1.1 Equipment

Determination of the relative impact of various significant bioprocess parameters that influence traditional batch propagation of kefir grains and daily kefir grain increase mass using the Taguchi design methodology requires the performance of a series of experiments. In order to ensure the highest quality as well as repeatability of raw experimental data, it is desired to perform those experiments (batch propagations of kefir grains in enriched milk under different bioprocess conditions) in computer controlled state-of-the-art laboratory reactor or fermentor.

Perhaps one of the most user-friendly and at the same time the most efficient high quality aforementioned equipment is heat flow reaction calorimeter RC1 (Mettler Toledo, Greifensee, Switzerland). Basically, the RC1 system is actually both — state-of-the-art computer controlled, electronically safe-guarded bench-scale ‘model’ of a batch/ semi-batch reactor or fermentor from pilot and/or industrial plant (automated lab reactor (ALR)) and at the same time a heat-flow reaction calorimeter. The RC1 system allows real time measurement, monitor and control of all important bioprocess parameters such as rotational frequency of the stirrer, temperature of reaction or fermentation media, reactor jacket temperature, pH value of reaction or fermentation media, mass concentration of dissolved oxygen, amount of added (dosed) material, etc. Primarily, it is designed for determination of the complete mass and heat balance over the course of the entire chemical reaction or physical transformation (e. g. crystallization, dissolution, etc.). In addition, using specific modifications, it can be employed for investigating thermal effects during bioprocess (Marison et al., 1998). This means that by using RC1 system it is possible to gain and/or determine wide range of process thermal data and constants such as specific heat capacity of reaction mixture, heat flow profile of the reaction or physical transformation, reaction enthalpy, maximum heat flow due to reaction or physical transformation, potential adiabatic temperature increase in case of cooling failure, heat accumulation, etc.. All obtained time-depended calorimetric data (heat flow data) can be further used for kinetic studies, etc. The RC1 system enables performance of chemical and also bio(chemical) reactions or physical transformation under different modes such as isothermal conditions, adiabatic conditions, etc. Using RC1 it is possible to perform distillations and reactions (transformations) under reflux with heat balancing. Last but not least, the RC1 system is a recipe driven (managed) which means that all process operations can be programmed or written by recipe beforehand and thus its maximum flexibility is assured. Finally, it is worldwide recognized as an industrial standard to gain safety data for a later scale-up to pilot or production plant.

As already stated, MBRs represent an important technical option for wastewater treatment and reuse, being very compact and efficient systems for separation of suspended and colloidal matter and enabling high quality, disinfected effluents to be achieved. A key advantage of these MBR systems is complete biomass retention in the aerobic reactor, which decouples the sludge retention time (SRT) from the hydraulic retention time (HRT), allowing biomass concentrations to increase in the reaction basin, thus facilitating relatively smaller reactors or/and higher organic loading rates (ORL). In addition, the process is more compact than a conventional activated sludge process (CAS), removing 3 individual processes of the conventional scheme and the feed wastewater only needs to be screened (1­3 mm) just prior to removal of larger solids that could damage the membranes (Figure 1).

Notwithstanding the advantages of MBRs, the widespread implantation is limited by its high costs, both capital and operating expenditure (CAPEX and OPEX), mainly due to membrane installation and replacement and high energy demand. This high energy demand in comparison with a CAS, is closely associated with strategies for avoiding/mitigating membrane fouling (70% of the total energy demand for iMBR) (Verrech et al., 2008; Verrech et al., 2010). Fouling is the restriction, occlusion or blocking of membrane pores or cake building by solids accumulation on the membrane surface during operation which leads to membrane permeability loss. The complexity of this phenomenon is linked to the presence of particles and macromolecules with very different sizes and the biological nature of the microbial suspensions, which results in a very heterogenic system. Meanwhile, the dynamic behaviour of the filtration process adds a particular complication to the fouling mechanisms (Le-Clech et al., 2006). Furthermore, permeability loss can also be caused by channel clogging, which is the formation of solid deposit in the voids of the membrane modules due to local breakdown of crossflow conditions (Figure 2). In addition, there are other operational problems, such as the complexity of the membrane processes (including specific procedures for cleaning), the tendency to form foam (partly due to excessive aeration), the smaller sludge dewatering capacity and the high sensitivity shock loads.

For the immersed configuration, the operating strategy to control membrane fouling, ( impacting directly or indirectly on CAPEX and OPEX) includes the following:

i. selecting an appropriate permeate flux,

ii. scouring of membrane surface by aeration,

iii. applying physical cleaning techniques, like backflushing (when permeate is used to flush the membrane backwards) and relaxation (when no filtration takes place), and

iv. applying chemical cleanings protocols, with different frequency and intensity (maintenance cleaning and recovery cleaning).

The fist concern, selecting an appropriate permeate flux, is determined by the classical trade­off problem: at higher fluxes CAPEX decreases while OPEX increases. High fluxes are desirable to reduce the membrane required (i. e. reduce CAPEX), however, membrane fouling increases with flux, which results in a higher membrane scouring demand and more frequent cleaning to control membrane fouling (i. e. increase OPEX). Furthermore, the correlation between membrane fouling and flux is not only influenced by hydrodynamics and cleaning protocols but also by feedwater characteristics and biological conditions. As a result, deciding a flux value depends on the analysis of empirical data obtained from pilot and full-scale experiments or available in the recent literature.

The second concern is membrane scouring. Ever since the iMBR appeared, air sparging has been widely used to mitigate fouling by constant scouring of the membrane surface (Cui et al., 2003) or by causing lateral fibre movement in HF configuration (Wicaksana et al., 2006). While the membrane fouling has been studied and mathematically modelled in classic filtration regimes (crossflow and dead-end) (e. g. Foley, 2006), the effect of turbulence induced by gas sparging in iMBR systems is still being assessed (Drews, 2010). As is well known, it has a clear contribution to minimizing the fouling problem, and therefore, a deeper understanding is extremely important in order to optimise aeration mode and rate, which has been proved to be one of its major operational costs.

The third concern is related to methods of physical cleaning (relaxation and backflushing) that have been incorporated as standard operation mode in MBRs. These techniques have successfully been proved to remove reversible fouling caused by pore blocking or sludge cake. For backflushing, the key parameters in the design of physical cleaning have been identified as frequency, duration, the ratio between these two parameters and its intensity (Le-Clech et al., 2006), and the same key parameters are expected for relaxation (with the exception of intensity). However, there is a knowledge gap in the inter-relationships between those parameters and the imposed permeate flux, especially when comparing both methods to obtain the same water productivity (Wu et al., 2008).

Finally, the fourth concern is chemical cleaning. Chemical cleaning is required when fouling cannot be removed by membrane surface scouring or physical cleaning methods. Although there are several types of chemical reagents used in membrane cleaning, in most full-scale facilities, two types of chemical reagents are commonly used: oxidants (e. g. NaOCl) for removing organic foulants (e. g. humic substances, proteins, carbohydrates), and organic acids (e. g. citric) for removing inorganic scalants. Basically, two objectives are pursued in the addition of chemical reagents: maintaining membrane permeability and permeability recovery. Maintenance cleaning is applied routinely via a chemically enhanced backflush where the reagent, at moderate concentration, is introduced with the permeate. In contrast, recovery cleaning is applied when the membrane permeability decreases until reaching non­operative values. The procedure consists of taking off the modules or draining off the membrane tanks to allow the membranes to be soaked in high concentrated reagents. Each MBR supplier has his own protocols which differ in concentrations and methods. Given its impacts on membrane lifetime and therefore on OPEX, there has recently been a growing interest in studying the influence of chemical cleaning procedures on membrane permeability maintenance and recovery (Brepols et al., 2008; Ayala et al., 2011). However, at the moment, the optimization of chemical cleaning protocols is far from being fully resolved.

Determination of metals (Ca, Cu, Fe, K, Mg, Mn and Zn) content was performed with flame atomic absorption spectrometry (F-AAS) by calibration curve method. Atomization of samples proceeded in the flame acetylene/air; rate of injection of samples into the flame was 4.5 mL min-1. Wavelengths used for the metals determination were 422.7, 285.2, 766.5, 213.9, 324.8, 279.5 and 248.3 nm for Ca, Mg, K, Zn, Cu, Mn and Fe, respectively. Determination of all metals content was performed with atomic spectrometer VARIAN SpectrAA 110. Limits of detection (LOD) and limits of quantification (LOQ) of the metals determination are given in Table 4. Determination of Cd, Zn and Cu was performed with flame atomic absorption spectrometry in samples prepared with dry decomposition. Atomization of samples was proceeded in the flame acetylene/air; rate of injection of samples into the flame was 4.5 mL min-1. Wavelengths used for the metals determination were 228.8, 324.8 and 213.9 nm for Cd, Cu and Zn, respectively. Determination of Pb was performed with atomic absorption spectrometry with electrothermic atomization by Varian SpectrAA 280Z atomic absorption spectrometer furnished with GTA 120 electrothermic atomizer at wavelength 283.3 nm.

1.1.3 Determination of dry weight

Dry matter of straw samples was determined by drying at 105 0C in a laboratory oven and of that grain at 130 0C to constant weight (ISO 612).

Many elements play a double role in the physiology of living organisms; some are indispensable, while most of them are toxic at elevated concentrations. The concern on the potential toxic effects of metal ions has been increasing in recent years. As a result of industrial activities and technological development, heavy metals released into the environment pose a significant threat to environment and public health because of their toxicity, accumulation in the food chain and persistence in nature.

In the sixties of last century the importance of controlling the concentration of toxic metal ions in waters for human use became apparent after the Four Big Pollution Diseases of Japan, a group of manmade diseases all caused by environmental pollution due to improper handling of industrial wastes by Japanese corporations.

Two of the Four Big Pollution Diseases of Japan, Minamata (1932-1968) and Niigata disease (1965), were due to mercury poisoning. The first one, first discovered in Minamata in 1956, is a neurological disease characterized by ataxia, numbness in the hands and feet, general muscle weakness, narrowing of the field of vision and damage to hearing and speech, and in extreme cases, insanity, paralysis, coma and death. This poisoning was caused by the release of methyl mercury in the industrial wastewater from the Chisso Corporation’s chemical factory. The highly toxic mercury has been bio-accumulated in shellfish and fish in Minamata Bay and the Shiranui Sea, and human and animals deaths continued over more than 30 years. In March 2001, 2265 victims had been officially recognized (1784 of whom had died) and, in addition, individual payments of medical expenses and a medical allowance had been provided to 10072 people in Kumamoto, Kagoshima and Niigata for their mercury related diseases (http://www. nimd. go. jp/english/index. html).

In the semiarid regions, water and salinity stresses are increasingly becoming primary limiting environmental conditions which restrict successful establishment of crops. Sorghum is tolerant of low input levels and essentially for areas that receive too little rainfall for most other grains (Table 7). Increased demand for limited fresh water supplies, increasing use of marginal farmland, and global climatic trends, all suggest that dry land crops such as sorghum will be of growing importance to feed the world’s expanding
populations. Generally lower water demands for sorghum than maize, versus their equal ethanol yields, suggests that sorghum will be of growing importance in meeting grain-based biofuels needs. In many tropical and temperate countries where sugarcane cannot be grown, a growing interest is being focused on the potential of sweet sorghum to produce bio­ethanol feed stocks (Avant, 2008) specially that salinity and drought tolerance are major features of sweet sorghum with low water requirements for high yields. One of the main reactions to drought stress is closing of stomata. The C4 plant such as sweet sorghum, in opposite to the C3, are able to utilize very low concentration of carbon dioxide which enables them to assimilate CO2 even during considerable stomatal closure (El Bassaru, 1998). This might be one of the probable reasons for the difference in resistance to stress between both plant groups. Photosynthesis is a complex process; therefore, it is possible that a number of elements in the C3 and the C4 may differ in resistance to drought.

Sweet sorghum

About 4 months.______________

One season in temperate and two or three seasons in tropical area.

All types of drained soil.

12000 m3/h__________________

Little fertilizer required; less pest and disease complex;

easy management.____________

54 — 69 tons._________________

7 — 12%.____________________

6 — 8 tons/ha.________________

3000 L/ha.

Very simple; both manual and through mechanical harvested.

Table 7. Comparison of Sugarcane, Sugar Beet, and Sweet Sorghum in Iran (Almodares & Hadi, 2009).

Many countries have been trying to prepare a regulatory limit to the use of organic wastes, such as food wastes or sludge, into crop production system in the light of their rapid increase. The regulatory system for the agricultural use of organic waste in South Korea is defined as soil concentration limits for potentially toxic elements (PTEs) to safeguard human health and crop yields. Despite legal limits, the damage of crop in the agricultural soil frequently occurs with organic waste for long-term application and with sub-quality compost made from sewage sludge.

The control system in the application of sludge to farmland varies according to country (Table 1). In South Korea, the control system for the application of sludge to farmland primarily depends upon heavy-metal concentrations that are similar to those in developed countries. Legally allowed limit values for PTEs— such as copper (Cu), zinc (Zn), chromium (Cr), cadmium (Cd), lead (Pb) and nickel (Ni) —were 400, 1,000, 250, 5, 130 and 45 mg kg-1, respectively, under the Fertilizer Management Act in South Korea (Anon., 2010a).

The control system for soil intoxication limit levels primarily depends upon heavy-metal concentration. The limit levels in South Korea are Cu 50, Zn 300, Cr 4, Cd 1.5, Pb 100 and Ni 40 mg kg-1 under the Soil Environmental Conservation Act (Anon., 2007). In Japan, Cu must be less than 125 mg kg-1, Cr 0.05 mg l-1 or less, Cd 0.4 mg kg-1 or less and Pb 0.01 mg l-1 or less (Ministry of the Environment Government of Japan, 1994).

In many countries, current rules for controlling the use of organic wastes on agricultural land have been criticized because they apparently do not take into consideration of the potential adverse effects of inorganic heavy metals and organic compounds produced in organic waste-treated soils on soil organisms (McGrath, 1994). The regulatory limit to the application of industrial waste on farmland only depends upon the level of PTEs in South Korea. However, PTEs limit may not be an adequate regulation protocol since organic wastes contain lots of inorganic and organic contaminants (Ministry of Agriculture, Fisheries and Food [MAFF], 1991).

An overall assessment of the soil contamination caused by inorganic and organic compounds of organic waste has been, therefore, attempted by ascribing qualitative description of the apparent risk and developing the integrated hazard assessment system (Hembrock-Heger, 1992). Available options for dealing with sludge include application to agricultural land, incineration, land reclamation, landfill, forestry, sea disposal and biogas. Of these, the application to agricultural land is the principal way for deriving beneficial uses of organic sludge by recycling plant nutrients and organic matter to soil for crop production (Coker et al., 1987). Also, agricultural use provides a reliable cost-effective method for sludge disposal. Recycling (81.7%) is the largest means of waste disposal, with 11.1% land deposition, 5.2% incineration and 2.0% sea disposal in South Korea (Anon., 2009). As an alternative way of waste disposal, the Fertilizer Management Act was revised to make it possible to apply industrial and municipal wastes into farmland in December 1996 in South Korea (Anon., 2006).

Table 1. Criteria of the inorganic pollutants in compost or sewage sludge for application to the arable land in 14 selected countries

As noted in the experimental procedure, all stages were performed using the same sequence of filtration and relaxation (450 s and 30 s, respectively). The experimental period was divided into five phases, each one operated at constant permeate flux. Membrane fouling was followed by measuring transmembrane pressure (TMP) evolution with operation time (Figure 11). Each phase finished when a pre-established TMP was reached.

The initial period (Figure 11) showed a high rate of fouling (0.011 Pa/s) despite working with relatively low permeate flux (20-23 l/h m2) and without reaching a high concentration of MLSS. This could be attributed to the initial biomass development until it obtained a high level of biological degradation. During this period, it was expected that microcolloidal and soluble species would have caused irreversible pore blocking, as a result of their small size (Di Bella et al., 2006). Afterwards, we assume that the developed biomass reaches steady — state conditions and degrades most of the colloidal and soluble matter. Therefore, feedwater characteristics and the level of physiological biomass seem to have a significant effect of fouling propensity.

Carotenoids are synthesized in nature by plants and many microorganisms. In addition to very few bacterial carotenoids with 30, 45, or 50 carbon atoms, C40-carotenoids represent the majority of the more than 600 known structures. Two groups have been singled out as the most important: the carotenes which are composed of only carbon and hydrogen; and the xanthophylls, which are oxygenated derivatives (Frengova & Beshkova, 2009). In the
later, oxygen can be present as OH groups, or as oxy-groups or in a combination of both (as in astaxanthin). Hydroxy groups at the ionone ring may be glycosylated or carry a glycoside fatty acid ester moiety. Furthermore, carotenoids with aromatic rings or acyclic structures with different polyene chains and typically 1-methoxy groups can also be found. Typical fungal carotenoids possess 4-keto groups, may be monocyclic, or possess 13 conjugated double bonds (Britton et al., 1998).

All carotenoids are derived from the isoprenoid or terpenoid pathway. Carotenoids biosynthesis pathway commonly involves three steps: (i) formation of isopentenyl pyrophosphate (IPP), (ii) formation of phytoene and (iii) cyclization and other reactions of lycopene (Armstrong & Hearst, 1996). Before polyprenyl formation begins, one molecule of IPP must be isomerized to DMAPP. Condensation of one molecule of dimethylallyl diphosphate (DMADP) and three molecules of isopentenyl diphosphate (IDP) produces the diterpene geranylgeranyl diphosphate (GGDP) that forms one half of all C40 carotenoids. The head to head condensation of two GGDP molecules results in the first colorless
carotenoid, phytoene. As Figure 9 shows, phytoene synthesis is the first committed step in C40-carotenoid biosynthesis (Britton et al., 1998; Sandmann, 2001). Subsequent desaturation reactions lengthen the conjugated double bond system to produce neurosporene or lycopene (Schmidt-Dannert, 2000).

Following desaturation, carotenoid biosynthesis branches into routes for acyclic and cyclic carotenoids. In phototrophic bacteria acyclic xanthophylls spheroidene or spheroidenone and spirilloxanthin, respectively are formed (Figure 9). Synthesis of cyclic carotenoids involves cyclization of one or both end groups of lycopene or neurosporene. Typically, P — rings are introduced, but formation of є-rings is common in higher plants and carotenoids with у-rings are found, for example, in certain fungi. Most cyclic carotenoids contain at least one oxygen function at one of the ring carbon atoms. Cyclic carotenoids with keto-groups at C4(C4′) and/or hydroxy groups at C3(C3′) (e. g. zeaxanthin, astaxanthin, echinenone and lutein) are widespread in microorganisms and plants (Schmidt-Dannert, 2000).

The contribution of each functional group can be evaluated by chemical treatment. It consists in carrying out chemical reactions that selectively block different functional groups on the sorbent surface. The most common chemical modifications are esterification of carboxylic and phosphate groups, methylation of amines, and modification of mercapto groups. Carboxylic groups can be alkylated by reaction with methanol or ethanol in acidic media, while amines by reaction with formaldehyde and formic acid. Alkylation of both functional groups prevents their participation in metal biosorption, thus reducing the biosorption efficiency.

Chemical treatments were also used to selectively extract different compounds, such as fats or polyphenols, in order to improve metal sorption. A report on the application of these methods can be found in a work of Nurchi at al., 2010.

2.1.1 Total yield

Total yield of potato tubers was significantly modified by the examined factors and their interaction (table2). The highest yields of potato tubers were harvested from the objects

fertilized with a mixture of white clover with Italian ryegrass, white clover, and phacelia both plowed down in autumn, and left till spring in the form of mulch. Only after Italian ryegrass applying total yield of potato tubers was significantly lower than recorded on control object. Straw fertilization also significantly modified the yield of potato tubers. At the sub-block with straw, potato tuber yield was significantly lower than recorded at the sub-block without straw. An interaction has been noted, which shows that the highest yield of potato tubers were obtained from the object fertilized with a mixture of white clover with Italian ryegrass and white clover with straw, and the smallest from control object, without intercrop fertilization.