Aspergillus niger DSM 26641 was exposed to 60Co g-radiation to enhance the b-1,4-endoxylanase activity, restrict colony growth and improve robustness of pellets. The first promising mutant obtained after g-radiation of the fungal spores at 50-2000 Gy showed a restricted colony growth and an 82% enhancement in b-1,4-endoxylanase activity. The mutant was subjected to a second round of g-radiation at 1400 Gy generating a mutant with double the b-1,4-endoxylanase activity compared to the native strain. The selected final mutant, deposited as Aspergillus niger DSM 28712, showed a maximal saccharification activity of 26 U·ml-1 on xylan based broth, 48 U·ml-1 on lignocellulose hydrolysate and 375 U·ml-1 on lignocellulose hydrolysate supplemented with yeast extract and mineral salts.

As the microbrewery industry expands, disposal of brewery wastewater is becoming more of a concern, both for brewery operators and for local municipal wastewater authorities. Brewery wastewater is characterized as containing high strength organics and high variability in both organic and hydraulic loading. This high variability increased the challenge of treating brewery wastewater properly. Therefore, it is significant for optimizing the operation condition for the small-scale wastewater treatment system. This study conducted a batch study and a field study for optimizing a craft brewery on-site wastewater treatment system, which is equipped with two moving bed biofilm reactors (MBBR). In the batch study, a two-factor Box-Wilson central composite design (CCD) was adopted to find optimum biomethane production conditions for the digestion of brewery wastewater with a dairy manure inoculum. The effects of two major influencing factors of temperature (T) (25-49°C) and brewery wastewater concentration (BWC) (2-9 g VS/L) on biochemical methane potential (BMP) (CH₄ yield) and CH₄ maximum production rate (Rmax) were evaluated by applying response surface methodology (RSM). All of the trials presented a high organic removal efficiency with volatile solid (VS) 82-91%, soluble chemical oxygen demand (sCOD) 77-88%, and total chemical oxygen demand (tCOD) between 47% -76%. The experiment result suggested that the first-order kinetic rate constant and biogas content (methane percentage in the biogas) can be affected by the temperature. The mesophilic regime had the highest average rate constant, and the psychrophilic regime rate constant was significantly lower than the mesophilic and thermophile regime. The conditions in the thermophile range present a high variability for the first-order rate constant. The methane ratio in the biogas increased and stabilized by the operation time. Mesophilic and thermophilic regimes obtained a stabile biogas content around 25 days, and the psychrophilic regime spent extra time to stabilized. At the end of the anaerobic digestion, the psychrophilic, mesophilic, and thermophilic regimes had an average methane percentage of 47%,65%, and 67% respectively. Optimum BMP and Rmax were achieved under conditions of 49 °C and BWC of 5g VS/L. Correspondingly, the BMP and Rmax were 141.40 mL CH₄/g VS added and 36.5 mL CH₄/ day, respectively. However, by pursuing stability the preferable operational condition T=35℃ and BWC=5 g/L is recommended, at this condition methane yield is 110.07 CH₄/g VS added and maximum methane daily production is 28.06 CH₄/ day, which is similar to the maximum result. In field study, an on-site brewery wastewater treatment system equipped with two MBBR reactors was evaluated from October 12th, 2018 to February 10th, 2020 in Beau`s All-Natural Brewing Company, Vankleek Hill, Ontario, Canada. The aim of the study was to characterize the wastewater production (flow and organic loading rate), evaluate the operating conditions and performance of the MBBR system, and recommend improvements. Discharge from the brewery is highly variable for both organic and hydraulic loading with flow balancing recommended. The MBBR full-scale reactors operated at relatively stable conditions at a surface area loading rate (SALR) of less than 25 g/m2.d and dissolved oxygen (DO) greater than 2mg/L. Kinetic rate constants for suspended growth and attached growth biomass in the reactors were found to be similar at 0.0764-0.0908 h-1, however, a much larger attached growth mass in the reactors suggests that only a fraction of the attached growth biofilm is active. Effluent recycle was shown to be effective at controlling filamentous bacteria (type-0041) sludge bulking, reducing suspended solid concentration, and sCOD concentration.

This project combines nonwoven technology, biological technology and food waste management and seeks for this feasibility to use fungal microfiber (FM) as a binder for lyocell pro-duction and the characterizations for possible applications. Rhizopus delemar was cultivated apple pomace in liquid-state fermentation to obtain mycelia biomass. The biomass was later blended in a kitchen blender for one minute. The blended FM was later mixed with 6 mm lyocell fibre at different FM dry weight percentage and water to make nonwoven webs by wet-laid method. The feasibility of using fungal microfiber as a binder for lyocell nonwovens was confirmed in this study. It is not possible to make nonwoven webs using lyocell short fibre without any binder applied. With 5%_FM, the tensile strength of lyocell nonwoven webs reached 0.0989 MPa. A clear increasing tensile strength was recorded as the increasing of FM weight per-centage and resulted a highest tensile strength at 9.38 MPa when applying 60%_FM. The re-sult of water contact angle proved that the increasing FM could decrease the hydrophobicity of nonwoven samples. Abrasion test showed that FM could improve the abrasion resistance of the lyocell nonwoven samples. Porosity test showed that lyocell nonwoven samples with a higher FM ended up with smaller mean flow pore size diameter (MFP) that nonwoven samples with 65%_FM has an average MFP at 7.26 m m. The SEM images reviled that FM bonded nonwoven webs had a fibrous structure, which is due to binding effect of fungal microfiber on lyocell short fibers. These characterizations have demonstrated the mechanism of using fungal microfiber as a binder for lyocell nonwovens in this project. In this thesis project, FM bonded lyocell nonwoven webs showed a great potential on the application of nonwoven applications such as interior materials or filtration materials.

In this work Rhizopus Delemar was grown on an apple pomace medium, a waste product from the juice industry. The apple pomace was pre-treated with the enzyme invertase to hydrolyse the sucrose available in the waste to glucose and fructose, which are digestible by the fungus. Combination of invertase pre-treatment and yeast extract supplementation, resulted in highest biomass growth which was 4.3 ± 0.5 g/l biomass. The fungal cell wall was separated from fungal biomass using an alkali treatment. A hydrogel was formed from the cell wall material and used for spinning of filaments using dry gel spinning. The average dry weight percentage of the gel was 11.6 ± 1.3 %. The gel was spun through a needle to a collecting rotating surface to make filaments. The filaments were easy to spin and to collect continuous fibres. The spun filaments had a rubber-like texture. All the tested filaments had an ultimate tensile strength approximately 2-3 MPa and 10 – 12% elongation at break. The conclusion of this work is that it is possible to produce fibers from fungi grown in apple waste and that it is possible to improve fungal growth using invertase and yeast extract. The tensile strength of the filaments needs further improvement to compete with other materials used in woven fabrics.

Hammarby Sjöstadsverk is a pilot plant operated by IVL Swedish Environmental Research Institute and Royal Institute of Technology (KTH) which treats municipal wastewater. Different processes are used of which one is a Sequential Batch Reactor (SBR) with activated sludge process. The activated sludge process is an effective biological municipal wastewater treatment process. Foaming and floating sludge is a significant problem for municipal wastewater treatment plants globally, that appears from time to time and a full understanding of the process causing this phenomenon is still not available. The problem can have multiple causes such as imbalance of microorganisms, nutrients, toxicity, pH, DO, temperature, denitrification, nitrification etc. The scope of this thesis project is to find out which microorganism’s cause foaming and bulking and how to control it. For this investigation, the Hammarby Sjöstadsverk pilot wastewater treatment plant in Stockholm was utilized. The laboratory facilities at KTH were used during thesis work. Laboratory methods were used to cultivate and isolate filamentous organisms from foam, sludge and inlet wastewater samples to investigate by microscopy and see which microorganisms exist in the activated sludge process were performed. General cultivation media for bacteria and fungi were used for culturing microorganisms. Isolation of some filamentous forms was based on colony morphology on an agar plate and microscopy. Microscopic observation of foam and isolates were performed, comparing cell morphology with literature studies. The sludge showed very few types of protozoa. The isolated and cultivated samples were fungi, yeast-like fungi and bacteria. The organisms are most likely the reason for foaming and bulking. Other reasons were also investigated by comparing retrieved data from IVL with literature studies; the process levels such as pH, DO, temperature and nutrients proved to be low or varying. The reason to why fungi and yeast-like microorganisms habitate in the basin depend on a low pH level average of 6.7. A method for effective settling is to withstand a cutting-pump to the container. This was tested during this project and resulted in increased levels of floating sludge during a period of time. Hence this is not a recommended treatment solution. Suggested treatment methods for further studies are to introduce protozoa to treat foaming and bulking, or by raising the pH and DO. This has not been covered within this project. / Hammarby Sjöstadsverk är en testanläggning som drivs av IVL Svenska Miljöinstitutet och KTH som består av ett avloppsreningsverk i pilotskala. Verket behandlar kommunalt avloppsvatten i en Sekvenserad Batch Reaktor (SBR) med aktiv slam processen. Den aktiva slamprocessen är en effektiv biologisk reningsmetod av avloppsvatten. Skumning och flytslam är ett stort problem för avloppsreningsverk globalt sett. Orsaken till dessa problem kan vara många, som t.ex. obalans av mikroorganismer, näringsämnen, toxicitet, pH, löst syre, temperatur, denitrifikation, nitrifikation osv. Målet med projektet är att ta reda på vilka organismer orsakar skumning och flytslam hos Hammarby Sjöstadsverk i Stockholm. Vad som orsakar dessa samt vilka kontrollmetoder som fungerar mot skumning och flytslam. Under projektet användes laboratoriet på KTH campus för att kultivera och isolera filamentösa organismer för att undersöka dessa under mikroskop och även se vilka mikroorganismer faktiskt existerar i skummet och flytslammet. Ympning av bakterier och fungi användes med hjälp av media. Isolering av vissa filamentösa organismer baserades på dess morfologi på agar plattor. Mikroskopiska observationer av skummet och isoleringar utvärderades och jämfördes med relevant litteratur om cellmorfologi. Resultaten av isolaten var fungi, jäst-liknande fungi och bakterier. Organismerna är mest sannolikt anledningen till skumning och flytslam. Andra anledningar så som datajämförelser med litteraturstudier visade att processens parametrar så som pH, löst syre, temperatur och näringsämnen visades vara låga eller varierande. Anledningen till att exempelvis fungi och jäst-liknande organismer trivdes berodde på låga pH nivåer som i snitt var 6.7. En föreslagen metod för att gynna sedimentering är att inte använda en skärpump till bassängen. Detta testades under projektets gång och resulterade under en period i ökade nivåer av flytslam. Därmed är denna metod inte att föredra. Ett förslag till en biologisk metod introduceras med att tillföra protozoer för att behandla skumning och flytslam, eller att höja pH och löst syre i bassängen. Detta har dock inte undersökts inom ramen för detta projekt.

Nitrogen, when present in excess, can cause eutrophication in waterways, which may result in hypoxia and the desertion or death of aquatic life. As nitrogen continues to pollute our water, wastewater discharge limits are becoming more stringent with effluent limits based on preserving receiving waters. This project took place at the Hampton Roads Sanitation District's, Chesapeake-Elizabeth Wastewater Treatment Plant; a High-Rate Activated Sludge (HRAS) plant with no primary clarifiers operating at an SRT of 1.5" 2 days without biological nitrogen removal (BNR). BNR is considered more cost-effective than comparable chemical and physical processes, but it requires considerable resources to meet increasingly strict discharge limits. As these limits decrease, the resource requirement increases, making them no longer cost-effective. By 2021 HRSD anticipates the plant will be included in a bubble permit, resulting in a total nitrogen (TN) effluent target of approximately 5-8 mg/L. Conventional BNR plants remove carbon and nitrogen simultaneously, which requires both increased volume (capital costs) and aeration energy demand (operating costs). As an alternative, HRSD is pilot testing an A/B process; a two-sludge system comprised of a carbon removal stage followed by a nitrogen removal stage. The very high rate, low dissolved oxygen (DO) A-stage could reduce the organic load, allowing the B-stage to perform BNR within the existing reactor volume and eliminating the need for primary clarifiers. However, improper control of the carbon removal system can lead to carbon and alkalinity deficiencies, which results in poor nitrogen removal. This is mediated by employing a short-cut nitrogen removal technology. A novel aeration strategy based on set-points for reactor ammonia, nitrite and nitrate concentrations with the aim of maintaining equal effluent ammonia and nitrate + nitrite (NOx) concentrations was successfully employed. The goal was to inhibit nitrite-oxidizing bacteria (NOB) so the nitrification process stopped at nitrite. This helps promote an effluent with equal parts ammonia and nitrite, which is amenable to anammox polishing to achieve low effluent nitrogen concentrations. NOB suppression has been successfully applied in sidestream anaerobic digestion waste streams because NOB out-selection is favored in warm, nitrogen-rich conditions. However, the cold, dilute conditions of continuous mainstream processes are not favorable to NOB out-selection. The mechanisms employed to achieve sidestream NOB out-selection are not reasonable for mainstream applications. This study employed operational and process control strategies to aggressively out-select NOB based on optimizing the chemical oxygen demand (COD) input, imposing transient anoxia, aggressive solids retention time (SRT) operation approaching ammonia oxidizing bacteria (AOB) washout, and a dissolved oxygen concentration (DO) of 1.5 mg O2/L during aeration. This pilot-scale study demonstrated that when run aggressively, the proposed online aeration control is able to out-select NOB in mainstream conditions and provide relatively high nitrogen removal without supplemental carbon and alkalinity at a low hydraulic retention time (HRT). Successful full-scale implementation would promote improved water quality that is economically sustainable. The ability of two different process configurations (full intermittent aeration and Modified Ludzak-Ettinger [MLE]) to achieve high nitrite accumulation and nitrogen removal efficiencies in four equal volume tanks in series followed by a cone-bottom clarifier in a pilot scale biological nitrogen removal (BNR) process (V=0.61 m3) was evaluated. All four biological reactors were equipped with a variable speed mixer, a 17.7 cm membrane disc diffuser, and a Hach LDO probe. Aeration capacity in all four tanks allowed the system to be operated with or without a defined anoxic zone. Both processes utilized a novel aeration strategy based on set-points for reactor ammonia, nitrite and nitrate concentrations with the aim of maintaining equal effluent ammonia and NOx concentrations. The B-stage had a variable HRT (2-7 hours) and a variable influent flow rate. When operating in the MLE configuration, an internal mixed liquor recycle (IMLR) line returned nitrified mixed liquor from the last aerobic reactor to the anoxic reactor using a peristaltic pump at a rate between 200-450% of the influent flow. When IMLR was used the first tank was not aerated. RAS from the clarifier was returned to the anoxic zone at 100% of the influent flow. SRT was controlled by wasting solids from the last aerobic tank. The wasting was automated to maintain desired SRT. The nitrite accumulation ratio (NAR), NO2- -N/(NO2- -N+ NO3- -N), was best under full intermittent aeration, achieving 0.43+0.10 at a 3 hour HRT and influent carbon to ammonia ratio (COD/NH4+-N) of 7.9+1.4. As an MLE, the NAR decreased with increasing internal mixed liquor return (IMLR); at IMLR of 200%, 325% and 450%, the NAR was 0.20+0.04, 0.17+0 and 0.14+0.03, respectively. The MLE did, however, improve the overall TIN removal efficiency compared to operation where all reactors were intermittently aerated. The TIN removal efficiency was best under MLE operation, increasing as the IMLR and influent COD/NH4+-N increased. When the IMLR was 200%, 325% and 450%, the TIN removal efficiencies were 76.4+4.0%, 80.2+0% and 86.3+5.0%, respectively, which corresponded to an influent COD/NH4+-N and HRT of 9.2+0.8 and 4 hr, 9.8+0.4 and 6 hr, and 10.3+1.2 and 6 hr, respectively. In addition to process operation, key issues of filamentous bulking were assessed. Concrete solutions to this continual issue are not available as the unique features of each plants influent and process dynamics prohibit the formulation of a universal solution. Filaments observed throughout this study included Type 0041, Type 0675, Type 0803, Nocardia, Thiothrix I and Thiothrix II. Type 0041 and Type 067 were observed throughout the study and are typical of BNR systems; they arguably do not contribute to settling issues. Type 0803 filaments are linked to low F/M, high SRT systems. It was present at the start of the experiment and then no longer detected. Nocardia made a brief appearance on day 72 causing temporary foaming issues. This was fixed by vacuuming the surface of the clarifier daily and may be attributed to the high surface area to volume ratio present in pilot-scale systems. Thiothrix I and Thiothrix II were observed after day 93, however, never as the dominant species. Thiothrix related bulking was observed in the A-stage (Miller et al, 2012), which was attributed to high sulfide and organic acids in the influent raw wastewater during high temperature periods and carryover of sulfide and Thiothrix from the over-sized A-stage clarifier. The goals of this evaluation were to identify favorable parameters of common filaments and establish their impacts on the system. Typically an SVI of 150 mL/g indicates good settling. Overall the study experienced good settling (128.3+36.3 mL/g), indicating that operating under different influent substrate concentrations and process configurations did not result in poor settling. / Master of Science

Banana peels account for about 61 million tons of waste each year globally. The aim of this project was to investigate the possibility of using banana peels as a substrate to cultivate edible filamentous fungi. The peels were subjected to physical and thermal pretreatments while variables such as changes in the medium pH, biomass concentration, fungal strain dependence, and protein content of the fungal biomass were analyzed. The experiments were carried out in three phases. The purpose of phase I was to identify which of the four fungal strains among Neurospora intermedia, Aspergillus oryzae, Rhizopus oryzae, and Rhizopus oligosporus could grow in a medium containing ball-milled banana peel powder (BPP) only. In phase II, the best performing strains from phase I in terms of biomass concentration, i.e., A. oryzae and R. oryzae, were cultivated using banana peel broth (BPB) obtained from thermal pretreatment of BPP. During this phase, the impact of medium supplementation with yeast extract was also assessed. The biomass yield for A. oryzae and R. oryzae 2.9 g/L and 1.6 g/L, respectively, yeast supplementation compared to 2.7 g/L and 0.7 g/L, respectively, without supplementation. In phase III, the experiments performed in phase II without yeast extract supplementation were scaled up, after which protein analysis was performed. A crude protein content of 8.82% was determined for A. oryzae, while in R. oryzae, a higher value of 21.1% was obtained. The protein content from both fungal strains was much higher than that present in the BPP, which was 4.8 g/L. The results showed the potential of using banana peel as a substrate to produce edible fungal biomass with higher protein content and thus has potential applications as animal feed or human food. Further studies are needed to optimize the process in order to raise the fungal biomass yield as well as increase the protein content of the biomass. In addition, comprehensive characterization of the fungal biomass would reveal other important components, such as the amino acid profile.

The circular economy is about rethinking the definition of waste into resource. Tempeh boiling water is cheap and would otherwise be washed into the river and pollute the water which would affect the environment badly. Tempeh boiling water is going to be used as substrate to produce mycoprotein with the fungi Rhizopus oligosporus. This study is about making a mycoprotein nugget and evaluate it with a sensory evaluation and to evaluate the protein content in the nugget and chemical oxygen demand of the boiling water. The sensory evaluation will have 65 panellists to assess the liking of the nugget with two control samples. The purpose of this project was to evaluate the potential of tempeh boiling water for the circular economy as substrate. To produce high mycoprotein nugget that will be accepted by the community. Assess the protein content in the mycoprotein nugget and assess the carbon used by the fungi with chemical oxygen demand analyse. The target group for evaluating the fungal nugget was students studying at Gadjah Mada University, Yogyakarta, Indonesia. The project was done in multiple following stages: Finding best formula of mycoprotein, mycoprotein production, sensory evaluation, and protein analyse with Kjeldahl method and COD analyse of the boiling water.The result of this study is that the mycoprotein nugget were not liked nor disliked with the average score of 3,9 out of 7. The overall characteristics (appearance, colour, texture, and taste) were 4,0 out of 7. The COD before and after fermentation were 6,6 g/L. The most COD were removed by pre-treatment of the boiling water from 172 to 121 g/L.The protein content of the mycoprotein nugget were 23,8%. The social aspect to produce healthy foods to a low cost at the same time improve water quality by removing foods for toxic microorganisms.

No description available.

"2003". / Thesis (PhD)--Macquarie University, Division of Environmental & Life Sciences, Department of Biological Sciences, 2004. / Bibliography: leaves 136-160. / Introduction: The Antarctic environment; Antarctic inhabitants; Microfungi; Identification of microfungi; Physiological factors affecting Antactic microfungi; Flow cytometry and microfungi; Hydrolytic enzymes of industrial interest; Isolation of genes from microfungi; Aims of this study -- Materials and methods: Fungal strains and cultivation conditions; Molecular identification of fungal isolates; Fungal physiology; Hydrolase activity of secreted proteins; Gene cloning and expression -- Results and discussion: Microfungal identification; Physiological factors affecting Antarctic microfungi; Activity in microfungi when grown on solid media; Characterisation of hemicellulases from selected Antarctic microfungi; Cloning of an Antarctic Penicillium allii lipase gene and its expression in Trichoderma reesei -- Conclusions and future prospects. / The Antarctic occupies that region of the planet that falls below the 60th parallel of South latitude. Although it has been frequented by adventurers, journeyman scientists and tourists for the past 100 years, the Continent has remained virtually unoccupied. The intense cold, the absence of human occupation and the limited range of local higher animal species have combined to create the impression that the Continent is virtually devoid of life. -- Although the microbiota of the Antarctic has attracted some small level of attention in the past, the examination of filamentous microfungi has been largely overlooked and fallen to a small group of dedicated investigators. In this study it will be shown that far from being an insignificant component of the Antarctic network, microfungi represent a potentially large and so far untapped bioresource. -- From just 11 bryophyte samples collected at four sites in the Ross Sea/Dry Valleys region of Southern Antarctica, some 30 microfungal isolates were recovered. Using molecular techniques, the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA (nrDNA) was sequenced to reveal no less than nine unique microfungal species. For only two of these species did the ITS sequence data produce a 100% match with records held on the public databases. This investigation also highlighted the problems inherent in the traditional morphological identification system which are now being perpetuated in the molecular database records. -- A set of seven notionally identified isolates obtained from ornithogenic soil samples gathered in the Windmill Islands in Eastern Antarctica (offshore from the Australian Antarctic Division's Casey Station) were also subjected to molecular identification based on ITS sequence data. Each of the seven isolates was identified as a unique species; six were cosmopolitan in nature and the one remaining bore very little resemblance at the molecular level to any of the recorded species although it was provided with an epithet commonly used in the identification of Antarctic microfungal species. -- To evaluate their potential as a bioresource, samples of Antarctic microfungi were examined to determine if the same physiological factors common to mesophilic species also applied to their Antarctic analogues. It is known that when placed under stress, trehalose can act as a protectant against cold (cryoprotection) and dehydration in mesophilic yeasts and fungi. The level of trehalose produced by the Antarctic isolates and their mesophilic analogues when subjected to stress was compared. A similar comparison was made for the production of glycerol which is well established as a compatible solute providing protection to mesophilic species against osmotic stress. Only in the case of trehalose production by an Antarctic Embellisia was there any indication that either of these two compounds could play a significant role in providing protection to the Antarctic fungi against the rigours of their environment, which leaves open to question what in fact does. -- In the course of investigating the means by which Antarctic microfungi guard against the damage which can ensue when subjected to oxidative stress, flow cytometry was introduced as an investigatory tool. It was established that there is a window of opportunity during which flow cytometry can be used to undertake a detailed analysis of the early stages of fungal growth from germination through hyphal development. -- Of major significance in determining the potential of Antarctic microfungi as a resource is their ability to produce new and novel enzymes and proteins. The microfungal isolates were screened for hydrolytic activity on solid media containing indicative substrates and proved to be a fruitful source of enzymes active over a range of temperatures. A detailed characterisation of two hemicellulases, β-mannanase and xylanase, secreted into a liquid medium by a subset of the Antarctic fungi and a high producing mesophilic reference strain permitted direct comparisons to be made. It was shown that the maximum hemicellulase activity of the Antarctic strains occurred at least 10°C and as much as 30°C lower than that of the reference strain and that mannanase activity for two of the Antarctic isolates exceeded 40% of their maximum at 0°C. These assay results highlight the potential of Antarctic microfungi to yield novel cold-active enzymes. -- As a final measure of the capacity of the Antarctic to yield novel enzymes from its microfungal stock, a lipase gene was selected as a target for isolation and expression in a heterologous fungal host. Using PCR techniques, the gene of interest was isolated from an Antarctic isolate of Penicillium allii, transformed into the mesophilic production host Trichoderma reesei and the active protein successfully produced in the growth medium. The recombinant lipase was assayed and found to exhibit novel characteristics consistent with a cold-adapted enzyme. / Mode of access: World Wide Web. / 186 leaves ill