Research Journal of Recent Sciences ______ ______________________________ ______ ____ ___ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 234 - 240 (201 3 ) Res.J. Recent .Sci. International Science Congress Association 234 Studies on the G eneration of B iogas from C ollagen H ydrolysate obtained from C hrome S havings by A lkaline H ydrolysis: A G reener D isposal M ethod Pati Anupama and Chaudhary Rubina School of Energy a nd Environmental Studies, Devi Ahilya University, Khandwa Roa d, Indore , MP , INDIA Available online at: www.isca.in Received 30 th Octo ber 2012 , revised 2 8 th December 2012 , accepted 29 th January 201 3 Abstract The leather industry produces a significant amount of chromium bearing h azardous waste. Solid waste disposal is increasingly becoming a huge challenge to tanners due to paucity of landfill sites and strict environmental legislations worldwide. Hence, finding a holistic solution to the tannery solid waste disposal problem is a challenge for researchers. Protein based solid wastes acquire much attention due to its high value. In this work, proteinous matter recovered from chrome shaving through chemical process was feed to the anaerobic digester to generate biogas. Two different modes of alkaline chromed shaving dust hydrolysis i.e. using lime and KOH followed by neutralization with HCl and H 3 PO 4 subsequently. The Full scale investigations conducted to evaluate the performance of anaerobic digestion of collagen hydrolysate. The fe ed and overflow of both digesters were monitored for various parameters such as t otal solids %, a sh content %, v olatile f atty a cids and c hemical o xygen d emand (COD). COD reduction efficiency 34 - 46 % and 45 - 54% was observed in lime - HCl and KOH - H 3 PO 4 digeste rs respectively. Gas generation is increased 30% in KOH - H 3 PO 4 digester as compared to Lime - HCl and CO 2 % in KOH - H 3 PO 4 digester was in the range of 19 - 20% as compared to Lime - HCl were it was 30 - 40%. Hence, it shows that CO 2 produced by the acidogens is effe ctively utilized by the methanogens in producing methane gas and potassium and phosphates are the macronutrients to the microorganisms. Keywords: Chrome shavings, h ydrolysis, c ollagen hydrolysate, b iogas, l eather processing . Introduction The global e nvironment is gradually deteriorating as a result of the socio - economic activities of mankind. Among them, processing industries causes adverse changes in the environment, where, leather industry is one of them. Leather and environment can be described as two sides of the coin despite the leather industry making traceable and visible impacts on socio - economic through employment and export earning; the industry has gained a negative image in society owing to the resulting pollution 1 . In the tanning industr y, raw skin is transformed into leather by means of a series of chemical and mechanical operations. Environmental challenges from leather processing arise from both the nature and the quantum of wastes discharged 2 - 5 . There are about 1600 tanneries located in India and are distributed mainly in four states, viz. Tamil Nadu, West Bengal, Uttar Pradesh and Punjab with a processing capacity of 0.9 million tons of raw hides and skins 6 . The leather - making process generates substantial quantities of solid, liquid , and gaseous wastes 7 f igure 1. Many cleaner processing approaches aimed at the reduction of liquid and solid wastes proved to be economically and environmentally beneficial 8 - 15 . Chromium has been used as a predominant tanning agent approximately 85% of a ll leather produced in the world. However, due to the uniqueness of the chromium tanning and properties that chrome tanning confers to the resulting products 16 - 18 . It has been estimated that about 0.8 million tons of chrome shavings could be generated per year globally. This waste is partly used in the manufacture of leather board, but most are normally disposed of in landfill sites, wasting all the contained resources 19 - 20 . This waste presents low compaction ability and density, therefore presenting high land filling costs per mass unit. Furthermore, the cost of disposal increases as the hazardous nature of the waste increases. The literature suggests that chromium - tanned leather waste may be managed through more sustainable technological alternatives, nam ely by wet treatments. Hydrolysis of shavings with alkalis such as CaO, NaOH and MgO at moderate temperatures and oxidative de - chroming were studied to recycle chrome tanned solid waste 21 - 30 . In present study, process developed to separate collagen hydrol ysate and chromium by alkaline hydrolysis. Collagen hydrolysate is feed for anaerobic digester, and chromium after further processing is recycled as basic chromium sulphate and reused in tanning operation. The obtained hydrolysate is protein rich and has a n approx imate . C:N ratio of 3:1. Experiments were conducted to evaluate the anaerobic digestion of collagen hydrolysate after two different modes of alkaline hydrolysis i.e. using lime and KOH followed by neutralization with HCl and H 3 PO 4 subsequently. The efficiency of protein separation from chrome shaving by both the method has been studied. Collagen hydrolysate from both the process was used as feed in anaerobic digester and gas generation was measured. Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 234 - 240 (201 3 ) Res.J.Recent.Sci International Science Congress Association 235 Figure - 1 Molecular structure of chrome - tanned l eather and shavings Material and M ethods Fresh chrome leather solid waste – chrome shaving was collected from a leading commercial tannery in India. All the chemicals used for the analysis were of analytical grade. Characterization of Chrome shavings: T he collected chrome shavings were characterized for moisture, chrome content, inorganic ash, nitrogen, oils and fats, pH and apparent density as per standard procedures 31 - 37 table 1. Table - 1 Characteristics of chrome shaving* Parameters Values % Moisture content 24.1  0.6 % Cr 2 O 3 3.12  0.12 % Inorganic ash 5.21  0.11 % Nitrogen 16.4  0.1 % Oils and fat 1.5  0.05 pH (10% aqueous leather) 3.4  0.2 Apparent density 0.91 g/mL *based on dry weight , Number of replicates 3 Hydrolysis of chrome shavings : Colla gen hydrolysates were prepared by treating shaving dust with lime and KOH separately in aqueous media at 90 - 95 0 C temperature for 4 hrs. The hydrolysates after cooling were filtered and neutralized to 6.8 - 7.2 pH with HCl and H 3 PO 4 respectively . To maintain uniformity both hydrolysates were diluted to 3 % t otal s olids. Analysis results of both the hydrolysates are summarized in table - 2. Biomethanation of collagen hydrolysate : Two separate anaerobic continuous digesters of 20 liters effective volume each star ted with starter culture of bottom sludge of existing biomethanation plant . After incubation for 3 days KOH - H 3 PO 4 and lime - HCl hydrolysate were charged in lab scale anaerobic digester. Initially, 500 ml feeding/day started andtemperature maintained at arou nd 32 - 35 0 C throughout the experiments. Gas volume measured every day using water displacement columns. Input and output samples of both digesters monitored for pH, chemical oxygen demand, total solids, % ash content, volatile f atty a cids and density of mic robial population. Analysis result of feed and overflow of both digesters were given in table - 3. Table - 2 Analysis result of KOH - H 3 PO 4 and Lime - HCl hydrolysates Sr. No. Test Parameter KOH - H 3 PO 4 Hydrolysate Lime - HCl hydrolysate 1. pH 7.12 7.06 2. % Total solids 3.23 3.12 3. % Ash content 20.1 16.3 4. VFA (as such) 1260 1138 5. SS mg/L (as such) 1240 971 6. VSS mg/L (as such) 240 155 7. Sulphate mg/L (as such) 2163 1025 8. COD mg/L (as such) 27190 26241 Table - 3 Analysis result of feed and overflow o f both hydrolysates digester Test Parameter KOH - H 3 PO 4 Lime - HCl Feed Overflow Feed Overflow PH 7.12 7.76. 7.06 7.5 % Total solids 3.23 1.02 3.12 1.24 % Ash content 20.1 52.6 16.3 46.5 VFA (as such) 1260 3726 1138 2178 COD mg/L (as such) 27190 14388 2 6241 17220 Microbial population Moderate growth Heavy growth Scanty growth Moderate growth Microbial Study : Microbial population in both samples varied in terms of number and type as seen under microscope and was confirmed through biochemical tests of h ydrolysis, decarboxylation and deamination by bacteria. Recovery and reuse studies : Protein and chromium derived from chrome shavings were used as feed in anaerobic digester and chrome tanning in re - chroming process of leather making. The chromium bearing residue of chromium (III) hydroxide was treated with sulphuric acid till pH 2.8, left overnight and the basicity of the chromium (III) sulphate estimated by standard procedures 37 - 38 . Results and Discussion Characterization of Chrome shavings: The charac teristics of chrome shaving from a c ommercial tannery are given in t able 1. The amount of moisture, chromium (as Cr 2 O 3 on dry weight basis), % inorganic ash, % nitrogen, %oils and fat present in the Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 234 - 240 (201 3 ) Res.J.Recent.Sci International Science Congress Association 236 chrome shavings are 24.1, 3.12, 5.21, 16.4 and 1.5, respe ctively. The pH of aqueous leather and apparent density of chrome shavings used in this study was 3.4 and 0.94 g/ml. Optimization of hydrolysis of collagen hydrolysates: It was observed that under aerobic conditions putrefaction of KOH - H 3 PO 4 gelatin is f aster than Lime - HCl gelatin. Both hydrolysates were kept in a thermostat at 30 - 35 o C temperature for almost two weeks and changes in parameters like pH, v olatile f atty a cids, s uspended s olids and v olatile s uspended s olids were studied after every four days figure 2 - a, 2 - b, 2 - c, 2 - d. Spectrophotometric turbidity analysis confirmed that the growth of bacteria is faster in KOH - H 3 PO 4 hydrolysate. Increase in v olatile s uspended s olids also confirms faster growth of micro - organisms in KOH - H 3 PO 4 hydrolysates as mor e and more total dissolved solids gets hydrolyzed and converted to v olatile s uspended s olids by bacteria. Analysis results are given in table - 4 . Gas Analysis : Gas volume was monitored for almost 6 months and an increase of average 25 - 30% biogas in KOH - H 3 P O 4 digester was observed. One month analysis results of both digesters are shown in figure 3. Gas samples of both digesters checked for CO 2 and H 2 S % using Gastec Standard Tube Detector System from Gastec Corporation 6431 Fukaya, Ayase - Shi, Kanagawa 252, J apan using tube no. 2HH for CO 2 and 4HH for H 2 S. Throughout the experiment CO 2 % in KOH - H 3 PO 4 digester was found 25 - 35% against Lime - HCl route were it was 30 - 40%, whereas H 2 S was 2 - 3% and 1 - 2% respectively. The CO 2 result of KOH - H 3 PO 4 digester further co nfirmed in a 1 ton digester of similar feed with improved digester design having more surface area contact and was observed the similar result as mentioned above. Increase of average 25 - 30% biogas observed in KOH - H 3 PO 4 digester as compared to Lime - HCl oper ated under same experimental conditions. Table - 4 Analysis of both KOH - H 3 PO 4 andLime - HCl hydrolysates Sr. No. Parameter KOH - H 3 PO 4 gelatin Lime - HCl gelatin 1 st day 4 th day 8 th day 12 th day 16 th day 1 st day 4th day 8 th day 12 th day 16 th day 1 pH 7.12 7.85 7.96 8.15 8.11 7.06 7.48 7.46 7.48 7.33 2. VFA 1260 2377 1985 1462 40 1138 183 400 610 1340 3. SS 1240 1340 1 691 1821 5691 971 1014 1116 1343 1431 4. VSS 240 1120 1744 1785 1791 155 217 288 290 301 Table - 5 Microbial Study of both digester overfl ow Bacterial arrangements Enzyme produced Chemical nature Gram positive rods in chains Protease producing Deaminating Gram positive rods singly arranged Protease producing Deaminating Gram positive very short rods with spores Protease producing Deamin ating Gram positive very thin long rods Protease producing Deaminating Gram positive rods in clusters - Deaminating Gram positive short rods with rounded ends Protease producing Deaminating Figure 2 - a pH of KOH - H 3 PO 4 and Lime - HCl digester Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 234 - 240 (201 3 ) Res.J.Recent.Sci International Science Congress Association 237 Figure 2 - b VFA of KOH - H 3 PO 4 and Lime - HCl digester Figure 2 - c SS of KOH - H 3 PO 4 and Lime - HCl digester Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 234 - 240 (201 3 ) Res.J.Recent.Sci International Science Congress Association 238 Figure 2 - d VSS of KOH - H 3 PO 4 and Lime - HCl digester Figure 3 Research Journal of Recent Sciences ______ _ _ _______________________________ ______________ _ ________ ISSN 2277 - 2502 Vol. 2 ( ISC - 2012 ), 234 - 240 (201 3 ) Res.J.Recent.Sci International Science Congress Association 239 Volume of gas in KOH - H 3 PO 4 and Lime - HCl digester Microbial Study : Analysis confirmed the growth of bacteria is faster in KOH - H 3 PO 4 hydrolysate. This is further confirmed by increase in Volatile Suspended Solids value. It seems negatively charged phosphate ion plays an important role in energy metabolism. It is an important part of biologically impor tant molecules ATP and nucleic acid. The amount of phosphate that dissolves depends primarily on the amount of acid that themicroorganisms produce in their metabolic processes. Microorganisms secrete protease enzymes that hydrolyze proteins and polypeptide s to amino acids, which are transported in the cells and catabolized. The first step in amino acid utilization is deamination, the removal of the amino group from an amino acid. This is often accomplished by transamination (amino group is transferred from an amino acid to an alpha keto acid acceptor). The organic acid resulting from deammination can be co nverted to Pyruvate, Acetyl - CoA , TCA cycle intermediate and eventually oxidized in the TCA cycle to release energy. It can be used as a source of carbon fo r the synthesis of cell constituents. Bacterial count of diluted samples of same concentrations of both digesters showed variations with higher count in KOH - H 3 PO 4 digester. In both digesters gram positive rod shaped bacteria of varying arrangements are fo und and it is observed that their growth is faster in KOH - Phosphate digester. This contributes to high speed putrefaction of KOH - Phosphate gelatin. Excess nitrogen from deamination may be excreted as ammonium ions, thus making the medium alkaline. The bact erial arrangements and their chemical nature have given in table5.Since it is difficult to maintain strict anaerobic conditions in Lab. Methanogens are not considered in this study. Inference related to methanogens was made indirectly by methane gas readin gs. Reuse of chromium in tanning process : Protein and chromium derived from chrome shavings were used as feed in anaerobic digester and chrome tanning in re - chroming process of leather making. The chromium bearing residue of chromium (III) hydroxide was treated with sulphuric acid till pH 2.8, left overnight. The chromium (III) sulphate solution was used in tanning as a 40% replacement for basic chromium sulphate (BCS) salt as per conventional procedures and the leathers evaluated against control leathers tanned using conventional chromium salts. Conclusion Waste management approaches are gaining importance in all the industries for sustainability. In this study, an attempt has been made to extract and reuse protein and chromium from chrome shaving throu gh two different chemical hydrolysis methods. Thus growth of micro - organism is faster in KOH - H 3 PO 4 gelatin than the Lime - HCl gelatin during storage for few days in aerobic conditions, which ultimately helped in more gas production during anaerobic digestio n. Indirectly, faster the degradation of shaving dust hydrolysate more is the volatile suspended solids produced which is readily utilized my methanogens. This is reflected in the methane gas increase with reduced CO 2 %. Thus it is concluded that chromed l eather waste hydrolysis and neutralization using KOH - H 3 PO 4 mode is more feasible than Lime - HCl method.Extracted chromium is reused in tanning process. References 1. Thanikaivelan P. Rao J. R., Nair B.U. and Ramasami T. Progress and recent trends in biotechno logical methods for leather processing, Trends in Biotechnology, 22(4) , 181 - 188 (2004) 2. Gaidau C., Niculescu M., Stepan E., Taloi D. and Filipescu L., Additives and advanced biomaterials obtained from leather industry by - products, Rev. 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