A review of volatile fatty acids production from organic wastes: Intensification techniques and separation methods.

High value-added products from organic waste fermentation have garnered increasing concern in modern society. VFAs are short-chain fatty acids, produced as intermediate products during the anaerobic fermentation of organic matter. VFAs can serve as an essential organic carbon source to produce substitutable fuels, microbial fats and oils, and synthetic biodegradable plastics et al. Extracting VFAs from the fermentation broths is a challenging task as the composition of suspensions is rather complex. In this paper, a comprehensive review of methods for VFAs production, extraction and separation are provided. Firstly, the methods to enhance VFAs production and significant operating parameters are briefly reviewed. Secondly, the evaluation and detailed discussion of various VFAs extraction and separation technologies, including membrane separation, complex extraction, and adsorption methods, are presented, highlighting their specific advantages and limitations. Finally, the challenges encountered by different separation technologies and novel approaches to enhance process performance are highlighted, providing theoretical guidance for recycling VFAs from organic wastes efficiently.

Synergetic effect on fouling alleviating of membrane distillation in urine resource recovery by thermally activated peroxydisulfate pretreatment.

Given that the spontaneous precipitation of minerals caused by urea hydrolysis and abundant organic compounds, membrane fouling became a major obstacle for urine recovery by membrane distillation (MD). Herein, this study developed a combined system (TAP-MD) by integrating thermally activated peroxydisulfate (TAP) and MD process to inhibit membrane fouling and improve separation efficiency. Based on the TAP-MD system, the separation performance was improved significantly, improving nutrient recovery efficiency and quality of reclaimed water. More than 80% of water could be recovered from urine, and about 94.13% of total ammonia nitrogen (TAN), 99.02% of total nitrogen (TN), 100% of total phosphate (TP), and 100% of K+ were rejected. The mechanism for alleviating urine-induced fouling was systematically and intensively studied. With TAP pretreatment, the TAN concentration of pretreated urine was kept at a low level steadily and the pH was at neutral or weakly acidic. Hence, inorganic scaling represented by carbonate and phosphate precipitates were significantly inhibited by creating unfavorable solvent environment for crystallization with TAP pretreatment. Additionally, aromatic proteins were found as the main organic foulants. According to the secondary structure of protein, the proteins were degraded by the cleavage of peptide bonds by TAP pretreatment. Meanwhile, the hydrophilicity of protein increased, which reduced the hydrophobic interaction of protein and membrane surface and thus alleviated protein-induced membrane fouling. This study revealed the inorganic and organic foulants in urine that caused membrane fouling and demonstrated the mechanism of membrane fouling alleviation by TAP-MD system. The experimental results will be instrumental in better understanding the mechanisms of membrane fouling induced by urine and optimize MD process for resource recovery from urine.

Dissemination of antibiotic resistance genes through fecal sewage treatment facilities to the ecosystem in rural area.

Infection of antibiotic-resistant pathogens mostly occurs in rural areas. In this paper, the dissemination of antibiotic resistance genes (ARGs) through fecal sewage treatment facilities to the ecosystem in a typical rural area is investigated. Household three-chamber septic tanks (TCs), household biogas digesters (BDs), wastewater treatment plants (WWTPs), vegetable plots, water ponds, etc. Are taken into account. The relative abundance of ARGs in fecal sewage can be reduced by BDs and WWTPs by 80% and 60%, respectively. While TCs show no reduction ability for ARGs. Fast expectation-maximization microbial source tracking (FEAST) analysis revealed that TCs and BDs contribute a considerable percentage (15-22%) of ARGs to the surface water bodies (water ponds) in the rural area. Most ARGs tend to precipitate in the sediments of water bodies and stop moving downstream. Meanwhile, the immigration of microorganisms is more active than that of ARGs. The results provide scientific basic data for the management of fecal sewage and the controlling of ARGs in rural areas.

Non-negligible greenhouse gas emissions from non-sewered sanitation systems: A meta-analysis.

Current methods for estimating sanitation emissions underestimate the significance of methane emissions from non-sewered sanitation systems (NSSS), which are prevalent in many countries. NSSS play a vital role in the safe management of fecal sludge, accounting for approximately half of all existing sanitation provisions. We analyzed the distribution of global NSSS and used IPCC accounting methods to estimate the total methane emissions profiles from these systems. Then, we examined the literature to establish the level of uncertainty associated with this accounting estimate. The global methane emissions from NSSS in 2020 was estimated to as 377 (22-1003) Mt CO2e/year or 4.7% (0.3%-12.5%) of global anthropogenic methane emissions, which are comparable to the greenhouse gas (GHG) emissions from wastewater treatment plants. NSSS is the major option for open defecation and is expected to increase by 55 Mt CO2e/year after complete open defecation free. It is time to acknowledge the GHG emissions from the NSSS as a non-negligible source.

Operating status of public toilets in the Hutong neighborhoods of Beijing: An empirical study.

The provision of sanitation services for fast-growing urban populations is one of the world's urgent challenges. Hutong neighborhoods in Beijing, capital of China, cannot be rebuilt due to the protection of historical heritage, while residents still need to keep the habit of defecating in public toilets. One hundred public toilets with non-sewered sanitation in the Hutong neighborhoods of Beijing were visited to investigate the actual operating status in response to the "toilet revolution" campaign. The fault tree approach was used to identify the barriers toward a decent and environment-friendly public toilet and evaluate potential risks from the malfunction of various components. Four subsystems are defined and elaborated to calculate the fault possibility. These subsystems are environment- and user-friendly, regarded as ancillary facilities, and used for fecal sludge (FS) management. Statistical analysis of targeted cases indicated that fault probabilities of environmental considerations, user-friendly considerations, ancillary facilities, FS management are calculated as 0.79, 0.96, 0.96, and 0, respectively. The subsystems were weighted using a Delphi method concept. Results showed that the well operation ratio of Beijing Hutong public toilets is only 32%, and the sanitation service value chain can be further optimized. This study also provides references for other countries, which are dedicated to promoting urban sanitation and public health.

Using system dynamics to assess the complexity of rural toilet retrofitting: Case study in eastern China.

Rural toilet retrofitting (RTR) is a complex, dynamic system that is affected by many factors and the positive/negative feedback relationships between subsystems and variables. Traditional technologies and management methods face challenges in fundamentally describing and solving problems in RTR. To bridge this gap, this study utilizes system dynamics and causal loop diagrams to explain such problems based on data collected from the stakeholders of the RTR in Jiaozhou from 2018 to 2019. Specifically, this study examines the RTR system from the perspectives of household users, wastewater treatment plants, local governments, grassroots promoters, operation and maintenance personnel, toilet supplier and construction teams, and fecal sludge end users. The factors and processes involved in RTR are identified, and the feedback and relationships among its major stakeholders are established. Results show that the motivation of farmers to engage in RTR is a key variable that affects their final decisions regarding retrofitting and maintaining toilet functions. Meanwhile, the important variables related to the feedback and relationships among the major stakeholders of RTR are mostly focused on policies, subsidies, technology, satisfaction, and cooperation. A scientific analysis method and the updated RTR plan for toilet revolution are then formulated to promote the implementation of RTR in developing countries.

CRISPR-Local: a local single-guide RNA (sgRNA) design tool for non-reference plant genomes.

SUMMARY: CRISPR-Local is a high-throughput local tool for designing single-guide RNAs (sgRNAs) in plants and other organisms that factors in genetic variation and is optimized to generate genome-wide sgRNAs. CRISPR-Local outperforms other sgRNA design tools in the following respects: (i) designing sgRNAs suitable for non-reference varieties; (ii) screening for sgRNAs that are capable of simultaneously targeting multiple genes; (iii) saving computational resources by avoiding repeated calculations from multiple submissions and (iv) running offline, with both command-line and graphical user interface modes and the ability to export multiple formats for further batch analysis or visualization. We have applied CRISPR-Local to 71 public plant genomes, using both CRISPR/Cas9 and CRISPR/cpf1 systems. AVAILABILITY AND IMPLEMENTATION: CRISPR-Local can be freely downloaded from http://crispr.hzau.edu.cn/CRISPR-Local/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Investigation on methane yield of wheat husk anaerobic digestion and its enhancement effect by liquid digestate pretreatment.

There are few studies on the use of wheat husk for anaerobic digestion until now. In this study, the BMP (biochemical methane potential) of wheat husk without pretreatment and with liquid digestate (LD) soaking pretreatment was investigated. It was demonstrated that wheat husk could be potentially used as a single feeding substrate for anaerobic digestion with a methane yield of 68 mL/g VS and this yield could be enhanced to a maximum of 130.0 mL/g VS using LD soaking pretreatment, which increased the methane production by 91%. Although the particle size (8, 5, 2 mm) and soaking pretreatment time (1, 3, 5 d) affected the biogas production, the influence of particle size was negligible when the pretreatment time was 5 days. Further analysis of the microbial community showed that the LD soaking pretreatment did not change the predominant microbial communities in the digestion. Therefore, LD pretreatment was effective in enhancing the performance of wheat husk anaerobic digestion.

Toilet revolution in China.

The wide-spread prevalence of unimproved sanitation technologies has been a major cause of concern for the environment and public health, and China is no exception to this. Towards the sanitation issue, toilet revolution has become a buzzword in China recently. This paper elaborates the backgrounds, connotations, and actions of the toilet revolution in China. The toilet revolution aims to create sanitation infrastructure and public services that work for everyone and that turn waste into value. Opportunities for implementing the toilet revolution include: fulfilling Millennium Development Goals and new Sustainable Development Goals; government support at all levels for popularizing sanitary toilet; environmental protection to alleviate wastewater pollution; resource recovery from human waste and disease prevention for health and wellbeing improvement. Meanwhile, the challenges faced are: insufficient funding and policy support, regional imbalance and lagging approval processes, weak sanitary awareness and low acceptance of new toilets, lack of R&D and service system. The toilet revolution requires a concerted effort from many governmental departments. It needs to address not only technology implementation, but also social acceptance, economic affordability, maintenance issues and, increasingly, gender considerations. Aligned with the ecological sanitation principles, it calls for understanding issues across the entire sanitation service chain. Public-private partnership is also recommended to absorb private capital to make up the lack of funds, as well as arouse the enthusiasm of the public.

Catalytic reduction of nitrate in secondary effluent of wastewater treatment plants by Fe(0) and Pd-Cu/γ-Al2O3.

Total nitrogen, in which NO3(-) is dominant in the effluent of most wastewater treatment plants, cannot meet the requirements of the Chinese wastewater discharge standard (<15 mg/L), making nitrate (NO3(-)) elimination attract considerable attention. In this study, reductant iron (Fe(0)) and γ-Al2O3 supported palladium-copper bimetallic catalysts (Pd-Cu/γ-Al2O3) were innovatively used for the chemical catalytic reduction of nitrate in wastewater. A series of specific operational conditions (such as mass ratio of Pd:Cu, catalyst amounts, reaction time and pH of solution) were optimized for nitrate reduction in the artificial solution, and then the selected optimal conditions were further applied for investigating the nitrate elimination of secondary effluent of a wastewater treatment plant in Beijing, China. Results indicated that a better catalytic performance (74% of nitrate removal and 62% of N2 selectivity) could be obtained under the optimal condition: 5 g/L Fe(0), 3:1 mass ratio (Pd:Cu), 4 g/L catalyst, 2 h reaction time and pH 5.1. It is noteworthy to point out that nitrogen gas (N2) predominated in the byproducts without another system to treat ammonium and nitrite. Therefore, the chemical catalytic reduction combining Fe(0) with Pd-Cu/γ-Al2O3 could be regarded as a better alternative for nitrate removal in wastewater treatment.

Low-temperature methanation of fermentation gas with Ni-based catalysts in a multicomponent system.

A large amount of greenhouse gases, such as carbon dioxide and methane, are released during the production process of bioethanol and biogas. Converting CO2 into methane is a promising way of capturing CO2 and generating high-value gas. At present, CO2 methanation technology is still in the early stage. It requires high temperature (300-400 â„ƒ) and pressure (> 1 MPa), leading to high cost and energy consumption. In this study, a new catalyst, Ni-Fe/Al-Ti, was developed. Compared with the activity of the common Ni/Al2O3 catalyst, that of the new catalyst was increased by 1/3, and its activation temperature was reduced by 100℃. The selectivity of methane was increased to 99%. In the experiment using simulated fermentation gas, the catalyst showed good catalytic activity and durability at a low temperature and atmospheric pressure. Based on the characterization of catalysts and the study of reaction mechanisms, this article innovatively proposed a Ni-Fe/Al-Ti quaternary catalytic system. Catalytic process was realized through the synergism of Al-Ti composite support and Ni-Fe promotion. The oxygen vacancies on the surface of the composite carrier and the higher activity metals and alloys promoted by Fe accelerate the capture and reduction of CO2. Compared with the existing catalysts, the new Ni-Fe/Al-Ti catalyst can significantly improve the methanation efficiency and has great practical application potential.

What makes residents participate in the rural toilet revolution?

•Motivation of Chinese rural residents to participate in RTR identified•Motivational model of Chinese rural resident participating in RTR is constructed•Model includes intrinsic and extrinsic factors, and impacts of RTR policy•RTR policy may influence rural residents' final decision to participate RTR.

New insights into membrane fouling during direct membrane filtration of municipal wastewater and fouling control with mechanical strategies.

Direct membrane filtration (DMF) technology achieves energy self-sufficiency through carbon recovery and utilization from municipal wastewater. To control its severe membrane fouling and improve DMF technology, targeted research on fouling behaviour and mechanisms is essential. In this study, a DMF reactor equipped with a flat-sheet ceramic membrane was conducted under three scenarios: without control, with intermittent aeration, and with periodic backwash. This system achieved efficient carbon concentration with chemical oxygen demand below 50 mg/L in permeate. Membrane fouling was dominated by intermediate blocking and cake filtration. And reversible external resistance accounted for over 85 % of total resistance. Predominant membrane foulants were free proteins, whose deposition underlies the attachment of cells and biopolymers. Backwash decreased the fouling rate and increased fouling layer porosity by indiscriminately detaching foulants from the membrane surface. While aeration enhanced the back transport of large particles and microbial activity, causing a relatively thin and dense fouling layer containing more microorganisms and ß-d-glucopyranose polysaccharides, which implies a higher biofouling potential during long-term operation. In addition, aeration combined with backwash enhanced fouling control fivefold over either one alone. Therefore, simultaneous operation of backwash and other mechanical methods that can provide shear without stimulating aerobic microbial activity is a preferred strategy for minimizing membrane fouling during DMF of municipal wastewater.

Transport and deposition of solid phosphorus-based mineral particles in urine diversion systems.

The deposition of solid phosphorus-based mineral particles is a common problem in urine diversion systems, which occurs in transport systems, particularly in horizontal pipelines. In this work, particle deposition behaviour in turbulent flow in a 3D horizontal pipe was simulated by using the Euler-Lagrange method. The effects of particle diameter, particle density, particle shape factor and fluid flow velocity on particle deposition behaviour were investigated. The results showed that the deposition rate increased by 9.92%,6.88% and 6.88% with increasing particle diameter (10-90 µm), particle density (1400 kg/m3-2300 kg/m3), and particle shape factor (0.2-1), respectively. For particles with larger diameters (>90 µm) or larger density (>2300 kg/m3), the deposition rate of these particles was almost reached 100%. It was found that gravitational sedimentation was the dominant deposition mechanism in low fluid flow velocity range (0.1-0.5 m/s). As fluid flow velocity increased (>0.5 m/s), turbulent fluctuation became the dominant factor that affected particle motion behaviour, whereas the effect of gravitational sedimentation on particle deposition behaviour declined significantly, and the increase in fluid flow velocity no longer significantly affects deposition rate. It was found that the deposition rate decreased by 29.13% as the fluid flow velocity was increased from 0.1 m/s to 0.5 m/s, while the corresponding deposition rate only decreased by 14.24% when the fluid flow velocity was increased from 0.5 m/s to 2 m/s. The optimal flow velocity was found to range between 0.75 and 1.25 m/s, which may mitigate the deposition of mineral solids in urine diversion systems.

Machine-learning-aided application of high-gravity technology to enhance ammonia recovery of fresh waste leachate.

Stripping is widely applied for the removal of ammonia from fresh waste leachate. However, the development of air stripping technology is restricted by the requirements for large-scale equipment and long operation periods. This paper describes a high-gravity technology that improves ammonia stripping from actual fresh waste leachate and a machine learning approach that predicts the stripping performance under different operational parameters. The high-gravity field is implemented in a co-current-flow rotating packed bed in multi-stage cycle series mode. The eXtreme Gradient Boosting algorithm is applied to the experimental data to predict the liquid volumetric mass transfer coefficient (KLa) and removal efficiency (η) for various rotation speeds, numbers of stripping stages, gas flow rates, and liquid flow rates. Ammonia stripping under a high-gravity field achieves η = 82.73% and KLa = 5.551 × 10-4 s-1 at a pH value of 10 and ambient temperature. The results suggest that the eXtreme Gradient Boosting model provides good accuracy and predictive performance, with R2 values of 0.9923 and 0.9783 for KLa and η, respectively. The machine learning models developed in this study are combined with experimental results to provide more comprehensive information on rotating packed bed operations and more accurate predictions of KLa and η. The information mining behind the model is an important reference for the rational design of high-gravity-field-coupled ammonia stripping projects.

Potential of food waste hydrolysate as an alternative carbon source for microbial oil synthesis.

Volatile fatty acids (VFAs) have great potential as cheap raw materials in microbial oil synthesis and reducing the cost of substrates is essential for the development of microbial oil biosynthesis. In this study, the food waste hydrolysate and synthetic VFAs media were both used as substrate to synthesis microbial oil. The optimal short-chain VFAs ratio for microbial oil synthesis is 20:5:5 and increasing the proportion of propionic acid is the key to obtaining odd fatty acids. The hydrolysate obtained from food waste under the total solid condition of 2:1 and pH 5 is the most suitable medium for microbial oil synthesis. The biological products obtained from food waste hydrolysate were comparable to synthetic VFAs media, obtaining a 34.02% of lipid content. Results prove that food waste hydrolysate has great potential as the available feedstock for microbial oil synthesis and a promising application value in food waste recycling.

From Newtonian to non-Newtonian fluid: Insight into the impact of rheological characteristics on mineral deposition in urine collection and transportation.

The deposition of phosphorus-based mineral solids in urine diversion systems has been one of the main challenges for the large-scale practical applications of urine source separation. Accurate rheological characterization of urine slurry is of high importance for its practical flow performance. The rheological data of urine slurry was obtained using a narrow gap rotating rheometer. Based on current pipe flow theories and the obtained rheological data of urine slurry, the transition velocity was determined. The impacts of solid concentration and temperature on the rheological behavior of urine slurry were investigated in this study. Urine slurry behaved as a Newtonian fluid at low solid concentration. By contrast, urine slurry changed from Newtonian to non-Newtonian fluid with the increase in solid concentration, demonstrating a shearing thinning behavior and yielding stress fluid. The impact of temperature on the apparent viscosity of the urine slurry was described using an Arrhenius-type function. Moreover, the impact of solid concentration and temperature on the transition velocity was quantified, which indicated that the non-Newtonian behavior of the urine slurry in the compression settling region has a significant impact on the pipe flow behavior, leading to the formation of a compressed layer on the bottom of the pipe. The targeting understanding of transition velocity is particularly useful for the practical design and optimization of urine piping system, especially on how to mitigate pipe blockages. Based on the evaluation of different piping systems, this work proposed several potential urine collection and transportation modes.

Characterization of microplastics in the septic tank via laser direct infrared spectroscopy.

Microplastics (MPs) are emerging pollutants that have been widely detected in the atmosphere, hydrosphere, lithosphere, and biosphere. Such wide spread of MPs indicates that the effective control in different environmental sectors is in an urgent need, and the first step in meeting this need is to identify the occurrence of MPs in the relevant environment. However, research on MPs in septic tanks has not been reported so far. This study investigated the distribution characteristics of MPs in septic tanks with a size detection limit of as low as 20 µm detected by laser direct infrared spectroscopy. Results showed that the number of MPs in the septic tank was reached 2803 (1489-4816) particles/g dry sludge, and the amount detected in the sediments was one order of magnitude higher than that in the scums. A total of 36 types of MPs were found in the septic tank, and 26 types were found in both sediments and scums, but the type in the scums was 21% higher than that in the sediments. The size was mostly 20-100 µm, accounting for 86.3% and 91.2% in the sediments and scums, respectively. Four shapes of MPs were detected in the septic tank, namely, fiber, bead, granule, and fragment. Our study revealed that septic tanks are both sinks and sources of MPs, which are reflected in the fact that MPs are not only large in number but also abundant in types. Thus, significant attention should be paid to septic tank-based microplastic pollution, which may lead to environmental and health risks without proper control and management.

Stabilization of source-separated urine by heat-activated peroxydisulfate.

Source-separated urine is an attractive fertilizer due to its high nutrient content, but the rapidly hydrolysis of urea leads to ammonia volatilization and other environmental problems. Urine stabilization, which meanly means preventing enzymatic urea hydrolysis, receives increasing attention. Accordingly, this study developed a technique to stabilize fresh urine by heat-activated peroxydisulfate (PDS). The effect of three crucial parameters, including temperature (55, 62.5, and 70 °C), heat-activated time (1, 2, and 3 h), and PDS concentration (10, 30, and 50 mM) that affect the activation of PDS in urine stabilization were investigated. Nitrogen in fresh urine treated with 50 mM PDS at 62.5 °C for 3 h existed mainly in the form of urea for more than 22 days at 25 °C. Moreover, the stabilized urine could remain stable and resist second contamination by continuous and slow pH decrease due to PDS decomposition during storage. Less than 8% of nitrogen loss in stabilized urine was detected during the experiment. The investigation of nitrogen transformation pathway demonstrated that urea was decomposed into NH4+ by heat-activated PDS and further oxidized to NO2- and NO3-. The nitrogen loss during treatment occurred via heat-driven ammonia volatilization and N2 emission produced by synproportionation of NO2- and NH4+ under acid and thermal conditions. Overall, this study investigated an efficient approach of urine stabilization to improve urine utilization in terms of nutrient recovery.

Enhanced lipid production by Yarrowia lipolytica cultured with synthetic and waste-derived high-content volatile fatty acids under alkaline conditions.

BACKGROUND: Volatile fatty acids (VFAs) can be effective and promising alternate carbon sources for microbial lipid production by a few oleaginous yeasts. However, the severe inhibitory effect of high-content (> 10 g/L) VFAs on these yeasts has impeded the production of high lipid yields and their large-scale application. Slightly acidic conditions have been commonly adopted because they have been considered favorable to oleaginous yeast cultivation. However, the acidic pH environment further aggravates this inhibition because VFAs appear largely in an undissociated form under this condition. Alkaline conditions likely alleviate the severe inhibition of high-content VFAs by significantly increasing the dissociation degree of VFAs. This hypothesis should be verified through a systematic research. RESULTS: The combined effects of high acetic acid concentrations and alkaline conditions on VFA utilization, cell growth, and lipid accumulation of Yarrowia lipolytica were systematically investigated through batch cultures of Y. lipolytica by using high concentrations (30-110 g/L) of acetic acid as a carbon source at an initial pH ranging from 6 to 10. An initial pH of 8 was determined as optimal. The highest biomass and lipid production (37.14 and 10.11 g/L) were obtained with 70 g/L acetic acid, whereas cultures with > 70 g/L acetic acid had decreased biomass and lipid yield due to excessive anion accumulation. Feasibilities on high-content propionic acid, butyric acid, and mixed VFAs were compared and evaluated. Results indicated that Y X/S and Y L/S of cultures on butyric acid (0.570, 0.144) were comparable with those on acetic acid (0.578, 0.160) under alkaline conditions. The performance on propionic acid was much inferior to that on other acids. Mixed VFAs were more beneficial to fast adaptation and lipid production than single types of VFA. Furthermore, cultures on food waste (FW) and fruit and vegetable waste (FVW) fermentate were carried out and lipid production was effectively improved under this alkaline condition. The highest biomass and lipid production on FW fermentate reached 14.65 g/L (Y X/S: 0.414) and 3.20 g/L (Y L/S: 0.091) with a lipid content of 21.86%, respectively. By comparison, the highest biomass and lipid production on FVW fermentate were 11.84 g/L (Y X/S: 0.534) and 3.08 g/L (Y L/S: 0.139), respectively, with a lipid content of 26.02%. CONCLUSIONS: This study assumed and verified that alkaline conditions (optimal pH 8) could effectively alleviate the lethal effect of high-content VFA on Y. lipolytica and significantly improve biomass and lipid production. These results could provide a new cultivation strategy to achieve simple utilizations of high-content VFAs and increase lipid production. Feasibilities on FW and FVW-derived VFAs were evaluated, and meaningful information was provided for practical applications.