Bio-ethanol from water hyacinth biomass: An evaluation of enzymatic saccharification strategy

Biomass feedstock having less competition with food crops are desirable for bio-ethanol production and such resources may not be localized geographically. A distributed production strategy is therefore more suitable for feedstock like water hyacinth with a decentralized availability. In this study, we have demonstrated the suitability of this feedstock for production of fermentable sugars using cellulases produced on site. Testing of acid and alkali pretreatment methods indicated that alkali pretreatment was more efficient in making the sample susceptible to enzyme hydrolysis. Cellulase and β-glucosidase loading and the effect of surfactants were studied and optimized to improve saccharification. Redesigning of enzyme blends resulted in an improvement of saccharification from 57% to 71%. A crude trial on fermentation of the enzymatic hydrolysate using the common baker’s yeast Saccharomyces cerevisiae yielded an ethanol concentration of 4.4 g/L.     

Green synthesis of silver nanoparticles using cellulose extracted from an aquatic weed; water hyacinth

As part of the desire to save the environment through “green” chemistry practices, we herein report an environmentally benign synthesis of silver nanoparticles (Ag-NPs) using cellulose extracted from an environmentally problematic aquatic weed, water hyacinth (WH), as both reducing and capping agent in an aqueous medium. By varying the pH of the solution and reaction time, the temporal evolutions of the optical and morphological properties of the as-synthesised Ag-NPs were investigated. The as-synthesised cellulose capped silver nanoparticles (C–Ag-NPs) were characterised using Fourier transform infrared spectroscopy (FTIR), ultraviolet–visible spectroscopy (UV–vis), transmission electron microscopy (TEM) and high resolution transmission electron microscopy (HRTEM). The maximum surface plasmon resonance (SPR) peak decreased as the pH increased indicating that an increase in the pH of the solution favoured the formation of smaller particles. In addition, instantaneous change in the colour of the solution from colourless to brown within 5 min at pH 11 showed that the rate of reduction is faster at this pH compared to those at lower pH. The TEM micrographs showed that the materials are small, highly monodispersed and spherical in shape. The average particle mean diameters were calculated to be 5.69 ± 5.89 nm, 4.53 ± 1.36 nm and 2.68 ± 0.69 nm nm at pH 4, 8 and 11 respectively. The HRTEM confirmed the crystallinity of the material while the FTIR spectra confirmed the capping of the as-synthesised Ag-NPs by the cellulose. It has been shown therefore that based on this synthetic method, this aquatic plant can be used to the advantage of mankind.     

Nutrient limitation to the decomposition of water hyacinth ( Eichhornia crassipes)

The responses of decomposition to N and P supply were investigated in three leaf types of water hyacinth (Eichhornia crassipes (Mart.) Solms): dead green leaves collected from Donghu Lake; green, and brown leaves collected from outdoor tanks. The ratios of C:N, C:P, lignin:N and lignin:P were lowest in the green leaves collected from Donghu Lake, and highest in the brown leaves collected from outdoor tanks. Decomposition constant (k) of water hyacinth varied greatly, ranged from 0.006 to 0.099 d^–1. Leaf litters decayed most quickly within the initial two weeks during the experimental period, but decomposition rate decreased significantly in the following days. Decomposition and nutrient (N and P) release were fastest in the green leaves collected from Donghu Lake, intermediate in the green leaves collected from outdoor tanks, slowest in the brown leaves collected from outdoor tanks. Statistical analyses revealed that the effects of P-availability on decomposition rate and N, P release rate of the three litter types were significant, whereas the impacts of N-availability was insignificant (p > 0.05) except for the brown leaves collected from outdoor tanks. These results suggest that decomposition rate and nutrient content dynamics of water hyacinth differ with their growth habitats, and could partly be regulated by nutrient availability, especially by P-availability, in the environments.     

Biomass and photosynthetic productivity of water hyacinth (Eichhornia crassipes) as affected by nutrient supply and mirid (Eccritotarus catarinensis) biocontrol

The biological control of water hyacinth is affected by water nitrogen and phosphorus content and this was investigated experimentally at five levels of nutrient supply by measuring plant photosynthetic and growth responses, and mirid reproduction and herbivory of nutrient treated plants. Low nitrogen (2–0.2 mg L⁻¹) and phosphorus (0.2–0.01 mg L⁻¹) supply decreased hyacinth photosynthesis, growth and biomass accumulation relative to plants supplied 200 mg L⁻¹ N and 20 mg L⁻¹ P. This effect depended more on nitrogen supply than phosphorus supply. Chlorophyll fluorescence showed that the photosynthetic light reactions of low nutrient plants were affected and leaves had decreased chlorophyll content, density of functional photosystems II and dissipated a greater proportion of absorbed energy as heat. Gas exchange parameters showed reduced carboxylation efficiency, rates of RuBP regeneration and light saturated photosynthetic rates, but not quantum yields. Effects on photosynthesis translated into lower plant dry biomass. Mirid herbivory exacerbated the effects of low nutrients noted for chlorophyll fluorescence, gas exchange parameters and biomass accumulation, however, these effects were not always significant and there was no obvious correlation between the level of nutrients supplied and the effect of mirid herbivory. Low nutrient supply did, however, affect mirid performance reducing the number of adult insects, nymphs and herbivory intensity suggesting that in the long-term mirid populations would be significantly affected by water nutrient status.     

Genetic diversity of introduced populations of the water hyacinth biological control agent Eccritotarsus catarinensis (Hemiptera: Miridae)

Genetic bottlenecks can be deleterious to populations. In biological control, agent populations may be subject to severe bottlenecks during selection, importation and while in culture. The genetic variability of two collections of the water hyacinth biological control agent Eccritotarsus catarinensis Carvalho (Hemiptera: Miridae) was measured using Inter-simple Sequence Repeats (ISSR) and mtDNA cytochrome oxidase I (COI) sequences. The first collection (Brazilian) went through a bottleneck of a single gravid female, while the second collection (Peru) originated from 1000 individuals and has been maintained at a large size in culture. Two naturalised South African populations from the Brazilian collection were also sampled (Nseleni and Mbozambo). Polymorphism for ISSR was high in the Peruvian and two naturalised samples, but much less so in the Brazilian sample. The Peruvian population was shown to be highly differentiated from the Brazilian and its naturalised populations by high values of F_ST and Nei’s genetic distance, as well as in a Multidimensional Scaling (MDS) plot and an unrooted neighbour joining tree derived from Jaccard’s coefficient of similarity. In addition, sequencing of the COI region of the mitochondrial DNA revealed only two haplotypes, one Brazilian and one Peruvian, with a 5.2% sequence divergence, suggesting that recombination and not mutation is the cause of most variation in the ISSR regions. The results suggest that substantial genetic variation may be retained or recovered after a bottleneck. This may mitigate deleterious effects that are a concern for the fate of biological control agents after release.     

Abiotic and microbial decomposition of pre- and post-bloom leaves of water hyacinth (Eichhornia crassipes (mart.) Solms)

Rates of abiotic and microbial decomposition in pre- and post-bloom leaves of water hyacinth are determined under laboratory conditions. Decomposition in all types of hyacinth leaves is dominated by physical leaching in an initial phase of 4 days duration, and later by microbial processes. The largest part of physical leaching takes place within the first 4 h. Thereafter, the weight loss due to physical leaching declines exponentially. The weight loss by microbial decomposition is minimal in the initial phase but increases exponentially in the later phase. Pre-bloom leaves decompose significantly faster than post-bloom leaves, and post-bloom green leaves decompose faster than post-bloom brown leaves. The rate constants of abiotic decomposition are significantly higher in post-bloom leaves as compared with pre-bloom leaves, while microbial decomposition is significantly higher in pre-bloom leaves. After 30 days, the dry mass loss by abiotic and microbial decomposition is 15% and 55%, respectively, in pre-bloom leaves, 33% and 19% in post-bloom green leaves, and 24% and 6% in post-bloom brown leaves.     

Decomposition and mineralization of <Emphasis Type="Italic">Eichhornia crassipes</Emphasis> litter under aerobic conditions with and without bacteria

The water hyacinth (Eichhornia crassipes (Mart.) Solms.) plants in lakes and reservoirs have gained considerable attention in tropical and sub-tropical parts of the world due to its rapid growth. The amount of nutrients released from the dead plant materials is of particular interest. Thus, decomposition of water hyacinth plant parts under aerobic conditions was studied in the laboratory. Roots, petioles, and leaves of water hyacinth were enclosed separately in one litre polypropylene bottles which contained 500 ml of lake water. To study the influence of bacteria on the decomposition, antibiotics were added to half of the bottles. We observed that decomposition of leaves and petioles without antibiotics were relatively rapid through day 61, with almost 92.7 and 97.3% of the dry mass removed, respectively. Weight loss due to bacterial activities during 94 days decomposition was 22.6, 3.9, and 30.5% from leaf, petiole, and root litter. Decomposition of litter in lake water indicated that after 94 days 0.6, 0, and 0.6 g m^–2 of leaf, petiole, and root N was dissolved in leachate, while 23.1, 14.4, and 6.0 g m^–2 of leaf, petiole, and root N was either volatilized or remained as particulate organic N. Moreover, 0.2, 0, and 0.1 g m^–2 of leaf, petiole, and root P remained dissolved in the leachate, while 3.1, 3.4, and 1.1 g m^–2 of leaf, petiole, and root P was either precipitated or remained as particulate organic P. The carbon dynamics during the decomposition indicated that 7.4, 28.8, and 3.7 g m^–2 of leaf, petiole, and root C remained dissolved in the leachate after 94 days while 228.0, 197.6, and 107.4 g m^–2 of leaf, petiole, and root C was either diffused or remained as particulate organic C. These findings are useful for quantifying the nutrient cycles of very shallow lakes with water hyacinth under aerobic water environment. Further examination of the fate of the plant litter as it moves down in deep anaerobic water environment, is necessary to understand the leaching process better.     

Biogas production and saccharification of Salix pretreated at different steam explosion conditions

Different steam explosion conditions were applied to Salix chips and the effect of this pretreatment was evaluated by running both enzymatic hydrolysis and biogas tests. Total enzymatic release of glucose and xylose increased with pretreatment harshness, with maximum values being obtained after pretreatment for 10 min at 210 °C. Harsher pretreatment conditions did not increase glucose release, led to degradation of xylose and to formation of furfurals. Samples pretreated at 220 and 230 °C initially showed low production of biogas, probably because of inhibitors produced during the pretreatment, but the microbial community was able to adapt and showed high final biogas production. Interestingly, final biogas yields correlated well with sugar yields after enzymatic hydrolysis, suggesting that at least in some cases a 24 h enzymatic assay may be developed as a quick method to predict the effects of pretreatment of lignocellulosic biomass on biogas yields.     

Effect of physical pretreatment on dilute acid hydrolysis of water hyacinth (Eichhornia crassipes)

Effects of different physical pretreatments on water hyacinth for dilute acid hydrolysis process (121 ± 3 °C, 5% H₂SO₄, 60 min) were comparatively investigated. Untreated sample had produced 24.69 mg sugar/g dry matter. Steaming (121 ± 3 °C) and boiling (100 ± 3 °C) for 30 min had provided 35.9% and 52.4% higher sugar yield than untreated sample, respectively. The highest sugar yield (132.96 mg sugar/g dry matter) in ultrasonication was obtained at 20 min irradiation using 100% power. The highest sugar production (155.13 mg sugar/g dry matter) was obtained from pulverized samples. Hydrolysis time was reduced when using samples pretreated by drying, mechanical comminution and ultrasonication. In most methods, prolonging the pretreatment period was ineffective and led to sugar degradations. Morphology inspection and thermal analysis had provided evidences of structure disruption that led to higher sugar recovery in hydrolysis process.     

Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid

Enzymatic hydrolysis of pretreated lignocellulosic substrates has emerged as an interesting option to produce sugars that can be converted to liquid biofuels and other commodities using microbial biocatalysts. Lignocellulosic substrates are pretreated to make them more accessible to cellulolytic enzymes, but the pretreatment liquid partially inhibits subsequent enzymatic hydrolysis. The presence of pretreatment liquid from Norway spruce resulted in a 63% decrease in the enzymatic saccharification of Avicel compared to when the reaction was performed in a buffered aqueous solution. The addition of 15 mM of a reducing agent (hydrogen sulfite, dithionite, or dithiothreitol) to reaction mixtures with the pretreatment liquid resulted in up to 54% improvement of the saccharification efficiency. When the reducing agents were added to reaction mixtures without pretreatment liquid, there was a 13-39% decrease in saccharification efficiency. In the presence of pretreatment liquid, the addition of 15 mM dithionite to Avicel, α-cellulose or filter cake of pretreated spruce wood resulted in improvements between 25 and 33%. Positive effects (6-17%) of reducing agents were also observed in experiments with carboxymethyl cellulose and 2-hydroxyethyl cellulose. The approach to add reducing agents appears useful for facilitating the utilization of enzymes to convert cellulosic substrates in industrial processes.     

Enhanced saccharification of alkali-treated rice straw by cellulase from Trametes hirsuta and statistical optimization of hydrolysis conditions by RSM

A white rot fungus, identified as Trametes hirsuta based on morphological and phylogenetic analysis, was found to contain efficient cellulose degrading enzymes. The strain showed maximum endoglucanase (EG), cellobiohydrolase (CBH) and ß-glucosidase (BGL) activities of 55, 0.28 and 5.0 U/mg-protein, respectively. Rice straw was found to be a potentially good substrate for growth of T. hirsuta for cellulase production. Statistical experimental design was used to optimize hydrolysis parameters such as pH, temperature, and concentrations of substrates and enzymes to achieve the highest saccharification yield. Enzyme concentration was identified as the limiting factor for saccharification of rice straw. A maximum saccharification rate of 88% was obtained at an enzyme concentration of 37.5 FPU/g-substrate after optimization of the hydrolysis parameters. The results of a confirmation experiment under the optimum conditions agreed well with model predictions. T. hirsuta may be a good choice for the production of reducing sugars from cellulosic biomass.     

Cooperation of Aspergillus nidulans enzymes increases plant polysaccharide saccharification

Efficient polysaccharide degradation depends on interaction between enzymes acting on the main chain and the side chains. Previous studies demonstrated cooperation between several enzymes, but not all enzyme combinations have been explored. A better understanding of enzyme cooperation would enable the design of better enzyme mixtures, optimally profiting from synergistic effects. In this study, we analyzed the cooperation of several enzymes involved in the degradation of xylan, glucan, xyloglucan and crude plant biomass from Aspergillus nidulans by single and combined incubations with their polymeric substrate. Positive effects were observed between most enzymes, although not always to the same extent. Moreover, the tailor made cocktails formulated in this study resulted in efficient release of glucose from plant biomass. This study also serves as an example for the complex cooperation that occurs between enzymes in plant biomass saccharification and how expression in easily‐accessible hosts, such as Pichia pastoris, can help in revealing these effects.     

The biosynthesis of 8-phenylphenalenones from Eichhornia crassipes involves a putative aryl migration step

The first compounds of the novel 8-phenylphenalenone type were isolated from roots and leaves of Eichhornia crassipes (Pontederiaceae). The structures were elucidated by spectrometric methods including 1D and 2D NMR analysis. The incorporation of two molecules of [1-13C]phenylalanine provides experimental evidence for the biosynthesis of the 8-phenylphenalenones. A biosynthetic pathway via diarylheptanoid and 9-phenylphenalenone intermediates, including a 1,2-aryl migration, is proposed.     

Phytoremediation of mercury- and methyl mercury-contaminated sediments by water hyacinth (Eichhornia crassipes)

Phytoremediation has the potential for implementation at mercury- (Hg) and methylHg (MeHg)-contaminated sites. Water hyacinths (Eichhornia crassipes) were investigated for their ability to assimilate Hg and MeHg into plant biomass, in both aquatic and sediment-associated forms, over a 68-day hydroponic study. The suitability of E. crassipes to assimilate both Hg and MeHg was evaluated under differing phosphate (PO4) concentrations, light intensities, and sediment:aqueous phase contamination ratios. Because aquatic rhizospheres have the ability to enhance MeHg formation, the level of MeHg in water, sediment, and water hyacinth was also measured. Hg and MeHg were found to concentrate preferentially in the roots of E. crassipes with little translocation to the shoots or leaves of the plant, a result consistent with studies from similar macrophytes. Sediments were found to be the major sink for Hg as they were able to sequester Hg, making it non-bioavailable for water hyacinth uptake. An optimum PO4 concentration was observed for Hg and MeHg uptake. Increasing light intensity served to enhance the translocation of both Hg and MeHg from roots to shoots. Assimilation of Hg and MeHg into the biomass of water hyacinths represents a potential means for sustainable remediation of contaminated waters and sediments under the appropriate conditions.     

大水面放养水葫芦对富营养化湖泊水体可培养细菌群落结构的影响

【目的】了解大水面放养水葫芦对富营养化湖泊水体可培养细菌群落结构和多样性的影响。【方法】采用稀释平板法,分别对云南滇池紫根水葫芦放养区(ZW)、野生型普通水葫芦放养区(PW)、未放养水葫芦对照区(CK)水体中细菌进行分离,并对其16S r RNA序列进行分析。【结果】分别从ZW、PW、CK 3种水体分离得到54、49、40株菌落形态差异的细菌,Shannon-Wiener多样性指数分别为3.17、3.07、2.73,细菌数量分别为1.35×107、8.35×106、2.70×106 CFU/L。16S r RNA序列分析表明,ZW、PW、CK 3种水体可培养细菌主要包括变形菌门α亚群(Alphaproteobacteria,35.1%、32.4%和40%)、放线菌门(Actinobacteria,18.9%、32.4%和20%)、变形菌门β亚群(Betaproteobacteria,13.5%、5.9%和16.0%)、变形菌门γ亚群(Gammaproteobacteria,13.5%、14.6%和12.0%)、拟杆菌门(Bacteroidetes,13.5%、8.8%和8.0%)和厚壁菌门(Firmicutes,2.7%、5.9%和4.0%)。在属的水平上,3种水体仅有鞘氨醇盒菌属(Sphingopyxis)、红细菌属(Rhodobacter)、黄色杆菌属(Xanthobacter)、新鞘脂菌属(Novosphingobium)、鞘氨醇单胞菌属(Sphingomonas)、假单胞菌属(Pseudomonas)、微杆菌属(Microbacterium)、链霉菌属(Steptomyces)、黄杆菌属(Flavobacterium)、芽孢杆菌属(Bacillus)等10个属的细菌为共有菌属。【结论】大水面放养水葫芦提高了富营养化湖泊水体中可培养细菌的多样性,改变了细菌的群落结构。     

Kinetics of the pyrolytic and hydrothermal decomposition of water hyacinth

The kinetics of water hyacinth decomposition using pyrolysis and hydrothermal treatment was compared. With pyrolysis, initial vaporization occurred at 453 K as determined by thermogravimetric analysis, while initial solubilisation occurred at 433 K with subcritical hydrothermal treatment. The “kinetic triplet” was determined for the ranges of 423-483 K (range I) and 473-553 K (range II) using the Coats-Redfern method for both treatments. The calculated activation energies for ranges I and II were 110 and 116 kJ/mol for conventional pyrolysis and 145 and 90 kJ/mol for hydrothermal treatment. The similar activation energies for the two temperature ranges observed for pyrolysis implied that only hemicellulose decomposition occurred. For hydrothermal treatment, both hemicellulose and cellulose decomposition occurred in temperature range II, in which a notable lower activation energy was observed. This implied hydrothermal treatment was more suitable for conversion lignocellulosic biomass under these conditions.     

对水葫芦有杀草活性的菌株Ｓ63的分离筛选

采用水葫芦琼脂平板和氨氮平板初筛及点种于水葫芦茎处的方法,得到使水葫芦茎部病变黑斑的菌株,考察其对光照培养箱中培养水葫芦的生长抑制效果,获得对水葫芦生长有强抑制作用的菌株S63,鉴定为假单胞菌属。露天培养的试验结果表明,加入该菌液0.5%~2%(体积分数)的试验组发病率为41.2%~100%,高于对照组19.4%~33.3%的发病率;试验组叶片的丙二醛(MDA)含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)的酶活均显著高于对照组。表明该菌对水葫芦有一定的除草活性。     

Decomposition of young water hyacinth leaves in lake water

Rates of weight loss and release of nutrients during different phases of decomposition in young water hyacinth leaves were determined under laboratory conditions. The leaves decomposed solely by physical leaching during the initial 4-day phase and later by microbial processes. The largest part of weight loss and nutrient release by physical leaching took place within the first 4 h of incubation and thereafter the decomposition rate declined. Microbial processes decayed leaves at a significantly higher rate than that by physical leaching. The overall decay rate constants were related inversely and the release of nutrients directly to the levels of leaf additions in the lake water. The dissolved inorganic and organic nutrients were released chiefly by abiotic processes during the initial as well as later phases of decay. The release was significantly higher during the initial phase in comparison with that during the later phase. Microbes utilized only a small amount of nutrients that were released during decomposition of water hyacinth leaves. The % release of various elements from the decaying leaves was in the order of K > P > C > Na > N.     

Ecological and socio‐economic impacts of invasive water hyacinth (Eichhornia crassipes): a review

1. Water hyacinth (Eichhornia crassipes) is one of the world’s most invasive aquatic plants and is known to cause significant ecological and socio‐economic effects. 2. Water hyacinth can alter water clarity and decrease phytoplankton production, dissolved oxygen, nitrogen, phosphorous, heavy metals and concentrations of other contaminants. 3. The effects of water hyacinth on ecological communities appear to be largely nonlinear. Abundance and diversity of aquatic invertebrates generally increase in response to increased habitat heterogeneity and structural complexity provided by water hyacinth but decrease due to decreased phytoplankton (food) availability. 4. Effects of water hyacinth on fish are largely dependent on original community composition and food‐web structure. A more diverse and abundant epiphytic invertebrate community may increase fish abundance and diversity, but a decrease in phytoplankton may decrease dissolved oxygen concentrations and planktivorous fish abundance, subsequently affecting higher trophic levels. 5. Little is known about the effects of water hyacinth on waterbird communities; however, increases in macroinvertebrate and fish abundance and diversity suggest a potentially positive interaction with waterbirds when water hyacinth is at moderate density. 6. The socio‐economic effects of water hyacinth are dependent on the extent of the invasion, the uses of the impacted waterbody, control methods and the response to control efforts. Ecosystem‐level research programmes that simultaneously monitor the effects of water hyacinth on multiple trophic‐levels are needed to further our understanding of invasive species.