Production whey biotechnology

The disadvantages associated with anaerobic systems are the high capital cost, long start- up periods, strict control of operating conditions, greater sensitivity to variable loads and organic shocks, as well as toxic compounds [55]. The operational temperature must be maintained at about 33-378C for efficient kinetics, because it is important to keep the pH at a value around 7, as a result of the sensitivity of the methanogenic population to low values [48]. As ammonia- nitrogen is not removed in an anaerobic system, it is consequently discharged with the digester effluent, creating an oxygen demand in the receiving water. Complementary treatment to achieve acceptable discharge standards is also required. digester. It consists of a pond, which is normally covered to exclude air and to prevent methane loss to the atmosphere. Lagoons are far easier to construct than vertical digester types, but the biggest drawback is the large surface area required. In New Zealand, dairy wastewater [51] was treated at 358C in a lagoon (26,000 m3) covered with butyl rubber at an organic load of 40,000 kg COD per day, pH of 6.8-7.2, and HRT of 1-2 days. The organic loading rate (OLR) of 1.5 kg COD/m3day was on the low side. The pond's effluent was clarified and the settled biomass recycled through the substrate feed. The clarified effluent was then treated in an 18,000 m3aerated lagoon. The efficiency of the total system reached a 99% reduction in COD.

Figure 3 Simplified illustrations of anaerobic wastewater treatment processes: (a) anaerobic filter digester, (b) fluidized-bed digester, (c) UASB digester, (d) anaerobic lagoon/pond.

Completely stirred tank reactors (CSTR) are, next to lagoons, the simplest type of anaerobic digester. According the ORL rate ranges from 1-4 kg organic matter and the digesters usually have capacities between 500 and 700 m³ . These reactors are normally used for concentrated wastes, especially those where the polluting matter is present mainly assuspended solids and has COD values. Treatment of Dairy Processing Wastewaters slowest-growing bacteria involved in the digestion process. Ross found that the HRT of the conventional digesters is equal to the SRT, which can range from 15-20 days. This type of digester has in the past been to treat cheese factory wastewater. While 90% COD removal was achieved, the digester could only be operated at a minimum HRT of 9.0 days, most probably due to biomass washout.

Figure 4 Simplified illustrations of anaerobic wastewater treatment processes: (a) onventional digester, (b) Contact digester, (c) fixed-bed digester .

This type of digester has in the past been to treat cheese factorywastewater. While 90% COD removal was achieved, the digester could only be operated at aminimum HRT of 9.0 days, most probably due to biomass washout. The anaerobic contact process (Fig 4) was developed in 1955. It is essentially an nanaerobic activated sludge process that consists of a completely mixed anaerobic reactor followed by some form of biomass separator. The separated biomass is recycled to the reactor,thus reducing the retention time from the conventional 20-30 days to,1.0 days. Because thebacteria are retained and recycled, this type of plant can treat medium-strength wastewater(200-20,000 mg/L COD) very ef?ciently at high OLRs. The organic loading rate can varyfrom 1 to 6 kg/m³day COD with COD removal ef?ciencies of 80-95%. The treatmenttemperature ranges from 30-40A major dif?culty encountered with this process is the poorsettling properties of the anaerobic biomass from the digester ef?uent. Dissolved air ?otation [61] and dissolved biogas ?otation techniques [62] have been attempted as alternative sludgeseparation techniques. Even though the contact digester is considered to be obsolete there arestill many small dairies all over the world that use the system [63].The upflow anaerobic filter (Fig 3) was developed by Young and McCarty in 1969 and is similar to the aerobic trikling filter process. The reactor is field with inert support material such as gravel, rocks, coke, or plastic media and thus there is no need for biomassseparation and sludge recycling. The anaerobic ?lter reactor can be operated either as adown?ow or an up?ow ?lter reactor with OLR ranging from 1-15 kg / M 3 day COD and CODremoval ef?ciencies of 75-95%. The treatment temperature ranges from 20 to 35 C with HRTsin the order of 0.2-3 days. The main drawback of the up?ow anaerobic ?lter is the potentialrisk of clogging by undegraded suspended solids, mineral precipitates or the bacterial biomass.Furthermore, their use is restricted to wastewaters with COD between 1000 and 10,000 mg / L. Bonastre and Paris listed 51 anaerobic ?lter applications of which ?ve were used forpilot plants and three for full-scale dairy wastewater treatment. These ?lters were operated atHRTs between 12 and 48 hours, while COD removal ranged between 60 and 98%. The OLRvaried between 1.7 and 20.0 kg COD / M 3 day.

The wastewater, consistingof 80% washing water and 20% whey, had a COD of 17,000 mg/L. While the CSTR is very useful for laboratory studies, it is hardly a practical option for full-scale treatment due to the HRT limitation. anaerobic activated sludge process that consists of a completely mixed anaerobic reactor followed by some form of biomass separator. The separated biomass is recycled to the reactor, thus reducing the retention time from the conventional 20-30 days to ,1.0 days. Because the bacteria are retained and recycled, this type of plant can treat medium-strength wastewater (200-20,000 mg/L COD) very efficiently at high OLRs . The organic loading rate can vary from 1 to 6 kg/m3day COD with COD removal efficiencies of 80-95%. The treatment temperature ranges from 30-408C. A major difficulty encountered with this process is the poor settling properties of the anaerobic biomass from the digester effluent. Dissolved air flotation and dissolved biogas flotation techniques have been attempted as alternative sludge separation techniques. Even though the contact digester is considered to be obsolete there are still many small dairies all over the world that use the system and is similar to the aerobic trickling filter process. The reactor is filled with inert support material such as gravel, rocks, coke, or plastic media and thus there is no need for biomass separation and sludge recycling. The anaerobic filter reactor can be operated either as a downflow or an upflow filter reactor with OLR ranging from 1-15 kg/m3day COD and COD removal efficiencies of 75-95%. The treatment temperature ranges from 20 to 358C with HRTs in the order of 0.2-3 days. The main drawback of the upflow anaerobic filter is the potential risk of clogging by undegraded suspended solids, mineral precipitates or the bacterial biomass. Furthermore, their use is restricted to wastewaters with COD between 1000 and 10,000 mg/L . Bonastre and Paris listed 51 anaerobic filter applications of which five were used for pilot plants and three for full-scale dairy wastewater treatment. These filters were operated at HRTs between 12 and 48 hours, while COD removal ranged between 60 and 98%. The OLR varied between 1.7 and 20.0 kg COD/m3day. The expanded bed and/or fluidized-bed digesters are designed so that wastewaters pass upwards through a bed of suspended media, to which the bacteria attach . The carrier medium is constantly kept in suspension by powerful recirculation of the liquid phase. The carrier media include plastic granules, sand particles, glass beads, clay particles, and activated charcoal fragments. Factors that contribute to the effectiveness of the fluidized-bed rocess include: (a) maximum contact between the liquid and the fine particles carrying the bacteria; (b) problems of channeling, plugging, and gas hold-up commonly encountered in packed-beds are avoided; and (c) the ability to control and optimize the biological film thickness [57]. OLRs of 1-20 kg/m3day COD can be achieved with COD removal efficiencies of 80- 87% at treatment temperatures from 20 to 358C. Toldraйt al. [67] used the process to treat dairy wastewater with a COD of only 200-500 mg/L at an HRT of 8.0 hours with COD removal of 80%. Bearing in mind the wide variations found between different dairy effluents, it can be deduced that this particular dairy effluent is at the bottom end of the scale in terms of its COD concentration and organic load. The dairy wastewater was probably produced by a dairy with very good product-loss control and rather high water use. The upflow anaerobic sludge blanket (UASB) reactor was developed for commercial purposes by Lettinga and coworkers at the Agricultural University in Wageningen, The Netherlands. It was first used to treat maize-starch wastewaters in South Africa , but its full potential was only realized after an imp ressivedevelopment program by Lettingainthela e1970s [70,71]. The anaerobic contact process was developed in 1955. The upflow anaerobic filter was developed by Young and McCarty in settling properties of a granular sludge. The growth and development of granules is the key to the success of the UASB digester. It must be noted that the presence of granules in the UASB system ultimately serves to separate the HRT from the solids retention time (SRT). Thus, good granulation is essential to achieve a short HRT without inducing biomass washout. The wastewater is fed from below and leaves at the top via an internal baffle system for separation of the gas, sludge, and liquid phases. With this device, the granular sludge and biogas are separated. Under optimal conditions, a COD loading of 30 kg/m3day can be treated with a COD removal efficiency of 85-95%. The methane content of the biogas is between 80 and 90% (v/v). HRTs of as low as 4 hours are feasible, with excellent settling sludge and SRT of more than 100 days. The treatment temperature ranges from 7-408C, with the optimum treated a synthetic ice cream wastewater using the UASB process at HRTs of 18.4 hours and an organic carbon removal of 86% was achieved. The maximum OLR was 3.06 kg total organic carbon (TOC) per m3day. Cheese effluent has also been treated in the

UASB digester at a cheese factory in Wisconsin, USA . The UASB was operated at an HRT of 16.0 hours and an OLR of 49.5 kg COD/m3day with a plant wastewater COD of 33,000 mg/L and a COD removal of 86% was achieved. The UASB digester was, however, only a part of a complete full-scale treatment plant. The effluent from the UASB was recycled to a mixing tank, which also received the incoming effluent. Although the system is described as an UASB system, it could also pass as a separated or two-phase system, since some degree of pre- acidification is presumably attained in the mixing tank. Furthermore, the pH in the mixing tank was controlled by means of lime dosing when necessary. The effluent emerging from the mixing tank was treated in an aerobic system, serving as a final polishing step, to provide an overall COD removal of 99%. One full-scale UASB treatment plant [51] in Finland at the Mikkeli Cooperative Dairy, produces Edam type cheese, butter, pasteurized and sterilized milk, and has a wastewater volume of 165 million liters per year. The digester has an operational volume of 650 m3, which includes a balancing tank of 300 m3. The COD value was reduced by 70-90% and 400 m3biogas is produced daily with a methane content of 70%, which is used to heat process water in the plant. One of the most successful full scale 2000 m3UASB described in the literature was in the UK at South Caernarvon Creameries to treat whey and other wastewaters . The whey alone reached volumes of up to 110 kiloliters (kL) per day. In the system, which included a combined UASB and aerobic denitrification system, COD was reduced by 95% and sufficient biogas was produced to meet the total energy need of the whole plant. The final effluent passed to a sedimentation tank, which removed suspended matter. From there, it flowed to aerobic tanks where the BOD was reduced to 20.0 mg/L and the NH3-nitrogen reduced to 10.0 mg/L. The effluent was finally disposed of into a nearby river. The whey disposal costs, which originally amounted to Ј30,000 per year, were reduced to zero; the biogas also replaced heavy fuel oil costs. On full output, the biogas had a value of up to Ј109,000 per year as an oil replacement and a value of about Ј60,000 as an electricity replacement. These values were, however, calculated in terms of the oil and electricity prices of 1984, but this illustrates the economic potential of the anaerobic digestion process. The fixed-bed digester contains permanent porous carrier materials and by means of extracellular polysaccharides, bacteria can attach to the surface of the packing material and still remain in close contact with the passing wastewater. The wastewater is added either at the bottom or at the top to create upflow or downflow configurations. A downflow fixed-film digester was used by Caґnovas-Diaz and Howell to treat deproteinized cheese whey with an average COD of 59,000 mg/L. At an OLR of 12.5 kg COD/ m3day, the digester achieved a COD reduction of 90-95% at an HRT of 2.0-2.5 days. The deproteinized cheese whey had an average pH of 2.9, while the digester pH was consistently above pH 7.0 . A laboratory-scale fixed-bed digester with an inert polyethylene bacterial carrier was to treat cheese whey. The best results were obtained at an HRT of 3.5 days, with 85-87% COD removal. The OLR was 3.8 kg COD/m3day and biogas yield amounted to 0.42 m3/kg CODaddedper day. The biogas had a methane content of between 55 and 60%, and 63.7% of the calorific value of the substrate was conserved in the methane. In a membrane anaerobic reactor system (MARS), the digester effluent is filtrated by means of a filtration membrane. The use of membranes enhances biomass retention and immediately separates the HRT from the SRT. Li and Corrado (80) evaluated the MARS (completely mixed digester with operating volume of 37,850 L combined with a microfiltration membrane system) on cheese whey with a COD of up to 62,000 mg/L. The digester effluent was filtrated through the membrane and the permeate discharged, while the retentate, containing biomass and suspended solids, was returned to the digester. The COD removal was 99.5% at an HRT of 7.5 days. The most important conclusion the authors made was that the process control parameters obtained in the pilot plant could effectively be applied to their full-scale demonstration plant. A similar membrane system, the anaerobic digestion ultrafiltration system (ADUF) has successfully been used in bench- and pilot-scale studies on dairy wastewaters [81]. The ADUF system does not use microfiltration, but rather an ultrafiltration membrane; therefore, far greater biomass retention efficiency is possible. Separated phase digesters are designed to spatially separate the acid-forming bacteria and the acid-consuming bacteria. These digesters are useful for the treatment of wastes either with unbalanced carbon to nitrogen (C:N) ratios, such as wastes with high protein levels, or wastes such as dairy wastewaters that acidify quickly. High OLRs and short HRTs are claimed to be the major advantages of the separated phase digester. Burgess [82] described two cases where dairy wastewaters were treated using a separated phase full-scale process. One dairy had a wastewater with a COD of 50,000 mg/L and a pH of 4.5. Both digester phases were operated at 358C, while the acidogenic reactor was operated at an HRT of 24 hours and the methanogenic reactor at an HRT of 3.3 days. In the acidification tank, 50% of the COD was converted to organic acids while only 12% of the COD was removed. The OLR for the acidification reactor was 50.0 kg COD/m3day, and for the methane reactor, 9.0 kg COD/m3day. An overall COD reduction of 72% was achieved. The biogas had a methane content of 62%, and from the data supplied, it was calculated that a methane yield (YCH4/ CODremoved) of 0.327 m3/kg CODremovedwas obtained. Lo and Liao [83,84] also used separated phase digesters to treat cheese whey. The digesters were described as anaerobic rotating biological contact reactors (AnRBC), but can really be described as tubular fixed-film digesters orientated horizontally, with internally rotating baffles. In the methane reactor, these baffles were made from cedar wood, as the authors contend that the desired bacterial biofilms develop very quickly on wood. The acidogenic reactor was mixed by means of the recirculation of the biogas. However, it achieved a COD reduction of only 4%. More importantly, the total volatile fatty acids concentration was increased from 168 to 1892 mg/L. This was then used as substrate for the second phase where a COD reduction of up to 87% was achieved. The original COD of the whey was 6720 mg/L, which indicates that the whey was diluted approximately tenfold. Many other examples of two-phase digesters are found in the literature. It was the opinionof Kisaalita et al. [85] that two-phase processes may be more successful in the treatment of lactose-containing wastes. The researchers studied the acidogenic fermentation of lactose, determined the kinetics of the process [86], and also found that the presence of whey protein had little influence on the kinetics of lactose acidogenesis [87]. Venkataraman et al. [88] also used a two-phase packed-bed anaerobic filter system to treat dairy wastewater. Their main goals were to determine the kinetic constants for biomass and biogas production rates and substrate utilization rates in this configuration.

Land Treatment

Dairy wastewater, along with a wide variety of other food processing wastewaters, has been successfully applied to land in the past [3] Interest in the land application of wastes is also increasing as a direct result of the general move of regulatory authorities to restrict was postal into rivers, lakes, and the ocean, but also because of the high costs of incineration and landfilling [9]. Nutrients such as N and P that are contained in biodegradable processing wastewaters make these wastes attractive as organic fertilizers, especially since research has shown that inorganic fertilizers might not be enough to stem soil degradation and erosion in certain parts of the world. Land application of these effluents may, however, be limited by the presence of toxic substances, high salt concentrations, or extreme pH values [89]. It might be, according to Wendorff [7], the most economical option for dairy industries located in rural areas. Irrigation The distribution of dairy wastewaters by irrigation can be achieved through spray nozzles over flat terrain, or through a ridge and furrow system [7]. The nature of the soil, topography of the land and the waste characteristics influence the specific choice of irrigation method. In general, loamy well-drained soils, with a minimum depth to groundwater of 1.5 m, are the most suitable for irrigation. Some form of crop cover is also desirable to maintain upper soil layer porosity [30]. Wastewater would typically percolate through the soil, during which time organic substances are degraded by the heterotrophic microbial population naturally present in the soil [7]. An application period followed by a rest period (in a 1:4 ratio) is generally recommended. Eckenfelder reviewed two specific dairy factory irrigation regimes. The first factory produced cream, butter, cheese, and powdered milk, and irrigated their processing wastewaters after pretreatment by activated sludge onto coarse and fine sediments covered with reed and canary grass in a 1:3 application/rest ratio. The second factory, a Cheddar cheese producer, employed only screening as a pretreatment method and irrigated onto Chenango gravel with the same crop cover as the first factory, in a 1:6 application/rest ratio.

Specific wastewater characteristics can have an adverse effect on a spray irrigation system thatshouldalsobe considered. Suspendedsolids, for instance, may clog spraynozzlesandrender the soil surface impermeable, while wastewater with an extreme pH or high salinity might be detrimental to crop cover. Highly saline wastewater might further cause soil dispersion, and a subsequent decrease in drainage and aeration, as a result of ion exchange with sodium replacing magnesium and calcium in the soil [31]. According to Sparling et al. [15] there is little published information relating the effect that long-term irrigation of dairy factory effluent may have on soil properties. Based on the irrigation data Degens et al. [91] and Sparling et al. [15] investigated the effect that long-term dairy wastewater irrigation can have on the storage and distribution of nutrients such as C, N, and P, and the differences existing between key soil properties of a long-term irrigation site (22 years) and a short-term irrigation site (2 years). Degens et al. [9] reported that irrigation had no effect on total soil C in the 0-0.75 m layer, although redistribution of C from the top 0-0.1 m soil had occurred, either as a result of leaching caused by the irrigation of highly alkaline effluents, or as a result of increased earthworm activity. The latter were probably promoted by an increased microbial biomass in the soil, which were mostly lactose and glucose degraders. It was also reported that about 81% of the applied P were stored in the 0-0.25 m layer compared to only 8% of the total applied N. High nitrate concentrations were measured in the groundwater below the site, and reduced nitrogen loadings were recommended in order to limit nitrogen leaching to the environment [9]. In contrast to the results reported by Degens et al. (2000) for a long-term irrigated site, Sparling et al. [15] found no redistribution of topsoil C in short-term irrigated soils, which was probably the result of a lower effluent loading. Generally, it was found that hydraulic conductivity, microbial content, and N-cycling processes all increased substantially in long-term irrigated soils. Since increases in infiltration as well as biochemical processing were noted in all the irrigated soils, most of the changes in soil properties were considered to be beneficial. A decrease in N-loading was, however, also recommended [15].

1.3.6 Sludge Disposal

Different types of sludge arise from the treatment of dairy wastewaters. These include: (a) sludge produced during primary sedimentation of raw effluents (the amounts of which are usually low); (b) sludge produced during the precipitation of suspended solids after chemical treatment of raw wastewaters; (c) stabilized sludge resulting from the biological treatment processes, which can be either aerobic or anaerobic; and (d) sludge generated during tertiary treatment of waste- water for final suspended solid or nutrient removal after biological treatment. Primary sedimentation of dairy wastewater for BOD reduction is not usually an efficient process, so in most cases the settleable solids reach the next stage in the treatment process directly. An important advantage of anaerobic processes is that the sludge generated is considerably less than the amount produced by aerobic processes, and it is easier to dewater. Final wastewater polishing after biological treatment usually involves chemical treatment of the wastewater with calcium, iron, or aluminum salts to remove dissolved nutrients such as nitrogen and phosphorus. The removal of dissolved phosphorus can have a considerable impact on the amount of sludge produced during this stage of treatment. The application of dairy sludge as fertilizer has certain advantages when compared to municipal sludge. It is a valuable source of nitrogen and phosphorous, although some addition of potassium might be required to provide a good balance of nutrients. Sludge from different factories will also contain different levels of nutrients depending on the specific products manufactured. Dairy sludge seldom contains the same pathogenic bacteria load as domestic sludge, and also has considerably lower heavy metal concentrations. The recognition of dairy sludge as a fertilizer does, however, depend on local regulations. Some countries have limited the amount of sludge that can be applied as fertilizer to prevent nitrates from leaching into groundwater sources. According to the IDF , dairy sludge disposal must be reliable, legally acceptable, economically viable, and easy to conduct. Dairy wastewater treatment facilities are usually small compared to sewage treatment works, which means that thermal processes such as drying and incineration can be cost prohibitive for smaller operations. It is generally agreed that disposal of sludge by land spraying or as fertilizer isthe least expensive method. If the transport and disposal of liquid sludge cannot be done within reasonable costs, other treatment options such as sludge thickening, dewatering, drying, or incineration must be considered. Gravity thickeners are most commonly used for sludge thickening, while the types of dewatering machines most commonly applied are rotary drum vacuum filters, filter presses, belt presses, and decanter centrifuges.

Pollution prevention

Reduction of wastewater pollution levels may be achieved by more efficiently controlling water and product wastage in dairy processing plants. Comparisons of daily water consumption records vs. the amount of milk processed will give an early indication of hidden water losses that could result from defective subfloor and underground piping. An important principle is to prevent wastage of product rather than flush it away afterwards. Spilled solid material such as curd from the cheese production area, and spilled dry product from the milk powder production areas should be collected and treated as solid waste rather than flushing them down the drain [6]. Small changes could also be made to dairy manufacturing processes to reduce wastewater pollution loads, as reviewed by Tetrapak [6]. In the cheese production area, milk spillage can be restricted by not filling open cheese vats all the way to the rim. Whey could also be collected sparingly and used in commercial applications instead of discharging it as waste. Manual scraping of all accessible areas after a butter production run and before cleaning starts would greatly reduce the amount of residual cream and butter that would enter the wastewater stream. In the milk powder production area, the condensate formed could be reused as cooling water (after circulation through the cooling tower), or as feedwater to the boiler.Returned product could be emptied into containers and used as animal feed [6]. Milk and product spillage can further be restricted by regular maintenance of fittings, valves, and seals, and by equipping fillers with drip and spill savers. Pollution levels could also be limited by allowing pipes, tanks, and transport tankers adequate time to drain before being rinsed with water [8].

2. EXPERMENTAL PART

Milk whey is an important by-product of the dairy industry, nutrient-rich and potentially used as a growth medium for the production of commercial products. Many fermentation processes can be applied to whey, from using recombinant bacteria to mixed consortium. The latter often requires careful monitoring. Monitoring and control of biotechnological processes based on complex media is often expensive and tricky, and usually involve only physical-chemical parameters. When a mixed culture is involved, microbiology and molecular biology can provide a more complete view of biological variation within the bioreactor. In this work, a new monitoring technique was applied to an enriched mixed culture in a milk whey fermentation. The presence of the desired product PHA (polyhydroxyalkanoate), a biodegradable biopolymer, was evaluated throughout the SBB (Sudan Black B) staining. This cheap and easy method, usually employed in isolation of PHA storing bacteria, was useful in monitoring the fermentation process, not only to identify the recovery time but also for the assessment of the percentage of useful microorganisms present. Whey, a by-product of diary and cheese industry, constitutes the watery portion after the separation of fat and caseins from whole milk. Cheese whey is a surplus material produced in volumes almost equal to the milk processed in cheese manufactories, therefore its disposal as a waste causes serious pollution problems in the surrounding environment where itґs discarded. This is due to its enormous biochemical oxygen demand that is mainly caused by its high lactose content; as a consequence a large amount of industrial capital is requested for whey disposal. During the last years, the amounts of whey increased to such an extent that they cannot be simply used as animal feed as the most common application. To overcome these problems a sustainable alternative is to upgrade whey and its derivates to a resource for many value added industrial products, making whey not only a waste but also a valuable resource.

2.1 Production of PHAs using fermentation milk whey

Materials made from synthetic polymers are not biodegradable and are often improperly discarded. These materials are typically derived from petroleum-based plastics. Rapid progress in materials science technology has created new plastic products with favorable mechanical integrity and excellent durability. Nevertheless, plastic products usually have single-use applications, especially in food packaging and medical materials. Because these plastic products are not biodegradable, they are extremely persistent and accumulate in the ecosystem, resulting in a significant burden on solid waste management. For this reason European countries are trying to reduce oil consumption for plastic bags production, bringing it from the 100 to 60 million barrels/year . Polymers have a wide range of properties, which make them suitable for many uses and at the same time very difficult to replace. One of the most promising alternatives to oil produced polymers is the development of new biodegradable polymers ones. During recent years these materials have become more and more important on the worldwide market: the PlasticsEurope research group is expecting a yearly 16.6% increase in USA demand for biopolymers, for a total expected demand of 1.48. 105 tons in 2014 . Among the various types of biodegradable plastics, PHAs are the most studied, being recognized as completely biosynthetic and biodegradable, with zero toxic waste, and completely recyclable into organic waste. They are microbial polyesters produced by a wide range of microorganisms, under unbalanced growing conditions, mostly as intracellular storage compounds for energy and carbon. Their properties span a wide range, including materials that imitate thermoplastic properties and others that possess electrometric properties.

Nowadays PHA price is affected mainly by fermentation medium, extraction and purification costs (Bosco and Chiampo 2010). Many papers have addressed this issue in recent years (Lee 1996, Masani al. 2008, Loo et al. 2007).

Milk whey is now a high BOD waste but, since it has the proper composition for PHA production its use will serve a double purpose: to lower PHA production costs and to reduce the amount of waste produced by the cheese industry (Panesar et al. 2007, Jelen 2003, Kemp et al. 1989).

In this work, PHA production was carried out both in controlled and uncontrolled STR fermentation tests. A new monitoring approach was tested: the investigation through SBB staining technique of the PHA production.

2.1.1 Materials and methods

Biomass for PHA production was taken from a dairy plant activated sludge and enriched in a synthetic medium (Khardenavis et al. 2007). Biomass in exponential growth phase (about 75 hours) was collected and used as inoculum (10% v/v) for PHA production cultures in a 10 l bioreactor. K-mol fermentation medium (Bosco and Chiampo, 2010) with added deproteinized milk whey powder (Molkolac®, Milei GmbH) containing an initial lactose concentration of 20 g/l was employed. Tests were conducted either in uncontrolled and controlled conditions (30°C, pH 7±0,02). Cell growth was monitored by measuring the optical density (OD) at 620 nm (HP 8452A Diode Array Spectrophotometer). Culture broth samples were taken at different times, biomass was harvested by centrifugation (18000 rpm, 10 min, 4°C), in pre-weighted glass tubes and dried to constant weight at 60°C for 48 hours. The polymer was extracted from dried biomass according to the chloroform-hypochlorite method described by Hahn et al. (1994). PHA content (weight percentage) was defined as PHA concentration to cell concentration ratio. Plates were made starting from different fermentation time samples (10ml every 24 hours). Serial dilutions, 10-5 or 10-6 , were prepared using 0,9% NaCl solution; 100µl of diluted samples were spread on malt agar plates containing: malt extract 20g/l, D-glucose 20g/l, peptone 2g/l, agar 20g/l. After two to seven days incubation at 30°C, potential PHA producers were detected by SBB staining of the colonies. According to de Lima et al. (1999), a 0.02% SBB solution in ethanol 97% was gently spread over the plates, completely soaking them, incubated at room temperature for 30-60 min, then discarded and washed with ethanol 97%. PHA producers tend to be stained dark-blue or black, ьwhile negative PHA accumulators remains white or light-blue. Counting of the different kind of colonies (white and coloured) was performed.

2.2 Results and Discussion

The aim of this work is the evaluation of SBB staining as an effective method to optimize process parameters of PHA production fermentations. Staining was applied to uncontrolled and controlled STR batch fermentations (30°C, pH 7 ± 0,02); various tests were carried out with different initial lactose concentration and C/N ratio; in the current tests lactose concentration was 20 g/L and C/N ratio value was 50. OD analysis on uncontrolled and controlled fermentations showed different trends: faster growth of controlled culture is clear, stationary phases were comparable but reached at different times, 70 hours and 20 hours respectively (Figure 1). The lactose concentration, in fact, decreased significantly in the first 24 hours of controlled fermentation, reaching a value of 5.62 g/l and 1.3 g/l after 22 and 27 hours respectively, whereas was still 18,17 g/l at 28 hours of uncontrolled fermentation, becoming 10,72 g/l at 96 hours (Rella 2009 master thesis). Trends of ammonium consumption were also significantly different, its depletion occurred at 27 hours of controlled fermentation, while in uncontrolled one at 96 hours ammonium concentration was still 0,107 g/l. The maximum polymer yield, as well as the maximum concentration of biomass, obtained is much higher in controlled fermentation (41.20% g PHA/g dried biomass; 1.8 g/l) (Figure 2) than in uncontrolled one (38,01% g PHA/g dried biomass; 1.04 g/l) and is constant over time after reaching the stationary phase.

Figure 1: OD and lactose of uncontrolled and controlled cultures

To investigate the percentage of PHA producers, in each trial, microbiological analysis were performed. Through SBB staining is possible to make a cheap and easy qualitative estimate of the biopolymer content during fermentation. To evaluate PHA producers percentage present in the biomass during fermentation, samples were withdrawn at different times, plated on malt agar and incubated 2 to 7 days at 30°C. After SBB staining, coloured colonies counting returned the percentage of PHA producers. The plates presented different kind of colonies with distinct morphology and growth rate, roughly representative of the producers and non-producers population inside the mixed culture. During the culture's stationary phase, when PHA content was the highest and the carbon source was in excess respect to the nitrogen one, only half biomass in the reactor seemed constituted by biopolymer producers. Moreover, by plotting these percentage along with PHA percentage yields, and concentration, was possible to observe a good correlation between curves (Figure 2). This result confirms the hypothesis reported in a previous paper (Bosco et al. 2008). From controlled SBR fermentation plates, it's possible to remark that the ratio of PHA producers obtained in the stationary phase is different form the one obtained in the enrichment phase. This could suggest the presence of a limiting step either during the fermentation or the enrichment phase. The enrichment phase selectivity could be enhanced. Although initial pH is adjusted to 7.0, it isn't corrected during the preculture step: in this phase pH usually increases, selecting a biomass adapted to higher pH. Furthermore, a different carbon source was used in the enrichment and in the SBR tests (acetic acid and lactose, respectively) forcing microorganisms to metabolise different substrates, with different yields. In order to better evaluate these findings further investigation will be necessary. Comparing controlled and uncontrolled SBR plated samples, it becomes clear that same percentages of PHA-producers were achieved earlier in controlled SBR tests (46 and 70 hours respectively); the same can be said about nutrients consumption and maximum OD value.

Figure 2: %PHA producers, Yield % and PHA concentration of controlled fermentation

These values obtained by SBB staining reflect the similarity between yield values obtained with controlled and uncontrolled runs. The advantage of controls is that biomass concentration is higher, leading to a higher polymer production.

2.2.1 Conslusion of results

In this work were carried out two types of fermentation: uncontrolled and controlled one. Starting with the same medium composition (lactose 20 g/l, C/N 50) temperature and pH were set at 30°C and 7 respectively in controlled fermentation while only initial pH was set in uncontrolled one. In controlled fermentation stationary phase was reached at 20 hours, nutrients consumption depletion occurred earlier and yield was higher (41,2%) than in uncontrolled trials; from these results comes out that controlled process gives advantages in terms of shorter fermentation times. During the tests OD, pH, lactose, ammonium concentration were monitored; together with them, SBB staining technique was applied in order to investigate the trend of PHA producers presence. Exploiting this parameter allowed us to determine the moment with the highest PHA concentration. The application of SBB staining technique was successful, showing that PHA producers trend matches with PHA yield and concentration ones.

LIFE SAFETY

The wastewater-treatment industry has three major safety categories: confined-space entry; lockout/tagout; and personal protective equipment (PPE). All three safety concerns cover specific issues, and all are equally important. Methods of defense against some of these life-threatening conditions include air monitoring, proper ventilation, respiratory protection and fall protection.
Confined-space entry issues are closely monitored to ensure that employees are properly trained and follow the strict, OSHA-regulated wastewater-treatments facility guidelines.

In the wastewater-treatment industry, confined-space hazard awareness can mean the difference between life and death. Depending on individual sites, the following locations have the potential to be considered confined spaces in a wastewater-treatment facility: aeration basins,digesters,applicator machines, primary tanks, manholes, vaulted sampling pits.

Several of these locations are below ground level and have stair entry for access to routine maintenance, inspection, testing, sampling and repairs. The level of fall protection necessary depends on the facility, its required activities, and the job tasks being performed. Full-body harnesses, ladder-safety systems, tripods and hoists are among the more important fall-protection products. Although some of the above locations might not be deemed a confined space according to regulations, many facilities lean toward the side of safety and do treat them as confined spaces.

A permit-required confined space is defined as a confined space that:

· contains or has a potential to contain a hazardous atmosphere;

· contains a material that potentially could engulf an entrant;

Identifying and properly marking a confined space is a major step toward providing safety for wastewater-treatment facility employees. A wastewater-treatment facility presents employees with a variety of personal hazards. Employees depend on personal protective equipment to protect themselves from hazards and perform daily duties. PPE includes but is not limited to safety glasses, face shields, hard hats, gloves, foot protection, and durable and disposable chemical-protective clothing. Respirators and fall protection might also be required. However, respirators and fall protection fall under separate OSHA standards.

To properly determine what types of PPE employees need to help follow wastewater-treatment facility guidelines, the employer is required under the revised personal protective standard, 29 CFR 1910.132, to perform a hazard assessment or a walk-through survey of each work area and certify that it has been done. The survey should consider impact, penetration, compression (roll-over), chemicals, heat, harmful dust and light (optical) radiation. After the survey, the employer should select the proper PPE to suit the hazard.

CONCLUSIONS

As management of dairy wastes becomes an ever-increasing concern, treatment strategies will need to be based on state and local regulations. Because the dairy industry is a major water user and wastewater generator, it is a potential candidate for wastewater reuse. Purified wastewater can be utilized in boilers and cooling systems as well as for washing plants, and so on. Even if the purified wastewater is initially not reused, the dairy industry will still benefit directly from in-house wastewater treatment, since levies charged for wastewater reception will be significantly reduced. In the United Kingdom, 70% of the total savings that have already been achieved with anaerobic digestion are due to reduced discharge costs. The industry will also benefit where effluents are currently used for irrigation of pastures, albeit in a more indirect way.

All these facts underline the need for efficient dairy wastewater management. Before selecting any treatment method, a complete process evaluation should be undertaken along with economic analysis. This should include the wastewater composition, concentrations, volumes generated, and treatment susceptibility, as well as the environmental impact of the solution to be adopted. All options are expensive, but an economic analysis may indicate that slightly higher maintenance costs may be less than increased operating costs. What is appropriate for one site may be unsuitable for another. The most useful processes are those that can be operated with a minimum of supervision and are inexpensive to construct or even mobile enough to be moved from site to site. The changing quantity and quality of dairy wastewater must also be included in the design and operational procedures. From the literature it appears as if biological methods are the most cost- effective for the removal of organics, with aerobic methods being easier to control, but anaerobic methods having lower energy requirements and lower sludge production rates. Since no single process for treatment of dairy wastewater is by itself capable of complying with the minimum effluent discharge requirements, it is necessary to choose a combined process especially designed to treat a specific dairy wastewater.

All wastewater treatment systems are unique. Before a treatment strategy is chosen, careful consideration should be given to proper wastewater sampling and composition analysis as well as a process survey. This would help prevent an expensive and unnecessary or overdesigned treatment system. A variety of different local and international environmental engineering firms are able to assist in conducting surveys. These firms can also be employed to install effective patented industrial-scale installations for dairy processing wastewater treatment.

Therefore theme of my course work abouttreatment of dairy processing wastewaters and

REFERENCES

1. Russell, P. Effluent and waste water treatment. Milk Ind. Int. 1998, 100 (10), 36-39.

2. Strydom, J.P.; Mostert, J.F.; Britz, T.J. Effluent production and disposal in the South African dairy industry-a postal survey. Water SA 1993, 19 (3), 253-258.

3. Robinson, T. The real value of dairy waste. Dairy Ind. Int. 1997, 62 (3), 21-23.

4. Kessler, HG. (Ed.) Food Engineering and Dairy Technology; Verlag: Freisburg, Germany, 1981.

5. Odlum, C.A. Reducing the BOD level from a dairy processing plant. Proc. 23^rd Int. Dairy Cong., Montreal, Canada, October 1990.

6. Tetrapak. TetraPak Dairy Processing Handbook; TetraPak Printers: London, UK, 1995.

7. Wendorff, W.L. Treatment of dairy wastes. In Applied Dairy Microbiology, 2nd ed.; Marth, E.H., Steele, J.L., Eds.; Marcel Dekker Inc: New York, 2001; 681-704.

8. Steffen, Robertson, Kirsten Inc. Water and Waste-water Management in the Dairy Industry, WRC Project No. 145 TT38/89. Water Research Commission: Pretoria, South Africa, 1989.

9. Tamime, A.Y.; Robinson, R.K. (Eds.) Yoghurt Science and Technology; Woodhead Publishing Ltd: Cambridge, England, 1999.

10. Danalewich, J.R.; Papagiannis, T.G.; Belyea, R.L.; Tumbleson, M.E.; Raskin, L. Characterization of dairy waste streams, current treatment practices, and potential for biological nutrient removal. Water Res. 1998, 32 (12), 3555-3568.

11. Bakka, R.L. Wastewater issues associated with cleaning and sanitizing chemicals. Dairy Food Environ. Sanit. 1992, 12 (5), 274-276.

12. Vidal, G.; Carvalho, A.; Meґndez, R.; Lema, J.M. Influence of the content in fats and proteins on th anaerobic biodegradability of dairy wastewaters. Biores. Technol. 2000, 74, 231-239.

13. Andreottola, G.; Foladori, P.; Ragazzi, M.; Villa, R. Dairy wastewater treatment in a moving bed biofilm reactor. Wat. Sci. Technol. 2002, 45 (12), 321-328.

14. Strydom, J.P.; Britz, T.J.; Mostert, J.F. Two-phase anaerobic digestion of three different dairy effluents using a hybrid bioreactor. Water SA 1997, 23, 151-156.

15. Sparling, G.P.; Schipper, L.A.; Russell, J.M. Changes in soil properties after application of dairy factory effluent to New Zealand volcanic ash and pumice soils. Aust. J. Soil. Res. 2001, 39, 505-518.

16. Hwang, S.; Hansen, C.L. Characterization of and bioproduction of short-chain organic acids from mixed dairy-processing wastewater. Trans. ASAE 1998, 41 (3), 795-802.

17. Donkin, J. Bulking in aerobic biological systems treating dairy processing wastewaters. Int. J. Dairy Tech. 1997, 50, 67-72.

18. Samkutty, P.J.; Gough, R.H. Filtration treatment of dairy processing wastewater. J. Environ. Sci. Health. 2002, A37 (2), 195-199.

19. Ince,O. Performance of atwo-phase anaerobic digestion systemwhen treatingdairy wastewater. Wat. Res. 1998, 32 (9), 2707-2713.

20. Ince, O. Potential energy production from anaerobic digestion of dairy wastewater. J. Environ. Sci. Health. 1998, A33 (6), 1219-1228. Treatment of Dairy Processing Wastewaters

21. Torrijos, M.; Vuitton, V.; Moletta, R. The SBR process: an efficient and economic solution for the treatment of wastewater at small cheese making dairies in the Jura Mountains. Wat. Sci. Technol. 2001, 43, 373-380.

22. Malaspina, F.; Cellamare, C.M.; Stante, L.; Tilche, A. Anaerobic treatment of cheese whey with a downflow-upflow hybrid reactor. Biores. Technol. 1996, 55, 131-139.

23. Burton, C. FOG clearance. Dairy Ind. Int. 1997, 62 (12), 41-42.

24. Berruga, M.I.; Jaspe, A.; San-Jose, C. Selection of yeast strains for lactose hydrolysis in dairy effluents. Int. Biodeter. Biodeg. 1997, 40 (2-4), 119-123.

25. Robinson, T. How to be affluent with effluent. The Milk Ind. 1994, 96 (4), 20-2

26. Gough, R.H.; McGrew, P. Preliminary treatment of dairy plant waste water. J. Environ. Sci. Health. 1993, A28 (1), 11-19.

27. Droste, R.L. (Ed.) Theory and Practice of Water and Wastewater Treatment; John Wiley & Sons Inc: New York, USA, 1997.

28. Hemming, M.L. The treatment of dairy wastes. In Food Industry Wastes: Disposal and Recovery; Herzka, A., Booth, R.G., Eds.; Applied Science Publishers Ltd: Essex, 1981.

29. Graz, C.J.M.; McComb, D.G. Dairy CIP-A South African review. Dairy, Food Environ. Sanit. 1999, 19 (7), 470-476.

30. IDF. Balance tanks for dairy effluent treatment plants. Bull. Inter. Dairy Fed. 1984, Doc. No. 174.

Размещено на Allbest.ru