Endocellulase Production by <Emphasis Type="Italic">Cotylidia pannosa</Emphasis> and its Application in Saccharification of Wheat Bran to Bioethanol

For efficient bioconversion of lignocellulosic materials to bioethanol, the study screened 19 white-rot fungal strains for their endocellulolytic activity and saccharification potential. Preliminary qualitative and quantitative screening revealed Cotylidia pannosa to be the most efficient endocellulase producing fungal strain when compared to the standard strain of Trichoderma reesei MTCC 164. Ensuing initial screening, the production of endocellulase was further optimized using submerged fermentation to recognize process parameters such as temperature, time, agitation pH, and supplementation of salts in media required for achieving maximum production of endocellulase. The strain C. pannosa produced the maximum amount of endocellulase (8.48 U/mL) under submerged fermentation with wheat bran (2%) supplemented yeast extract peptone dextrose (YEPD) medium after an incubation time of 56 h at 30 °C and pH 5.0 at an agitation rate of 120 rpm with a saccharification value of 50.5%. The fermentation of wheat bran hydrolysate with Saccharomyces cerevisiae MTCC 174 produced 4.12 g/L of bioethanol after 56 h of incubation at 30 °C. The results obtained from the present investigation establish the potential of white-rot fungus C. pannosa for hydrolysis and saccharification of wheat bran to yield fermentable sugars for their subsequent conversion to bioethanol, suggesting its application in efficient bioprocessing of lignocellulosic wastes.     

Dual production of amylase and δ-endotoxin by Bacillus thuringiensis subsp. kurstaki during biphasic fermentation

This study examined the efficacy of a Bacillus thuringiensis (Bt) strain in producing amylase (EC 3.2.1.1) as a by-product without affecting its unique ability for producing δ-endotoxin, thus to establish a cultivation strategy for the dual production and recovery of both δ-endotoxin and amylase from the fermented medium with an industrial perspective. LB medium was individually supplemented (5 to 100%, wt/vol) with flour from six naturally available starchy stored foods (banana, Bengal gram, jack seed, potato, taro or tapioca); after initial fermentation (12 h), the supernatant in the medium obtained by centrifugation (1000 g, 10 min) was used for harvesting amylase and the resultant pellet was further incubated aseptically for the production of endospores and δ-endotoxin by solid-state fermentation. Maximum crude amylase activity (867 U/gram dry substrate, 12 h) was observed in potato flour-supplemented medium (10% wt/vol, 12 h), while the activity in LB control was only 4.36 U/mL. SDS-PAGE profile of the crude (supernatant), as well as partially purified (40–60% (NH₄)₂SO₄ fractionation) amylase showed that its apparent molecular mass was 51 kDa, which was further confirmed by native PAGE. The harvest of industrially significant extracellular amylase (probably α-amylase) produced as a byproduct during early growth phase would boost the economics of the Bt-based bio-industry engaged in δ-endotoxin production.     

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from <Emphasis Type="Italic">Aspergillus Fumigatus</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Chitosanase (CSN) from Aspergillus fumigatus has good thermal stability, wide pH range duration, and effective hydrolysis for chitosan. Inhere, CSN was successfully expressed in Escherichia coli followed by extracellular secretion under the guidance of an N-terminal signal peptide PelB, which effectively prompted its secretion out of E. coli cells. To facilitate its later purification, N-terminal or C-terminal 6xHis epitope tag was added to the PelB-CSN protein complex. Our results indicated that PelB-CSN without 6xHis-tag (PelB-CSN) or with N-terminal 6xHis-tag (PelB-CSN-N) can both be effectively secreted into the medium, while CSN with 6xHis-tag anchored at C-terminus was expressed as inclusion bodies. Process optimization strategies were further developed to improve the secretion efficiency of recombinant PelB-CSN-N in E. coli. Under the induction of 10 g/L lactose in shake-flask culture, the extracellular activity of CSN reached 6015 U/mL at 25 °C in TB medium containing 1 % glycine. Moreover, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 5-L fermenter, increasing the extracellular CSN activity to 14,000 U/mL in 2-day fermentation with the optimal addition of lactose and glycine.     

Biosynthesis of tannin acyl hydrolase from tannin-rich forest residue under different fermentation conditions

Caesalpinia digyna, a tannin-rich forest residue, was used as substrate for production of tannase and gallic acid. Media engineering was carried out under solid-state fermentation, submerged fermentation and modified solid state fermentation conditions for optimum synthesis of tannase and gallic acid (based on 58% tannin content in the raw material). Tannase vis-à-vis gallic acid recovery under modified solid-state fermentation condition was maximum. Conversions of tannin to gallic acid under solid-state fermentation, submerged fermentation and modified solid-state fermentation conditions were 30.5%, 27.5% and 90.9%, respectively. Journal of Industrial Microbiology & Biotechnology (2000) 25, 29–38. Received 02 November 1999/ Accepted in revised form 12 February 2000     

Formation of cellulases by co-culturing ofTrichoderma reesei andAspergillus niger on cellulosic waste

A cellulase system possessing high hydrolytic and -glucosidase activity was obtained by co-culturingTrichoderma reesei andAspergillus niger by a new approach using semi-solid fermentation of lignocellulosic materials. Various types of pretreatments were used for making the cellulose easily accessible to enzymatic attack. The optimal water content for maximum activity of the mixed fermentation was investigated. A more concentrated enzyme preparation could be obtained by semi-solid state fermentation than by conventional submerged fermentation.     

Discoloration of the azo dye Congo Red by manganese-dependent peroxidase from <Emphasis Type="Italic">Pleurotus sajor caju</Emphasis>

This paper describes the production of ligninolytic enzymes by the white-rot fungus Pleurotus sajor caju under solid-state fermentation conditions using a cost-effective medium consisting of agro-industrial wastes. From the different agro-industrial wastes tested (i.e. orange, banana, mango and cantaloupe peels), banana peels led to the highest manganese-dependent peroxidase (MnP) activity (6.3 U/mL on the 10 day). MnP from banana peel cultures was purified and applied to the discoloration of the azo dye Congo Red (CR). The optimum temperature, pH and enzyme concentration for maximum discoloration (i.e. 95% in 1 h) were found to be 35°C, 4.0, and 1.4 U/mL, respectively. In addition, the phytotoxicity (with respect to Sorghum vulgare and Phaseolus radiatus seeds) of CR was considerably reduced after the treatment of plant material with MnP produced by P. sajor caju. The products obtained after discoloration of CR were characterized using GC/MS as 8-amino naphthol 3-sulfonic acid, 3-hydroperoxy 8-nitrosonaphthol, p-p'-dihydroxybiphenyl. Therefore, this approach holds promise for the production and application of MnP from P. sajor caju on a larger scale.     

Effect of media components and morphology of <Emphasis Type="Italic">Bacillus natto</Emphasis> on menaquinone-7 synthesis in submerged fermentation

Vitamin K₂ (menaquinone or MK) plays an important role in blood clotting, cardiovascular disease, and anti-osteoporosis. A novel bacterial strain was isolated and identified as Bacillus natto based on 16SrDNA sequencing and LC-MS analysis. The objective of this study was to improve the extraction efficiency and productivity of MK-7 from B. natto. Acid-heating method efficiently disrupted B. natto cells for MK-7 extraction. Bacillus natto had a wide range of pH (5.0 ~ 9.0) for optimal growth. Its MK-7 yield was increased when rotation speed was increased to 200 rpm. The highest MK-7 yield was obtained when glycerol and soy peptone were used in the growth media. Batch fermentation was subsequently tested in 5 L bioreactor, which gave a high productivity of MK-7 (at 0.60 mg/L/h). A positive correlation between MK-7 yield and sporulation ratio was also found. This study provides valuable information on the extraction and production of menaquinone-7 from B. natto under submerged fermentation condition.     

Glucose oxidase ofAspergillus niger

The authors studied the effect of the various components of synthetic nutrient medium on glucose oxidase production in submerged cultivation ofAspergillus niger. It was found that the optimal glucose concentration was 3.5–6%. The only suitable source of nitrogen was nitrate nitrogen. If the medium contained ammonia nitrogen, glucose oxidase was not formed. The addition of citric acid to the medium very effectively stimulated theQ _{O 2} of the mycelium. Calcium added in the form of calcium nitrate had the same effect. A decrease in the Mg²⁺ ion concentration raised the activity of the enzyme, while inhibiting growth of the mycelium. If the initial pH was less than 4, glucose oxidase production was inhibited and did not start until the pH rose in the course of fermentation. Differences in the initial pH affected not only production of the enzyme, but also the formation of acids and the morphological appearance of the submerged mycelium. On the basis of the findings the synthetic medium for submerged cultivation ofAspergillus niger was modified, resulting in a 50–100% increase in glucose oxidase production as compared with the original medium.     

Using drug-loaded pH-responsive poly(4-vinylpyridine) microspheres as a new strategy for intelligent controlling of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> contamination in bioethanol fermentation

Bioethanol fermentation is usually contaminated by bacteria, especially lactic acid bacteria (LAB), thereby leading to decrease of bioethanol yield and serious economic losses. Nisin is safer for controlling of bacterial contamination than antibiotics that are widely applied in industry. Moreover, in LAB contaminative bioethanol fermentation system, consistently decreased pH value provides opportunity to realize pH value responsive material-based release of anti-bacteria substances for intelligent and persistent controlling of bacterial contamination. In this study, nisin was embedded into pH-sensitive poly(4-vinylpyridine) (P4VP) microspheres synthesized by suspension polymerization to realize intelligent controlling of Lactobacillus plantarum contamination in bioethanol fermentation. Chloramphenicol with the highest antimicrobial activity and excellent stability was chosen as the model drug to be embedded into P4VP microspheres to test the drug release behavior. The drug release curve of chloramphenicol-loaded P4VP microspheres showed sustained and pH-responsive release properties. The diameters of the microspheres ranged from 40 to 100 µm. The encapsulation efficiency of nisin into P4VP microspheres was 47.67% and the drug-loading capacity of nisin was 2.5%. Nisin-loaded P4VP microspheres were added into the simulated contaminative fermentation system, and successfully reversed the decline of bioethanol yield secondary to L. plantarum contamination. The results in this study indicated that L. plantarum contamination in bioethanol fermentation could be effectively controlled by nisin-loaded P4VP microspheres.     

The production and uses of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> as a microbial insecticide

Among invertebrate fungal pathogens, Beauveria bassiana has assumed a key role in management of numerous arthropod agricultural, veterinary and forestry pests. Beauveria is typically deployed in one or more inundative applications of large numbers of aerial conidia in dry or liquid formulations, in a chemical paradigm. Mass production is mainly practiced by solid-state fermentation to yield hydrophobic aerial conidia, which remain the principal active ingredient of mycoinsecticides. More robust and cost-effective fermentation and formulation downstream platforms are imperative for its overall commercialization by industry. Hence, where economics allow, submerged liquid fermentation provides alternative method to produce effective and stable propagules that can be easily formulated as dry stable preparations. Formulation also continues to be a bottleneck in the development of stable and effective commercial Beauveria-mycoinsecticides in many countries, although good commercial formulations do exist. Future research on improving fermentation and formulation technologies coupled with the selection of multi-stress tolerant and virulent strains is needed to catalyze the widespread acceptance and usefulness of this fungus as a cost-effective mycoinsecticide. The role of Beauveria as one tool among many in integrated pest management, rather than a stand-alone management approach, needs to be better developed across the range of crop systems. Here, we provide an overview of mass-production and formulation strategies, updated list of registered commercial products, major biocontrol programs and ecological aspects affecting the use of Beauveria as a mycoinsecticide.     

Selection for novel,acid-tolerant <Emphasis Type="Italic">Desulfovibrio</Emphasis> spp. from a closed Transbaikal mine site in a temporal pH-gradient bioreactor

Almost all the known isolates of acidophilic or acid-tolerant sulphate-reducing bacteria (SRB) belong to the spore-forming genus Desulfosporosinus in the Firmicutes. The objective of this study was to isolate acidophilic/acid-tolerant members of the genus Desulfovibrio belonging to deltaproteobacterial SRB. The sample material originated from microbial mat biomass submerged in mine water and was enriched for sulphate reducers by cultivation in anaerobic medium with lactate as an electron donor. A stirred tank bioreactor with the same medium composition was inoculated with the sulphidogenic enrichment. The bioreactor was operated with a temporal pH gradient, changing daily, from an initial pH of 7.3 to a final pH of 3.7. Among the bacteria in the bioreactor culture, Desulfovibrio was the only SRB group retrieved from the bioreactor consortium as observed by 16S rRNA-targeted denaturing gradient gel electrophoresis. Moderately acidophilic/acid-tolerant isolates belonged to Desulfovibrio aerotolerans-Desulfovibrio carbinophilus-Desulfovibrio magneticus and Desulfovibrio idahonensis-Desulfovibrio mexicanus clades within the genus Desulfovibrio. A moderately acidophilic strain, Desulfovibrio sp. VK (pH optimum 5.7) and acid-tolerant Desulfovibrio sp. ED (pH optimum 6.6) dominated in the bioreactor consortium at different time points and were isolated in pure culture.     

Evaluation of antioxidant and antiproliferative metabolites of <Emphasis Type="Italic">Penicillium flavigenum</Emphasis> isolated from hypersaline environment: Tuz (Salt) Lake by Xcelligence technology

The aim of the study is the determination of antioxidant and antiproliferative activities of fungal isolates’ metabolites belonging to Penicillium flavigenum isolated from Lake Tuz, Turkey. Evaluation of the antioxidant activity, the total phenolic content and antiproliferative effect were evaluated with DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging assay, Folin-ciocalteu method, Xcelligence real-time cell analysis. The total phenolic content of these isolates were found 62–82 mg/GAE. Ethyl acetate extracts from identified isolates, P. flavigenum, showed cytotoxic effects on A549, MCF7, Caco-2 cell lines. IC₅₀ values of P. flavigenum ethyl acetate extracts were found 96.7 μg/mL for A549, 33.4 μg/mL for MCF7, 43.4 μg/mL for Caco-2 and 97.3 μg/mL for 3T3. Phenolic acids in the extracts from P. flavigenum were identified with HPLC and GC-MS. Penicillium flavigenum is a new report for Turkey. According to these findings, fungi-related secondary metabolites are very important sources in terms of antioxidant and antiproliferative effects.     

Promoting acid resistance and nisin yield of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> F44 by genetically increasing D-Asp amidation level inside cell wall

Nisin fermentation by Lactococcus lactis requires a low pH to maintain a relatively higher nisin activity. However, the acidic environment will result in cell arrest, and eventually decrease the relative nisin production. Hence, constructing an acid-resistant L. lactis is crucial for nisin harvest in acidic nisin fermentation. In this paper, the first discovery of the relationship between D-Asp amidation-associated gene (asnH) and acid resistance was reported. Overexpression of asnH in L. lactis F44 (F44A) resulted in a sevenfold increase in survival capacity during acid shift (pH 3) and enhanced nisin desorption capacity compared to F44 (wild type), which subsequently contributed to higher nisin production, reaching 5346 IU/mL, 57.0% more than that of F44 in the fed-batch fermentation. Furthermore, the engineered F44A showed a moderate increase in D-Asp amidation level (from 82 to 92%) compared to F44. The concomitant decrease of the negative charge inside the cell wall was detected by a newly developed method based on the nisin adsorption amount onto cell surface. Meanwhile, peptidoglycan cross-linkage increased from 36.8% (F44) to 41.9% (F44A), and intracellular pH can be better maintained by blocking extracellular H⁺ due to the maintenance of peptidoglycan integrity, which probably resulted from the action of inhibiting hydrolases activity. The inference was further supported by the acmC-overexpression strain F44C, which was characterized by uncontrolled peptidoglycan hydrolase activity. Our results provided a novel strategy for enhancing nisin yield through cell wall remodeling, which contributed to both continuous nisin synthesis and less nisin adsorption in acidic fermentation (dual enhancement).     

Statistical Optimization of Culture Variables for Enhancing Agarase Production by <Emphasis Type="Italic">Dendryphiella arenaria</Emphasis> Utilizing <Emphasis Type="Italic">Palisada perforata</Emphasis> (Rhodophyta) and Enzymatic Saccharification of the Macroalgal Biomass

Agarase is a promising biocatalyst for several industrial applications. Agarase production was evaluated by the marine fungus Dendryphiella arenaria utilizing Palisada perforata as a basal substrate in semi-solid state fermentation. Seaweed biomass, glucose, and sucrose were the most significant parameters affecting agarase production, and their levels were further optimized using Box-Behnken design. The maximum agarase activity was 7.69 U/mL. Agarase showed a degree of thermostability with half-life of 99 min at 40 °C, and declining to 44.72 min at 80 °C. Thermodynamics suggested an important process of protein aggregation during thermal inactivation. Additionally, the enzymatic saccharification of the seaweed biomass using crude agarase was optimized with respect to biomass particle size, solid/liquid ratio, and enzyme loadings. The amount of biosugars obtained after optimization was 26.15 ± 1.43 mg/g. To the best of our knowledge, this is the first report on optimization of agarase in D. arenaria.     

Determination of mosquito Larvicidal potential of <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> Cry11Ba fusion protein through molecular docking

Mosquitoes spread deadly infections around the world. Since decades Bacillus thuringiensis (Bt) δ-endotoxins have been used successfully as a biopesticide for controlling mosquito larvae. However, over a few years, mosquito larvae have evolved tolerance against Bt δ-endotoxins, rendering them ineffective for mosquito control. Such a problem entails the development of improved toxins with enhanced toxicity, affinity towards a wide range of mosquito receptors and ability to overcome or delay the resistance buildup. In this study, using in silico tools, we aimed to design a fusion protein by fusing active region of Bt subsp. jegathesan Cry11Ba protein with Aedes aegypti TMOF (trypsin modulating oostatic factor). Using computational study, the fusion protein was validated and its mosquitocidal potential was determined through molecular docking against cadherin and aminopeptidase N midgut receptors of Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Molecular docking revealed that from Cry11Ba-TMOF fusion protein, domain II amino acids of Cry11Ba protein showed hydrogen bond interactions with cadherin and aminopeptidase N receptors of the targeted mosquitoes. These results conclude that Cry11Ba-TMOF fusion protein has a strong affinity for the receptors of Ae.aegypti, An.gambiae and Cx.quinquefasciatus. Thus the designed fusion protein can be used as a potent mosquitocidal agent for the control of targeted mosquitoes.     

Nicotine degradation enhancement by <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> ZCJ during aging process of tobacco leaves

Nicotine is a key harmful component of tobacco and cigarettes, and the development of low-nicotine cigarettes is of increasing importance in the market. The objectives of this study are to isolate native nicotine-degrading strains and evaluate their feasibility for nicotine reduction during the aging (or fermentation) of tobacco leaves. A novel nicotine-degrading strain was isolated and identified as Pseudomonas stutzeri ZCJ based on its 16S rDNA sequence and morphological-biochemical characteristics. In submerged cultures, P. stutzeri ZCJ could tolerate 4.5 g/L nicotine and completely degrade 1.5 g/L nicotine within 24 h at 37°C and pH 7.4. The addition of glucose (1 g/L) could improve nicotine degradation by P. stutzeri ZCJ in submerged cultures. After submerged culturing, the cell suspension of P. stutzeri ZCJ could be utilized to improve nicotine reduction in tobacco leaves during solid-state fermentation. The nicotine content of tobacco leaves decreased by as much as 32.24% after 7 days of solid-state fermentation by P. stutzeri ZCJ, suggesting the industrial application potential of the native strain to enhance nicotine degradation during the aging of tobacco leaves.     

苏云金芽孢杆菌δ—内毒素的杀虫机理及其增效途径

苏云金芽孢杆菌（Ｂａｃｉｌｕｓｔｈｕｒｉｎｇｉｅｎｓｉｓ,Ｂｔ）制剂是当前应用最广、最有效的微生物杀虫剂。Ｂｔ属于革兰氏阳性细菌,在形成芽孢的同时,产生伴孢晶体。伴孢晶体是Ｂｔ杀虫活性的主要来源,它可能由几种晶体蛋白即δ－内毒素组成。δ－内毒素的专一...     

Production and characterization of cellulase from <Emphasis Type="Italic">E. coli</Emphasis> EgRK2 recombinant based oil palm empty fruit bunch

Oil Palm Empty Fruit Bunch (OPEFB) is an abundant biomass resource in Indonesia, which contains 41.3 ~ 46.5% (w/w) of cellulose. This research examined the production of cellulase by the E. coli EgRK2 recombinant strain using an OPEFB substrate. The production of the enzyme was initially examined to identify optimum growth conditions, by observing the growth and activity of E. coli EgRK2 compared to its wild type. Our results showed that the optimum production time, pH and temperature of the recombinant growth and cellulase activity were achieved at 24 h, and at 7 and 40°C, respectively. Using these optimum conditions, the enzyme was produced, and experiments were carried out to examine the enzyme characteristics, produced from both strains, on hydrolysis of cellulose from OPEFB. Our results showed that the activity of the enzyme produced by the recombinant almost doubled compared to that of the wild type, although the optimum pH for both strains was pH 6. Higher activity was achieved by the recombinant compared to the wild type strain, and values were 1.905 and 1.366 U/mL, respectively. The optimum temperature for hydrolysis by cellulase occurred at 50°C for Bacillus sp. RK2, and 60°C for Bacillus sp. EgRK2. The Michaelis-Menten constant (Km) and maximum velocity (Vmax) for OPEFB degradation by E. coli EgRK2 were 0.26% and 1.750 μmol/mL/sec, which were significantly better values than those of the wild type. Control experiments for the degradation test using CMC also showed a better Vmax value for E. coli EgRK2 compared to the wild type, which is 2.543 and 1.605 μmol/mL/sec, respectively.     

Glucooligosaccharide production by <Emphasis Type="Italic">Leuconostoc mesenteroides</Emphasis> fermentation with efficient pH control,using a calcium hydroxide-sucrose solution

95.3% of the sucrose in a feed batch fermentation (300 g/L) was hydrolyzed by Leuconostoc mesenteroides subp. mesenteroides NRRL B-23188 glucansucrase. Further, the glucose of sucrose formed glucooligosaccharides (GOS) of degree of polymerization (DP) over 2, together with 91.6% of the maltose (200 g/L). Lime saccharate (lime sucrate) was used to control the pH during fermentation. The GOS products had DP between 2 and 7. When Streptococcus mutans mutansucrase (0.1 U/mL) reacted with 0.1% sucrose, addition of 0.1 ~ 10% GOS to the mutansucrase reaction digest resulted in a 56 ~ 90% reduction of mutan formation. GOS also reduced E. coli (72.2%) and Salmonella sp. (over 40.0%) growth, when 2.5% GOS was used as a single carbon source, compared to growth using glucose. The calculated glycemic index and glycemic load of GOS was 8 and 1, respectively, based on a 10 g carbohydrate serving. GOS was calculated to have 2.43 kcal/g. After a glucose tolerance test was performed using C57BL/6 mice, we found that mice treated with GOS showed a 59.4% lower increase in plasma glucose than those treated with maltose.     

Ecophysiological responses of native invasive woody <Emphasis Type="Italic">Juniperus virginiana</Emphasis> L. to resource availability and stand characteristics in the semiarid grasslands of the Nebraska Sandhills

Vegetation in grasslands is changing at an unprecedented rate. In the Nebraska Sandhills, this shift is attributed in part to encroachment of the woody species Juniperus virginiana. We investigated changes in resource availability and their feedback on seasonal trends in photosynthetic characteristics of J. virginiana trees scattered in open grasslands vs. a dense 57-year-old stand. Dense stand exhibited lower volumetric soil water content, NH₄ ⁺, NO₃ ^–, and δ¹³C, as well as foliage δ¹³C, δ¹⁵N, and N content, compared to grasslands. Water potential was higher in trees in grasslands compared to dense stand. J. virginiana in dense stand exhibited similar trends to trees in grasslands for net photosynthetic rate (P _N), stomatal conductance, transpiration, maximum photochemical efficiency of PSII, maximum carboxylation velocity, and maximum rate of electron transport. P _N peaked early summer and declined in the fall, with trees in open grasslands lagging behind those in dense stand. Plasticity of this species may place it at a competitive advantage in the Sandhills, further altering grasslands vegetation and ecosystem processes.