Artikel Penelitian Screening Variables in Reducing the Brown Color from the Filtrate of Heavy Metal ’ s Elimination

Heavy metals contamination is a major concern in the world, and also is in Indonesia. Many attempts have been done to reduce or even eliminate it from the environment, among other ways the use of agriculture waste, such as rice straw. Why use rice straw? Because it is available abundantly. Many studies showed that rice straw could adsorb heavy metals from polluted water, and it is cheap. The drawback of rice straw is the color of the filtrate is brown, so that it cannot be used for everyday or household purposes. An attempt using enzyme has been tried to reduce the brown color and it did reduce the brown color. Enzyme L-α-arabino-furanosidase is used in this study. However, as there are many variables used in the experiments, before optimization can be conducted, a screening needs to be carried out first. Type of enzyme (optimum temperature of 50oC and 70oC), incubation time and amount of enzyme, number time of washing, water for washing, place of the rice plantation (high land and low land), and size of straw, are the variables that need to be screened. The variables that gives the highest response in this study were enzyme-50, amount of enzyme : straw = 2 : 1 (10 ml of enzyme for each 5 g of straw), 1 hour incubation time, amount of washing : 5 x 5 ml, place of plant: low land, and size of straw: ground. As for the type of washing liquid, both either demineralised water or Pb solution were the same. However, the variables are still need to be reduced, and the experiment/study will be continued to optimize the reduced variables.


Introduction
Heavy metals contamination of ground and surface water is of growing concern in many parts of the world, particularly in developing countries in which large populations have to use these sources for drinking and cooking water.Indone sia is facing substantial heavy metal pollution of ground and surface water in industrialized ar eas.A study by Sutomo et al. [1] found that there was Pb in drinking water in an area in Yogya karta and its impact on children.Yuliandari et al. [2] reported that the blood samples of pregnant women, breast feeding mothers and children un der five years old in Kenjeran area (in Surabaya) contained heavy metals such as Cd, Hg, and Pb.Kohar et al. [3] in their study on the hair of au tistic and non autistic children and adults also found an interesting results, that the hair of the autistic children contained twice as much lead as the hair of non autistic adults, and the adults hair also contained twice as much lead as in the hair of non autistic children.Joel Nigg of Oregon Health & Science University in Portland points to lead as an ideal candidate to trigger the developmental disorder called ADHD.In his article, published in Current Directions in Psychological Science, Nigg offers a causal model in which lead attaches to sites in the brain's striatum and frontal cortex, and acts on the genes in these regions causing them to turn on or remain inactive.This disrup tion in brain activity alters cognitive control, and in turn results in hyperactivity and lack of vigi lance.
Many studies have been conducted to elimi nate heavy metals from water resources, such as flocculation, filtration, using activated charcoal, and ion exchange, precipitation by chemicals, etc.However, because of the high cost of these me thods, the development of a more costeffective and environmental friendly remediation system is necessary [4].In order to find more cost-effec tive and environmental friendly methods, several studies have been carried out, by using living and dried plants, and agricultural wastes, such as soybean hulls, sugarcane bagasse, rice hulls, rice straw, barley straw, rice milling by product, etc., treated or untreated [516].
On the other hand, Indonesia also has vast number of padi fields, and the production of rice is over 50 million tons per year, and the resulting rice straw is considered agricultural waste that is usually burnt in the fields, thus yielding a lot of smog.Yet, rice straw has not received much attention as a potential remover of heavy metals so far, and the results appear to be inconclusive.Kumar and Dara [6] compared rice straw and rice husk with sugar cane bagasse, onion skin and garlic skin and found onion skin to be the best ad sorption material.Marshall et al. [9] found the ad sorption capacity of rice straw for Zn to be lower than that of soybean hulls and cottonseed hulls, but higher than that of sugar cane bagasse.Lars en and Schierup [7] used three types of barley straw (Hordeum vulgare): (1) dried and ground in a mill, referred to as straw (untreated); (2) dried, ground, boiled, and washed three times in de mineralized water, referred to as straw (washed); (3) treated as in (2) and mixed with pulverized CaCO 3 (2 : 1), referred as straw (washed) + CaCO 3 .Other authors proposed soaking in 38% NaOH solutions [6,9,12,17].It has been postulated that water treatment and alkali treatment will remove lignin and hemicelluloses, respectively, thus making the adsorptive sites more easily and abundantly available.Another issue when using plant material or agricultural waste for heavy metal adsorption is whether to use the materi als as is, or to first subject them to modification by chemical treatment.Kohar and de Zeeuw, and their co workers [18] found that rice straw as is adsorbed Cd(II) and Pb(II) from aqueous solution substantially higher than that of activated carbon and no chemical pretreatment of the straw was necessary to yield optimum results.
The particle size experiment show that at lower concentration of Pb(II) in terms of mg Pb adsorbed/g straw the differences between par ticle sizes are less significant, and there is an op timum in the amount of Pb adsorption by straw.
Indrajati Kohar, Soediatmoko Soediman, Mario, Deby Vinolia, Ni Nyoman Tri Puspaningsih, and Leon Janssen The influence of particle size of straw can be de tected only at higher initial concentration of Pb solution [19].
Although straw has been a good adsorbent for heavy metals in solution there is a drawback in using it for cleaning polluted water from heavy metals, which is the brown colour produced when straw is soaked in water.The filtrate has brown colour, and of course it cannot be used in every day needs not to say as drinking water.Some means and materials have been used to clean the filtrate from the brown colour, such as bentonite, zeolite, sand, charcoal, and carbo adsorbent [20].Actually carbo adsorbent can be used in laborato ry experiment, when nylon was used as the filter.It could adsorb the brown colour, but to filter the carbo adsorbent was another problem.It passes ordinary filter, such as filter paper or cotton cloth, and will give some blackish colour in the water.That was why it cannot be used in large or me dium scale, especially for the purpose in a con taminated well in a village, where a large nylon filter will be very costly; so cotton cloth will be more suitable for this purpose.
A preliminary study using L-α-arabino-furanosidase enzyme has been conducted in eliminating the brown colour (which is due to lignin) of the straw's extract, and it showed a satisfactory re sult, which is extracting lignin from the straw and the end result is clear water with low concentra tion of heavy metal (Pb).So, it is imperative to continue the research using the enzyme.In this research the optimization of the process will be carried out by varying on the amount of enzyme (ml), time of incubation (h), number of wash ings, and the type and size of straw.However, due to the many variables which will be optimized, screening of variables will be exercised first.

Materials
Two different types of rice straw are investi gated: rice straw from highland, i.e. rice which is grown in mountainous areas (Trawas area in Mojokerto) (1) and rice straw from lowland, i.e. rice which is grown at low altitude (collected in Surabaya).Chemicals used were Pb standard so lution 1000 ppm (Merck), demineralised water (Ubaya's Chemical Laboratory), Pb acetate p.a. (Merck), and enzyme L-α-arabino-furanosidase (from Tropical Disease Center Laboratory-Air langga University).

Experimental design
Experimental design is used to screen: the type of enzyme used (enzymes with an optimum working temperature of 50 o C = enzyme50 or 70 o C = enzyme-70) (1); the relative amount of enzyme used (2); the time of incubation of straw with enzyme (3); the number of washings after the enzyme treatment (4); the size of the straw (5); place of plantation (highland and lowland) (6); and the type of water used for washing the straw: demineralised water vs artificial polluted water (7).First step of the experimental design is to screen the variables in two replications.Using the Minitab program, an order of the experiment is defined (Table 1).
As output parameter, the following parameters will be used as the responses: the absorbance of the brown colour (the colour can be determined by spectrophotometer, by the absorbance of the brown colour) (1) and % adsorbed of Pb (the re moval of lead can be determined by ICPS) (2).

Protocol of the research
Metal concentrations are measured by atomic emission spectrometry (AES), using an inducedcoupled-plasma instrument (Fisons, model 3410+ ARL, Valencia, California-USA), with argon as (reactant) mobile gas.The wavelength for Pb emission is 283.306nm.Standard curve of Pb solution: diluting standard solution of Pb (1000 ppm) into several concentrations (1, 2, 5, 10, and 15 ppm).Pb solutions as sample: dissolving Pb acetate p.a. and diluting it to make 5 ppm aque ous solution.

Pretreating rice straw with enzyme
Five grams of rice straw (ground, half length or whole length) is wetted with the enzyme (con centration of straw (g) : enzyme (ml) = 1 : 0; 1 : 4; 1 : 8; according to the experimental design's run), and is allowed at room temperature at certain times (also in line with the experimental design's run), then washed with 25 (5 x 5) ml deminer alised water or with artificial (Pb) contaminated water (certain number of times, in line also with the experimental design's run).The filtrate of these washings will show brown colour, and the intensities of the brown colour are measured by visible spectrophotometer to detect the increas ing of brown colour with the increasing of the en zyme used.

Removal of Pb(II) by pretreated rice straw
The remaining rice straw (of 2.5) is then air dried, and is used to adsorb Pb(II) from solution.Pretreated straw (0.5%) is soaked in Pb(II) so lutions for 1 hour, then filtered through a nylon filter (Millipore; 0.45 μm -to avoid any adsorb tion of Pb(II) by filter paper), and the remaining Pb(II) in the filtrate is determined by ICPS.The % of Pb removed from the solution and the mg of Pb adsorbed/g straw are calculated using equa tion below.All the data collected were analysed by means of Minitab program.

Determination of maximum wavelength of the brown color
Determination of the maximum wavelength of the brown color was carried out by scanning the filtrate of the straw on the UV-VIS Spectropho tometer at 400600 nm, and it was obtained that the λ max . of the brown color of the straw's filtrate was 473 nm.

The activity of the pretreated straw on the adsorption of Pb(II)
The results of the treatment of straw with L-αarabinofuranosidase is in Table 3.

Minitab data analyses of experiments using
25 ml of washing liquids Figure 2. showed that enzyme-70 removed more lignin than enzyme-50; the longer the in cubation time (5 h), the higher the absorbance; the more enzyme was added, the more lignin was removed; the more the washing, the absorbance was higher; rice straw from highland released more lignin; lignin was removed easier from ground straw; and the absorbance was higher when washed with demineralised water, than with Pb(II) solution.
Figure 3. explained that when enzyme-50 (black line) and enzyme-70 (red line) were at lev el -1 (lowland), the %Pb adsorbed by the resulted pretreated straw were about the same i.e. around 45%.When they were at level 1 (highland), the %Pb adsorbed by enzyme50pretreated straw was still around 45%, but that of enzyme70pre treated straw dropped drastically to around 20%.This phenomenom can be explained, by the fact that the straw from highland was collected after it had remained for sometimes on the rice fields, getting wet from rain and dried under the sun and open air, so the condition was more fragile than the straw form lowland, and because of that it lost its lignin content easier than the straw from low land did.As it was already studied, lignin also has metaladsorbing activity.line) was at point 1 (1 x washing), %Pb adsorbed was around 50%, while Pb adsorbed of enzyme 70-pretreated straw (red line) was 25%.However, when they were at level 5 (5 times washing) the %Pb adsorbed of both pretreated straw were about 40% and 45% respectively for enzyme70 and enzyme50pretreated straw.Figure 5. also showed that when enzyme-50 and enzyme70pretreated straw were obtained by treating it with 10 ml of enzyme, the %Pb adsorbed were 45% and 40% respectively.How ever, when 40 ml of enzyme was used, the %Pb adsorbed were 50% and 30% respectively.It also showed that there were a decrease in %Pb ad sorbed in line with the increase of the intensity of the brown colour, meaning the more lignin lost it also followed by the decrease in the metal ad sorption.
The above graph (Figure 6.) showed that when straw was incubated for 1 hour with either en zyme-50 (black line) or enzyme-70 (red line), the %Pb adsorbed on the resulted pretreated straw were 50% and 40% repectively.However, when the incubation time was raise to 5 hours, the %Pb adsorbed became 45% and 30% respectively.
Figure 7. showed that when straw was incu bated at 1 hour (black line) and at 5 hours (red line), and then was washed once, the %Pb ad sorbed of the resulted pretreated straw were 40% and below 35% respectively, while when they were washed 5 times, the %Pb adsorbed of both types of the resulted straw were about the same, i.e. around 45%.
Another experiment was also carried out.The amount of the washing liquid of 50 ml was com pared to that of 25 ml, and analysed by paired t test.It showed that the results for both treatment was not significantly different.So, for cost saving, 25 ml can be used to wash the straw.
The colour of the filtrate of ground straw tends to be browner than that of half length (± 30 cm long), however, the %Pb adsorbed relatively high er that that of half length straw, due to the larger surface area of the ground straw.Eventhough the %Pb adsorbed is higher, the half length straw is Screening Variables in Reducing the Brown Color from the Filtrate of Heavy Metal's Elimination Table 3.Activity of pretreated straw on the adsorption of Pb (washing with 25 ml of washing liquid) more preferable, as the colour of the filtrate was clearer.To increase the elimination of Pb from the water, the soaking of process can be repeated sev eral times with new pretreated straw, or by soak ing the half length straw with more enzyme, so that more lignin will be removed, and the filtrate will be clearer.
Based on statistical analyses, it can be con cluded that the type of enzyme, amount of en zyme: straw, incubation time, number of wash ings, place of plant, and size of straw posses some effect on the adsorption of heavy metal.Because type of washing liquid did not influence the ad sorption process, in other words, the results of    The variables that give the highest respond in this case were enzyme50, amount of enzyme : straw = 2 : 1 (10 ml of enzyme for each 5 g of straw), 1 hour incubation time, number of wash ings: 5 x 5 ml, place of plant: lowland, and size of straw: ground.As for the type of washing liquid, both either demineralised water or Pb solution were the same (difference but not significant).
Interaction between variables also influenced the adsorption of heavy metal, so when one of the interacted variable is changed, it will affect the ad sorption process.The adsorption process might Screening Variables in Reducing the Brown Color from the Filtrate of Heavy Metal's Elimination be a physical or chemical process.In a physical process the heavy metals might be trapped in the cellulose of the straw.Intermolecular hydrogen bonds of the cellulose cause the rigidness of the cellulose and form a wovenlike structure.With such a structure, the cellulose can trapp or ad sorb the heavy metal in its pores.
The optimum condition of L-α-arabino furanosidase are either 50°C or 70°C, however, in this research the temperature was set at ambient temperature, in order to simplify the application at larger scale at a later times, because for people especially in the village, more over in a remote area, it will not be convenience to incubate the enzyme at its optimum temperature, as it will be costly and more time consuming.
The composition of straw : enzyme was 1 : 2 and 1 : 8 (by weight).Straw : enzyme = 1 : 2 was used to study whether using a relatively small amount of enzyme would still be able to yield good metal adsorption, while straw : enzyme = 1 : 8 was chosen to see whether the amount of en zyme would affect the adsorption activity toward heavy metal (Pb).
One and five hours incubation time were cho sen to find the shortest incubation time which still would be able to give good results in adsorb ing Pb.After incubation period, the straw was washed using demineralised water and Pb solu tion (5 ppm).As the research will be continued with an application to clean contaminated water in the village, it will not be convenient for those people to use demineralised water, so a model of contaminated water (i.e. 5 ppm Pb solution) was used to wash the straw in this research.
To find an appropriate amount of washing, 1 and 5 times washing were chosen as a model, while the length or the size of the straw was used in 2 level, ground and half length (or about 30 cm long), because if the results is about the same, it will be more efficient and less time consuming for the villagers to use the straw without grinding it.However, it was also found that the influence of particle size of straw can be detected only at higher initial concentration of Pb solution [6].

Conclusion
The variables that gives the highest respond in this case were enzyme-50, amount of enzyme : straw = 2 : 1 (10 ml of enzyme for each 5 g of straw), 1 hour incubation time, amount of washing: 5 x 5 ml, place of plant: low land, and size of straw: ground.As for the type of washing liquid, both either demineralised water or Pb solution were the same (different but not significant).To be more practical and less costly, to make the surface area bigger, the straw can be punched or press or shredded.So grinding is not necessary.However, the variables are still need to be reduced, and the experiment/study will be continued to optimize the reduced variables.

Figure 2 . 3 .Figure 4 .Figure 5 .
Figure 2. Main effects plot for absorbance Figure 3. Interaction between types of enzyme vs places of plant

Figure 6 .
Figure 6.Interaction between types of enzyme vs time of incubation

Figure 7 .
Figure 7. Interaction between time of incubation vs amount of washing

Table 1 .
Details of the codesScreening Variables in Reducing the Brown Color from the Filtrate of Heavy Metal's Elimination