs)24. Compared with traditional OTU that clusters at 97% similarity, ASV has higher accuracy, equivalent to 99% similarity clustering. The QIIME2 feature-classifier plugin was then used to align ASV sequences to the pretrained GREENGENES 13_8 99% database (trimmed to the V3-V4 region bound by the 338F/806R primer pair for bacteria) and UNITE database (for fungi) to generate the taxonomy table25. Any contaminating mitochondrial and chloroplast sequences were filtered using the QIIME2 feature-table plugin. Based on the sequence number of the lowest sample, perform the resampling to make the sequence number equal for each sample. Due to the random nature of sequencing, ASVs specific to each sample in this study were present. To reduce the uncertainty introduced by the sequencing process, we filtered out rare ASVs with less than 0.001% of the total sequence volume.Data analysisIn this study, the data of fungi and bacteria were integrated and analyzed, and all microbial diversity appearing in the text represent the sum of all fungi and bacteria. Species richness is equal to the number of taxa, which is equal to the total number of all bacterial and fungal ASVs. The vegan package in R 4.2.1 was used to calculate the species richness of each sample based on the ASV feature table26. Using flask volume instead of area, SAR fitting was performed using a semi-logarithmic model, and its significance was tested. The semi-logarithmic model is the function S = c + b*logA, where S is species richness, A is area (in this case, volume is used instead), and b and c are fit parameters27.The microcosmic system in this study is hermetically sealed, and all microorganisms originate from a single portion of well-mixed paocai soup (ie species pool). The speciation process in the 60-day experimental system should be negligible due to the short experimental period. The extinction rate of a microcosm system is equal to the number of ASVs lost in the microcosm system compared to the species pool divided by the total number of ASVs in the species pool. The extinction rate is the number of extinct ASVs in each system compared to the species pool. Pearson correlation analysis was performed with volume as the independent variable and extinction rate as the dependent variable to determine the correlation between volume and extinction rate at each time point. When microorganisms of a microcosmic system disappear entirely or cannot be detected, the microcosm is recorded as an annihilated microcosm. The annihilation rate at a time point is equal to the number of microcosms annihilated at that time, divided by the total number of microcosms. The difference between the extinction rate and annihilation rate defined in this paper is that the extinction rate is for ASVs within each sample, and the annihilation rate is for microcosmic system at each sampling time point. The two indicators jointly characterize the local extinction of microorganisms from different perspectives. Non-linear regression with a bell-shaped form was performed with time as an independent variable and pH and annihilation rate as dependent variables, and regression lines were plotted based on R 4.2.1.According to the taxonomy table, bacterial ASVs were divided into acid-producing and non-acid-producing categories, and their extinction rates were calculated separately. The agricola, ggplot2, vegan and ggpubr packages were used to draw alpha diversity box plots and perform the Wilcoxon rank sum test for differences between groups26,28,29,30. Non-metric multidimensional scaling (NMDS) analysis was performed with the vegan package based on Bray–Curtis dissimilarity. In addition, the potential Kyoto Encyclopedia of Genes and Genomes (KEGG) orthologue (KO) functional profiles of microbial communities were predicted with PICRUSt31. Resistance-related genes were screened using the gene function predictions. The relationship between the relative abundance of resistance-related genes and the volume of the microcosm was analysed by Pearson correlation, and a forest map was plotted to present the results. More

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