3.1.1. Physical characteristics of samples

Bio-oil used in the single separation process was produced by the pyrolysis of Mongolian pine sawdust (Wang et al., 2008). Wang et al. (Guo et al., 2009b; Wang et al., 2009) carried out experimental research on molecular separation of the bio-oil, which was pre-treated by centrifugation and filtration to remove solid particles. Molecular distillation of the bio-oil at 50, 70, 100, and 130 °C, respectively, was investigated under a fixed pressure of 60 Pa. Under all of the tested conditions, the light fraction collected by the second condenser placed before vacuum pump was designated as LF, the middle fraction condensed by the internal condenser as MF, and the heavy fraction as HF.

The color of the distilled fractions becomes lighter while the residual fractions become darker. Under the four conditions, water was concentrated in the LFs, which had water contents of about 70 wt%. The LFs could not be burned because of their high water contents. The pH values of the LFs were in the range 2.13-2.17 as a result of their carboxylic acid contents. On the other hand, the HFs had the highest heating values and the lowest water contents, resulting in good ignitability but inferior fluidity. At a distillation temperature of 70 °C, the water content of the MF was as low as 2 wt%. The total mass of the bio-oil distillation fractions amounted to more than 97% of the bio-oil feed. With increasing temperature, the yield of the LF increased without any coking or polymerization problem. Water and volatile carboxylic acids were evaporated from the feedstock in the temperature range 50-130 °C under low pressure, and more carboxylic acids escaped from the liquid at higher temperature. However, on further increasing the temperature, this phenomenon was not so pronounced, due to more and more molecules of higher boiling point also being distilled. The yield of the distilled fraction increased with increasing distillation temperature. However, too high temperature may lead to decomposition of some chemical compounds in the crude bio-oil. Hence, there must be an optimum temperature to realize reasonable separation.

The relatively large surface area of microorganisms exposed to the environment where they live enables them to take up and assimilate nutrients readily and a to multiply at impressive rates. An important parameter is the specific growth rate, wich is often expressed as the mean doubling time, defined as the time required by the microbial population to douyble its cellular protein content. The doubling time varies widely depending on the microbial species, the nature of the substrate and the degree of adaption to the substrate.

The growth of a bacterial culture can be determined by measuring the increase of turbidity in the medium as optical density or OD. The most common way to do this is to compare the absorbance of the culture to inoculated medium by shining light with a wavelength of 600 nm through the culture. The more growth occurs, the more turbid the culture will become and more light will be absorbed. Using optical density gives indirect measurement of bacterial growth. It doesn’t tell anything about how many living cells are in the culture. This becomes especially important in stationary phase. Dead cells still absorb light. To determine the actual number of live bacteria the broth is diluted and plated on appropriate media and incubated. In theory, colonies arising on a plate originate from one single bacterium and give therefore an accurate number of the live cells in the culture at that time point.

Procedure:

The kinetics of selected bacterial growth, it is used 10 mL of 24 h culture of the strains and inoculated into 60 mL of nutrient broth, the following conditions: 35 °C and 100 rpm agitation. Samples were read every 30 minutes in a Spectronic 20D+ visible spectrophotometer at 600 nm, substituting readings transmittance (% T) in the following equation:

A = 2 — logM(%T)

Where A, is the absorbance.

The different phases of growth kinetics can be observed by plotting the log (% T) versus time [1].

An experiment was designed to determine different classes of metabolizable energy (AME, AMEn, TME, TMEn) in artemia meals [13] .For determination of metabolizable energy of artemia meal and comparison with fish meal, samples gathered from 3 regions include : Urmia Lake Artemia Meal(ULAM), Earth Ponds Artemia Meal (beside urmia lake)(EPAM) and Ghom Salt Lake Artemia Meal(GSLAM). Then samples dried, milled and used in a biological experiment with fish meal. 20 Rhode Island Red cockerels with approximately same live weight used in Sibbald assay with completely randomized design with 5 treatments and 4 repetitions for determination of AME, AMEn, TME and TMEn.

Results showed there were significant differences between treatments from standpoints of metabolizable energy (P<0.05). ULAM and FM had highest ME and EPAM and GLAM had lowest ME. The highest TME belong to FM and the lowest TME pertained to EPAM. Except to EPAM that had the lowest TMEn, other treatments didn’t have any differences between them.

In the batch fermentation process, the entire medium is removed from the fermentation vessel. The vessel is then thoroughly washed, cleaned and the new batch is started only thereafter. The bioreactor is initially loaded with fresh medium and inoculated with selected microorganism.

During the batch fermentation process, various physiological states of the microorganism

are observed (Figure 3):

a. Lag phase — Period where microorganisms adapt to the new environment.

b. Positive acceleration phase — Period of slow increase in the population

c. Logarithmic or exponential phase — Period of rapid rise in population due to availability of nutrients. The exponential phase may be described by the following equation:

dX/dt = Vх

Where x is the concentration of microbial biomass

t is time, in hours

and p is the specific growth rate in hours-1

d. Negative acceleration phase — Period in which there is a slow rise in population as the environmental resistance increases.

e. Stationary phase — Finally, growth rate becomes stable because mortality and natality rates become equal. During the stationary phase, the organism is still maintaining a certain metabolic activity, while some secondary metabolites are formed (products not associated with microbial growth).

f. Death phase — Finally, environmental stress causes a decrease in metabolic activity of yeast and autolysis.

Ecuadorian National Planning and Development Department, has determined a new energetic matrix where it is established the use of 5% ethanol in regular gasoline to be used in vehicles until 2020 in the whole country [29].

To accomplish this endeavor, in 2010 Ecuador’s fuel ethanol needs were 32’187,903 gallons; nonetheless, the ethanol production reached was just 1’195,427 gallons [30]. This is, only 3.71% of the national requirements were fulfilled, which demonstrates that there exists a huge unsatisfied demand of fuel ethanol, representing an interesting market for ethanol producers as seen on Figure 5.

ETHANOL PRODUCTION IN ECUADOR IN 2010

The price of ethanol per liter has been fixed in 0.76 Dollars by the Ecuadorian Government. This is a very competitive price that the sugar mills are interested in producing this
renewable fuel using molasses as feedstock; molasses are byproducts rich in sugars that represent a valuable source of energy in animal husbandry.

The RESETA Project (the acronym of Sustainable Sources for Ethanol) studies the second generation ethanol production from lignocellulosic materials as well as leftovers and industrial effluents. One of the objectives of such project is the development of a financial model based on a baseline survey on lignocellulosic materials in Ecuador [31]. By means of the data obtained in such survey, it was possible to determine that there are three candidate raw materials taken into account its amount, localization and availability. The sources are banana stems, sugar cane bagasse and empty fruit bunches form oil palm.

Laboratory analysis demonstrated that the high level of moisture in banana stems, as well as the final disposition of the biomass, which is very dispersed in the field, makes its use an unfeasible task in terms of economy. On the other hand, sugar cane bagasse is already been used by sugar cane mills to generate power, thus, just a few biomass of this kind is really available. Finally, empty fruit bunch from oil palms present good characteristics for being used as feedstock in an industrial scale biorefinery. Empty fruit bunches account for about 45% cellulose dry weigh; which is enough to provide the raw material to run about 22% gasoline cars in Ecuador (Table 1).

One of the key factors influencing the feasibility studies to install a biorefinery is the price of the raw materials. Currently, the lignocellulosic residues represent a big environmental problem in Ecuador as well as an unsolved trouble to farmers and industries. One of the most common fates for lignocellulosics is the composting or decomposition on soil to provide organic matter for cultures and as mulch in a number of crops. The uncontrolled decomposition generates lixiviates, as well as greenhouse gases that finally are disposed in the subsoil and the atmosphere [31]. It is necessary to provoke a change in the current situation that makes a real shift in terms of the use of the lignocellulosics. One strategy proposed by the RESETA project is pushing to the creation of a market for the lignocellulosics in sustainable and fair conditions for the owners of the biomass as well as for the biorefinery project to be successful. The establishment of companies where the owners of the biomass can participate as shareholders is one of the strategies proposed by our study.

In these terms, we have designed a financial analysis based on the data obtained by laboratory and field surveys. The factors taken into account were:

a. The implementation zone would be in La Concordia town (Latitude -0.000017°; Longitude — 79,383563°), which is located in a strategic point for its big oil palm production and oil mill concentration.

b. The raw material in terms of empty fruit bunch is about 640 MT per day.

c. Enzymes, chemicals, yeasts, are calculated in 0.62 Dollars/Gal of ethanol.

d. Other costs are about 0.45 Dollars/Gal, as it is detailed in Table 5. Additionally, it has been established an income per by products commercialization. These by products are represented by liquid and solid fertilizers.

e. Another issue to be taken into account is the energy savings by means of the use of power co-generated using biogas from the biorefinery’s processes.

A similar European project (i. e. PERESEO project from Spain) establishes that the cost for the construction of a biorefinery plant for 500 MT of biomass per day is about 12 to 15 million Euros. In terms of the RESETA project, we may need to process about 640 MT/day. The criteria for building up a biorefinery are to consider a future growth, so the capacity must be overestimated in about 30% [32]. Then, the investments may overcome in about 1.6 times those of the European PERSEO project. In terms of Dollars, the total investment is among 24.96 to 31.2 million Dollars. We will use the highest number for our calculations.

It has been determined the cost per liter ethanol from lignocellulosics from empty fruit bunch of oil palm in 0,49 Dollars, which is shown in Table 6.

In this calculation it has been taken into account a 1% growth in costs and incomes with the aim of using the whole installed capacity at the end of the project. Following in Table 7, it is shown the cash flow of the project, where the initial investment (31,2 million Dollars), the investment recovery period; the IRR and NPV are shown. Table 8 is a resume of such values.

With such calculations and with a discount rate of 13.76%, calculated in basis to the established formula shown next, we have attained an IRR biggest than the discount rate.

Discount Rate = Rf+ p(Rm — Rf) + Rp,

Rf: Risk of the treasure founds bonus in the USA. p: Sensitivity or risk of the Project in the market Rm: Stock market index.

Rp: Country-risk

This Project exhibits a positive NPV, combined with a reasonable IRR value; both values can be interpreted as indicators of a feasible project. This financial study doesn’t consider the externalities and the social benefits a biorefinery can give the surrounding populations and environment.

Commercial production of bioethanol deals with the biotechnological production from different feedstock. The selection of the most appropriate feedstock for ethanol production strongly depends on the local conditions. Due to the agro-ecological conditions, North American and European countries have based their ethanol industry on the starchy materials. In Brazil, sugarcane is the main feedstock for bioethanol production. World production of ethanol (all grades) in 2010 was nearly 70 billion litres (IEA, 2010). Although many countries produce ethanol from a variety of feedstocks, Brazil and the United States are the major producers of ethanol in the world, each accounting for approximately 35 percent of global production [4].

The theoretical yield of ethanol from sucrose is 163 gallons of ethanol per tonne of sucrose. Factoring in maximum obtainable yield and realistic plant operations, the expected actual recovery would be about 141 gallons per tonne of sucrose [5]. Using [6],[7] and [8] reports, average sugar recovery rates, one tonne of sugarcane would be expected to yield 70 L of ethanol and one tonne of sugar beets would be expected to yield 100 L of ethanol. One tonne of molasses, a byproduct of sugarcane and sugar beet processing, would yield about 260 L of ethanol. Corn had the highest ethanol yield per tonne feedstock (403 L/t), followed by wheat with 350 L/t [9]. A lower ethanol yield per tonne of feedstock was obtained for cassava compared to corn. The ethanol yield from starchy materials were basically higher than sugar containing material because of the higher amount of fermentable sugars (glucose) that may be released from the original starchy material [10].

The conversion of sugar containing material into bioethanol is easier compared to starchy materials and lignocellulosic biomass because previous hydrolysis of the feedstock is not required since this disaccharide can be broken down directly by the yeast cells [11]. Therefore, using raw sugar as a feedstock, one tonne would yield 500 L of ethanol while refined sugar would yield 530 L ethanol. Molasses, from either sugarcane or sugar beets, was found to be the most cost competitive feedstock. The table below summarizes the estimated ethanol production yield and conversion efficiency from starchy and sugar containing materials from all over the world, as well as research ethanol yield produced from lignocellulosic biomasses.

Bioethanol is currently produced from raw materials such as sugar cane, or beet or starch from cereals. Recent interest was on the low cost and abundant availability of lignocellulosic biomass as the potential feedstock for bioethanol production. Lignocellulosic biomass which includes agricultural and forestry residues and waste materials, has the advantage of providing a greater choice of potential feedstock that does not conflict with land-use for food production, and that will be cheaper than conventional bioethanol sources. Many researchers from around the world are now working on transforming lignocellulosic biomass such as straw, and other plant wastes, into "green" gold — cellulosic ethanol. Cellulosic ethanol, a fuel produced from the stalks and stems of plants (rather than only from sugars and starches, as with corn ethanol), is starting to take root in the United States.

The bioconversion of lignocellulosic biomass to monomeric sugars is harder to accomplish than the conversion of starch, presently used for bioethanol production. However, many countries are making efforts to utilize these lignocellulosic biomasses into ethanol; Sweden, Australia, Canada and Japan are planning to invest into lignocellulosic ethanol mill [21]. The highest ethanol yield from lignocellulosic materials was obtained using switchgrass, 201 L/t with 80% conversion efficiency. Ballesteros et. al [20] studied on ethanol conversion using woody material such as Populus nigra and Eucalyptus globule found that the yield of 145 L/t and 137 L/t feedstock and conversion efficiency ranging 59% — 64% was observed. The conversion efficiency for lignocellulosic materials was lower than the conversion efficiency obtained from sugar-containing material and starchy material.

The selection of the feedstock is in concordance with the interests of each country based on their availability and low cost. Because feedstocks typically account for greater than one — third of the production costs, maximizing the bioethanol yield is imperative [22].

Carbon dioxide explosion can also be used for lignocellulosic biomass pre-treatment. The method is based on the utilization of CO2 as a supercritical fluid, which refers to a fluid that is in a gaseous form but is compressed at temperatures above its critical point to a liquid like density. Supercritical carbon dioxide has been used as an extraction solvent for non­extractive purposes, due to some advantages such as availability at relatively low cost, non­toxicity, non — flammability, easy recovery after extraction, and environmental friendly [153]. Besides a liquid-like solvating power, supercritical carbon dioxide displays gas-like mass transfer properties [154].

Supercritical pre-treatment conditions can effectively increase substrate digestibility by removing lignin. Addition of co-solvents such ethanol can improve delignification. Supercritical carbon dioxide has been mostly used as an extraction solvent but it is being considered for non-extractive purposes due to its many advantages [155]. CO2 molecules are comparable in size to water and ammonia and they can penetrate in the same way the small pores of lignocelluloses. This mechanism is facilitated by high pressure. After CO2 explosive, pressure released, disruption of cellulose and hemicellulose structure is observed and consequently accessible surface area of the substrate to enzymatic attack increases [141].

2.2.2. Combination with ammonia fibre explosion (AFEX) time, and then the pressure is suddenly released, resulting in a rapid expansion of the ammonia gas that causes swelling and physical disruption of LCF fibres and partial decrystallization of cellulose. This swift reduction of pressure opens up the structure of lignocellulosic biomass leading to increased digestibility of biomass.

One of the main advantages of AFEX pre-treatment is no formation of some types of inhibitory by-products, which are produced during the other pre-treatment methods, such as furans in steam explosion pre-treatment.

AFEX has been studied for decreasing cellulose crystallinity and disrupt lignin — carbohydrates linkages [156]. Ammonia recovery and recycle is feasible despite of its high volatility [157] but the associated complexity and costs of ammonia recovery may be significant regarding industrial scale using of the AFEX pre-treatment [34, 158].

There are some disadvantages in using the AFEX process compared to some other processes. AFEX simultaneously de-lignify and solubilize some hemicellulose while decrystallizing cellulose, but does not significantly solubilize hemicellulose as acid and acid — catalyzed steam-explosion pre-treatments [159-161]. The AFEX produces only a pre-treated solid fraction, while steam explosion produces a slurry that can be separated in a solid and a liquid fractions [15]. Furthermore, ammonia must be recycled after the pre-treatment to reduce the cost and protect the environment [106, 158].

Willows can be planted according to two different layouts. In most North European countries (Sweden, UK, Denmark) and in the US, the most frequent planting scheme is the double row design with 0.75 m distance between the double rows and 1.5 m to the next double row, and a distance between plants ranging from 1 m to 0.4 m, corresponding to an initial planting density of 10,000 — 25,000 plants ha-1. The most common plantation density in these countries is currently around 15,000 (1.5 x 0.75 x 0.59 m) plants ha-1 [31]. This rectangular planting arrangement is used to facilitate field machine manoeuvres through the plantation site. Tractors overlap the double row and the wheels run in the wider strips between those rows [32]. In Quebec, a simpler willow planting design, similar to that used for poplar in short rotations, has been in use since initial trials with only minimal modifications. It consists of a single row design ranging from 0.33 m between plants on a row and 1.5 m between rows (20,000 plants ha-1) in the very first plantations, to 0.30 m on the row and 1.80 m between rows (18,000 plants ha-1) in newer willow plantations. Theoretically, this design facilitates weed control during the establishment phase (the first three years), and consequently willow rooting and growth. In fact, the design choice depends mostly on machinery available for planting and harvesting, since it has been clearly demonstrated that planting design has less impact than plant density and cutting cycle on the yield of Salix plants, due to their ability to take advantage of the space available to each stool [32]. The choice of planting density must take into account other ecological factors as well. On sites with appropriate water supply, plantation establishment and subsequent biomass production depend largely on agronomic considerations such as plant spacing and harvesting cycles. Many studies have reported a correlation between spacing and harvesting cycles. In general, maximum yields are achieved early in dense willow plantations, but wider-spaced plantations ensure the highest long-term biomass yield [33-34]. On the other hand, under short harvesting cycles, willow stands have a shorter duration, as they are likely to be more exposed to pathogens [35]. At present, most willow short-rotation stands in Quebec have a plantation density of about 16,000 to 17,000 cuttings ha-1 and are harvested every two to three years.

Planting material consists of dormant willow stem sections, either rods or cuttings, depending on the planting machinery to be adopted. In some countries, for example in the UK and in the USA, ‘step planters’ are the most commonly used machines. Willow rods 1.5­2.5 m long are fed into the planter by two or more operators, depending on the number of rows being planted. The machine cuts the rods into 18-20 cm lengths, inserts these cuttings vertically into the soil and firms the soil around each cutting. Step planters have been calculated to cover 0.6 ha/hr in a UK study. [31]. In Quebec, the most common planting machine is a cutting planter that uses woody cuttings (20-25 cm long) and may operate on 3 rows simultaneously (Figure 1).

Normally, a cutting planter inserts cuttings into the soil at a depth of about 18 cm. Based on empirical experience, this equipment can plant 3,600-4,000 cuttings per hour (1 ha of willow every 3-4 hours), although the duration of this operation may vary depending on several factors (site topography, soil type, plot shape, etc.). Planting material in Quebec is prepared by harvesting one-year-old stems (about 3 m long) in the autumn (i. e. when plants are dormant) of the year prior to planting. This material is wrapped in plastic film to avoid moisture loss, and stored in a refrigerator at -2 to -4°C. In spring, two to three weeks prior to planting, healthy willow rods 1-2 cm in diameter (with no symptoms of disease on bark or wood) are selected to prepare cuttings. Tips of stems bearing flower buds are first discarded. Then the rest of the whip is cut into 20-25 cm lengths using an adapted rotary saw and stored in boxes, ready to be planted (Figure 2).

Figure 2. Willow cuttings before planting

If cuttings are left in temperatures above 0°C, a break in their dormancy will occur, adventitious roots will develop and the buds may burst. This will lead to a reduction in water and nutrient content and consequently reduced viability. Thus, it is very important to plan the planting operation carefully in advance, calculating the number of cuttings that can be planted.

The time of planting varies according to meteorological and soil conditions. Planting should be undertaken as soon as possible in the spring, to allow plants to benefit from the high soil water content following snowmelt, and then to establish quickly and take maximum advantage of a long growing season. In addition, a late willow planting is also more subject to failure due to drought if a dry summer should occur. However, there are several additional factors that play an important role in determining the planting date. In order for soil preparation (e. g. harrowing) to begin in the spring, soil should be free from snow but not so muddy that soil structure could easily be damaged by tractors. The date at which such conditions are met vary considerably from year to year, but in southern Quebec, it usually falls during May, although late planting (up to mid-June) is possible and, in our experience, does not result in serious problems in plant establishment. Planting willow in the colder, northernmost regions of Quebec (e. g. Abitibi) may take place up to the beginning of July. In all of these situations, rapid colonisation by highly competitive weed species occurs on fertile sites, thus the use of appropriate residual herbicides is essential to maximize plant survival and early growth. Pre-emergence residual herbicide should be applied immediately upon completion of planting (within a maximum delay of 3-5 days). A mixture of two herbicides (2.30 kg Devrinol and 0.37 kg Simazine per hectare) has been effective on most of our plantations. Since the treatment must reach the zone of weed seed germination, most pre-emergence herbicides require mechanical incorporation (such as by a power tiller) as well as adequate irrigation or natural moisture (rainfall or snow) for best results. More recently, a new herbicde (SureGuard, a. i. flumioxazin) has received approval for pre-emergent use at the time of planting on poplar and willow (including planting stock production in the field, on both stoolbeds and bareroot beds).

The mechanism involved is based on the formation of covalent bonds between the inorganic support (silica) and cells in the presence of crosslinking agents. A said joint is needed for the modification of the support surface. The reaction requires the introduction of reactive organic groups on the silica surface for the attachment of cells to the support. As coupling agent generally used aminopropyl triethoxy silane; this organic functional group condenses with hydroxyl groups of the silica and the group as a result becomes available for covalent bond formation on the surface. Covalent bonds can also be established by treating the silica surface with glutaraldehyde or isocyanate.

The advantage of this method is that the support can be generated without the limitations on physical and chemical conditions imposed by the biocatalyst, which can be optimized by the characteristics of mechanical stability, porosity, strength of the support, etc.

When you want to form covalent bonds between the substrate and cells, the problem is how to promote adhesion of cells to relatively large surface without damaging its stability and resistance to washing. The support may have pores of greater diameter than the cell to allow the latter to penetrate the internal surfaces. Porous supports are used which are embedded by immersion in cell suspensions [60].

Another important matrix being used for immobilization for metal removal is silica. Silica- immobilized preparations offer advantage in terms of reusability and stability. The silica immobilized product is mechanically strong and exhibits excellent flow characteristics [68]. A silica immobilized algal preparation AlgaSORBR (Bio-Recovery Systems, Inc., Las Cruces, NM 80003, USA) which is being used commercially retains approximately 90% of the original metal uptake efficiency even after prolonged use (> 18 months) [57].

Carlos E. de M. Bicudo and Norma C. Bueno

Additional information is available at the end of the chapter http://dx. doi. org/10.5772/54685

1. Introduction

Popularly known as stoneworts, brittleworts, muskgrass, muskworts or bass-weeds, Characeae are among the largest and most complex green algae. All common names come from some characteristics these plants may exhibit, such as the brittle, limestone (calcium carbonate) exoskeleton that can form on the external surfaces of the plant (e. g. Chara vulgaris and Chara globularis) and, particularly, from the distinctive smell of stale garlic emitted by the plant when crushed. It is important to note, however, that most charophyte species do not accumulate lime in observable amounts. The widespread misinterpretation that Chara plants generally form lime is understandable, since the two very common species above thrive in shallow waters where they are readily collected, forming spectacular extensive growths, and may solidify directly into a marl layer or onto a curious tufa rock, a porous limestone formed by deposits from springs. Plants accumulating lime become gray or whitish and quite opaque, whereas the many species without evident lime are generally soft, nearly transparent, with a glassy brilliance and rich green color.

Charophytes forms a significant part of the submerged vegetation of both natural and artificial systems represented by lakes, ponds, ditches, streams, canals, bog-pools, concrete tanks, reservoirs and excavations such as gravel pits, and are found on all continents except Antarctica. They are common in the littoral region of oligotrophic to moderately eutrophic water bodies (Kufel & Kufel 2002), and some authors (e. g. Krause 1985) consider these macrophytes indicators of water quality. Nitella specimens predominate in mildly acid water as in igneous rock areas, whereas Chara’s predominate in hard waters, but this is not a rule. They are characteristic of a disturbed habitat where periodic drastic changes create less favorable conditions for the growth of other algal species. They are often the first plants to colonize newly dug or cleared ponds and ditches, and some species are characteristic of ephemeral water bodies which dry up completely in the summer. The fast maturing charophytes have an advantage over the slow-growing macrophytes in such habitats. Charophytes are usually at a competitive disadvantage in shallow, moderately productive

habitats, but tend to dominate in deeper water at low light intensities, particularly where the water has a high pH value. They are more often found in mesotrophic and eutrophic, hard water, calcium rich and low in phosphate waters. Charophytes may grow in silt, mud, peat or sand and they often form a dense carpet, known as a charophyte meadow, which restricts colonization by other macrophytes. The more common charophyte species do not die down during the winter. They have been recorded growing down to 60 m deep in clear water, but usually prefer depths between 1 and 10 m. In tropical countries such as Brazil, charophytes grow best in shallow water bodies, mostly in small reservoirs built for cattle disedentation, where they form dense carpets at the littoral zone of the reservoirs, usually at 20-40 cm depths. They may often grow intermingled with other macrophytes, mainly with water lilies (Nymphaea spp.), whose floating leaves they use to cut down the light intensity.

In size, they are generally moderately large, average shoots varying from 15 to 30 cm in height, but they may range from 5 mm to 2 m at extremes. Specimens of Chara hornemannii collected from the Rodrigo de Freitas Pond, in the city of Rio de Janeiro, ranged between 1.9­2 m tall.

The charophyte ‘plant’ or thallus is erect, central axis or ‘stem’ is branched and differentiated into a regular succession of nodes and internodes (Figure 1). Each node bears a whorl of branches of limited growth (the ‘leaves’ or branchlets), but branches capable of unlimited growth may arise axillary to the leaves. The axis consists of a chain of alternating

long and short cells, the single long cells forming the internodes and the short, discoid cells forming the nodes. The single axial intermodal cell is commonly 1-4 cm long, but they may reach 50-60 cm in Nitella cernua and Nitella translucens. The intermodal cell is commonly 0.1­0.3 mm broad, but in the last two species, it may reach 2-3 mm broad. The plant is anchored by non-pigmented, single-celled processes, with or without a differentiation into nodes and internodes, the rhizoids, which penetrate the soil or substrate.

The importance of charophytes is indirect, as food for migratory waterfowl, protection of fish fry, and as a nuisance in shallow waters of reservoirs and recreational areas. They may also be used for sulfur baths, cattle food, fertilizers, scouring and filtering agents, and even for supposed control of mosquito larvae.