Annals of Microbiology, 59 (4) 643-648 (2009)

Effect of caffeine and saponin on anaerobic digestion of food waste

Vidhya PRABHUDESSAI, Anasuya GANGULY, Srikanth MUTNURI*

Biological Sciences, Birla Institute of Technology and Science, Pilani, Goa campus, Goa, India

Received 15 June 2009 / Accepted 16 October 2009

Abstract - Food waste is the single largest component of the waste stream by weight. In the present work, we attempted to study the effect of caffeine and saponin on anaerobic fermentation of food waste to examine their potential influence on biogas production at 8% total solids (TS) content. Addition of caffeine at 50, 100, and 150 ppm to the food waste on the first day resulted in biogas production in 24 hours which normally comes on 4th day. The maximum biogas production of 408.5 ml/g TS was found at 100 ppm caffeine whereas 50 ppm and 150 ppm caffeine produced 359 ml and 336 ml in comparison with the control which showed 182 ml/g TS. This is the first study of effect of caffeine as stimulants in anaerobic environments. Addition of saponin had no beneficial effect; on the other hand, it inhibited the biogas production at 50, 100 and 150 ppm.

Key words: anaerobic digestion; biogas; food waste; caffeine; saponin.

INTRODUCTION

Environmental pollution is one of the greatest challenges of the 21st century. Biotechnological option is a sustainable approach which can ease the stress on depleted natural resources and growing energy demand. Anaerobic digestion is one of the better options available which can combine waste treatment with the recovery of useful byproducts and renewable biofuels. Anaerobic digestion would fulfill three basic needs i.e improvement in health and sanitation through pollution control, generation of renew-able energy and supply of digested materials as biofertilizers. Among the different types of wastes available, food waste is the single largest component of the waste stream by weight. The food waste which includes both cooked and uncooked leftovers constitutes the largest component of waste coming from the restaurants and fruit/vegetable markets, residences, restaurants, cafeterias etc. Anaerobic digestion provide better solutions to manage above mentioned wastes. Although the food waste could be composted aerobically, anaerobic digestion without any pre-treatment, with energy recovery, seems to be the most attractive method in terms of potential for recovery of value (Lastella et al., 2002; Zhang et al., 2007). The physical and chemical characteristics of organic wastes like pH, moisture content, total solids, volatile solids, particle size and biodegradability play an important role in anaerobic digestion process. Zhang et al. (2007) determined the average methane yield of food waste at 6.8 and 10.5 g VS/L were 425 ml and 445 ml/g VS/L after 28 days of digestion. There are several tech-

nologies that have been used to increase methane generation Possible techniques include pretreatment of the feedstock with heat, ultrasonic devices, or impact grinding; microbial stimulants; or co-digestion with other wastes. It has been reported by Singh et al. (2001) that the use of microbial additives with food waste increased gas production by 16%. Plant secondary metabolites namely caffeine and saponin which acts like metabolic stimulants are used in this study to see their effects on the anaerobic diges-tion of food waste. Plants produce various secondary compounds mainly for protecting themselves from insects, animals, fungi or bacteria. Besides tannins, saponins are the most widely occur-ring compounds. Few researches were conducted to evaluate the potential of secondary plant constituents as natural agents to manipulate rumen fermentation with mixed results (Wallace et al., 1994 Sliwinski et al., 2002; Wina et al., 2005). As the proc-esses that take place in rumens is similar to anaerobic digestion, we studied the effect of saponin on anaerobic digestion. We also selected caffeine for our study as it is the most powerful and addictive stimulant and this is found in coffee spent ground apart from tannins & polyphenols whose properties have not been uti-lized properly (Pandey et al., 2000).

MATERIALS AND METHODS

Food waste collection and analyses. The food waste was collected as source separated waste from the dining Hall of our institute. To gain an understanding of the compositional vari-ability and amount of food waste, daily and weekly sampling was performed. The food waste composition and variability of total solids was studied for a period of one month.

* Corresponding Author. Phone: 0091 832 2580125; Fax: 0091 832 2557033; E-mail: srikanth.mutnuri@gmail.com

644 V. PRABHUDESSAI et al.

Anaerobic digestion tests. The biodegradability of food waste from the institute mess was determined using batch anaero-bic digestion (AD) tests. The initial tests were to optimize the concentration of total solids. Samples used for this study was the composite samples which were prepared by mixing all food waste samples and then taking representative samples from the mixture. The representative samples were shredded in an electric blender and then used for AD tests. The AD tests were carried out in 0.5 L bottles at varying initial total solids loadings (5, 8, 9, 11 and 14%) each in duplicate. Cow dung was used as inoculum. The mixing ratio of food waste to cow dung was 80:20. After the inoculum and food waste were added, each digester was filled up to 400 ml with distilled water. The pH value was adjusted to 7.2 using 3 N NaOH. The digesters were tightly closed with a screw cap. To ensure anaerobic conditions, the headspace was purged with N2 gas. Each digester was manually mixed twice a day and the pH was maintained at 7.2 using 3 N NaOH.

Anaerobic digestion tests using microbial stimulant. From the tests described previously, 8% TS content was found to be optimum for biogas production. To test the effect of plant second-ary metabolite caffeine, similar batch tests like described before were set up at 8% TS content. One set of digestion served as control and the other set were spiked with 100 ppm of caffeine on the 4th day and 6th day of incubation. In another experi-ment, the effect of plant secondary metabolites like caffeine and saponin on biogas production from food waste was studied at 8% TS content in 0.5 L bottles. The different concentrations of caf-feine and saponin used were 50, 100, 150 ppm. In all the above mentioned experiments, the biogas production was measured by water displacement set up (Singh et al., 2001) after passing the biogas through 1M NaOH. Briefly each 500 ml bottle was used as a digester for this work. A tube was used to connect the digester with an inverted 250ml graduated measuring cylinder immersed in a 1000 ml beaker filled with water. Biogas produced was col-lected in the graduated cylinder connected with a water reservoir after passing through 1 M NaOH (to trap carbon dioxide), which allowed volumetric biogas measurements at atmospheric pres-sure.

Critical Micelle Concentration (CMC) of saponin. CMC value for saponin was determined by diluting the saponin until reaching the CMC which was determined by plotting the surface tension as a function of saponin concentration and then the surface tension at that point wherein there is no further decrease in surface ten-sion is called as CMC (Kim et al., 2000).

Substrate analysis. Aliquots of fresh and digested samples were drawn from each set of experiments, and analyzed for Total solids and Total Kjeldahl Nitrogen according to the standards methods of American Public Health Association (APHA, 1998). Chemical oxygen demand (COD) was determined as described by Raposo (2008).

Scanning Electron Microscopy (SEM). Fixation and dehydra-tion of anaerobic samples were done according to Wu et al., 2001. Briefly the samples from the anaerobic digesters were first fixed in 0.1 M phosphate buffer solution (PBS) containing 2.5% (v/v) glutaraldehyde and 4% (w/v) paraformaldehyde for at least 12 h at 4 °C and washed three times with 0.1 M PBS. The sam-ples were subsequently fixed in 0.1 M PBS containing 1% (w/v) osmium tetroxide for 2 h at 4 °C and washed three times with 0.1 M PBS. The samples were dehydrated with a series of etha-nol washes (50, 75, 85, 95 and 100%), and exposed to acetone

for 15 min twice. The dehydrated samples were dried using the critical point drier (Lab companion HP-300 drier) and then coated with platinum by an ion sputter JEOL JFC -1600 Autobine coater. Finally the platinum coated samples were observed in a JEOL JSM-6360 LV Scanning Electron Microscope with an accelerating voltage of 20 KV. This microscopic observation was carried out and photographed extensively to make sure that the predomi-nant microbial population was obtained.

RESULTS AND DISCUSSION

Characterization of food wasteThe average composition of the food waste that was source separated was analyzed. The food waste mainly consisted of raw vegetables 31%, cooked rice 17%, cooked vegetables 16% whereas pulses, roti, tea, coffea and milk accounted for 9.9, 9.7, 5, 4.3 and 3.3% respectively. Figure 1 shows the com-position of food waste. The average amount of food waste per day was approximately 376 kg/day. The TS content remained constant at an average of 26%. The advantage of food waste is their high moisture content which facilitates anaerobic digestion (Bouallagui et al., 2004). The physical and chemical characterisit-ics of the organic waste are important information for designing and operating anaerobic digesters, because they affect biogas production and process stability during anaerobic digestion. The Characteristics of food waste used in Anaerobic Digestion experi-ments is shown in Table 1. The C/N ratio of 32.4 of food waste matches with the characteristic of other reports on suitability of food wastes for anaerobic digestion (Kivaisi and Mtila, 1998; Zhang et al., 2007).

TABLE 1 - Characteristics of substrates

Parameter Food waste Cow dungpH 4.3 5.3Total solids (%) 26 22.5Chemical Oxygen Demand (mg/L) 162 76.8Total Kjeldahl nitrogen (g N/L) 5.47 2.7

Raw vegetables,

31.7

Rice, 16.6Cooked vegetables,

16.3

Pulses / dal, 9.9

Roti, 9.7

Tea, 5.0

Coffee, 4.3

Milk / curd, 3.3

Salad, 3.2

Oil, 0.1

FIG. 1 - Composition of food waste (%).

Ann. Microbiol., 59 (4), 643-648 (2009) 645

Methane productionAnaerobic experiments were set at varying total solids concentra-tions of 5, 8, 9, 11 and 14%. Among all the different TS concen-trations 8% TS yielded more biogas and 11% and 14% did not yield biogas production which indicates that dry digestion was not possible with respect to our case. The biogas production during the digestion of food waste is shown in Fig. 2. The maximum biogas yield of 573 ml/g TS was found at 8% of TS concentration with 18 days of digestion whereas at 9% TS 267 ml of biogas was produced. At 8 and 9% TS gas production increased until day 5. At 5% TS, biogas production was relatively low during the first three days of digestion, increased to reach peak on the fifth day

of digestion and then declined again. The results obtained were averages of duplicate analysis.

Anaerobic digestion tests using microbial stimulantsIn the second experiment, the effect of microbial stimulant, caf-feine (100 ppm) was evaluated on food waste and cattle dung mixture (80:20) at total solid concentration of 8%. The biogas production from food waste spiked with caffeine is shown in Fig. 3. It was observed that the addition of single dose of caffeine at the rate of 100 ppm on the fourth day of incubation resulted in increased gas yield 42 ml/g TS in comparison with control 26 ml/g TS. To confirm the effect of caffeine we spiked with addi-

0

100

200

300

400

500

600

700

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Digestion time (days)

Bio

gas

yiel

d (m

l/g T

S)

TS 5% TS 8% TS 9% FIG. 2 - Daily biogas yield of food waste during anaerobic digestion at different TS concentrations.

0

10

20

30

40

50

60

70

80

90

100

6yaD 4 yaDDigestion time (days)

Bio

gas

prod

uctio

n ra

te (

ml/l

.d)

0 ppm caffeine 100 ppm caffeine

FIG. 3 - Effect of caffeine on biogas production rate on day 4 and day 6.

646 V. PRABHUDESSAI et al.

tional 100 ppm of caffeine on the 6th day. After second spiking the biogas production increased by 16% in comparison with the control. Further biogas production was also evaluated using different concentrations of caffeine and saponin (50, 100 and 150 ppm). The addition of caffeine at the rate of 50, 100, 150 ppm to the food waste cow dung mixture on the first day of addition resulted in biogas production on day two itself with all the three concen-trations of caffeine whereas control (0 ppm caffeine) started biogas production on day four. The biogas production of food waste was found at an average of 367.8 ml/g TS at all the three concentrations of caffeine. The maximum biogas production of 408.5 ml/g TS was found at 100 ppm caffeine whereas 50 ppm

and 100 ppm caffeine produced 359 ml and 336 ml in compari-son with the control which showed 182 ml/g TS (Fig. 4). This is the first study of effect of caffeine as stimulants in anaerobic environments. Caffeine was selected for this study as it is an active com-pound, one of the nature’s most powerful and addictive stimu-lants. Caffeine is known to induce laccase enzyme and sclerotiza-tion in Rhizoctonia solani, Pseudomonas fluoresens interactions due to the triggering of calcium or heat shock signaling path-ways by bacterial metabolites as shown by Rollins and Dickman (1998). Crowe and Olsson (2001) found that laccase enzyme was induced 5 fold during caffeine driven sclerotization in R. solani. Ogunseitan (2002) reported caffeine induced production

0

50

100

150

200

250

300

350

400

450

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Digestion time (days)

Bio

gas

yiel

d (m

l/g T

S)

50 ppm 100 ppm 150 ppm o ppm

FIG. 4 - Effect of different concentrations of caffeine on biogas production.

40

50

60

70

80

0 28 67 188 288 450 590 1015 1788 3575

Saponin (ppm)

Sur

face

ten

sion

(m

N/m

)

FIG. 5 - Critical micelle concentration of saponin.

Ann. Microbiol., 59 (4), 643-648 (2009) 647

of haem associated peroxidase activity in Pseudomonas putida ATCC 700097. Various studies had shown that caffeine is known to induce different enzymes but phosphodiesterase enzyme is inhibited by caffeine, the stimulatory effect of this plant second-ary metabolite being the result of raised cAMP levels. cAMP, the cell signaling molecule is a general activator of cell activity. Contradicting reports are available on the effect of sapon-ins on methanogenesis. Guo et al. (2008) reported inhibition of methanogenesis in the presence of saponin probably due to interference with interspecies hydrogen transfer between the protozoa and associated methanogens. Hiroki et al. (2004) reported that the addition of saponin was effective for improving anaerobic digestion especially increasing the acidogenesis rate. In our studies addition of saponin inhibited biogas production. As Quillaya saponin used in our study is used as a biosurfactant in the biodegradation of hydrocarbons (Pijanowska et al., 2007) and surfactants above CMC usually inhibits bacterial growth, we measured the surface tension of saponin at different concentra-tions to calculate critical Micelle concentration (Fig. 5). The CMC for saponin was found to be above 1000 ppm indicating non toxicity of saponin at the concentrations used which was much below the CMC value.

Scanning Electron Microscopy (SEM)Anaerobic samples were observed with SEM. SEM was used to observe the main cellular morphology during anaerobic diges-tion in the presence of caffeine. Figure 6 showed the diversity of morphotypes found in the anaerobic digestion with rod like morphologies that could correspond to Methanobacterium and Methanobrevibacter (Yang et al., 2008).

CONCLUSION

The maximum biogas production is at an initial total solid rate of 8% with average yield of 413 ml/g TS. Addition of caffeine increased biogas production by 16% in comparison with the control. Caffeine which serves as a stimulant enhanced biogas production by potentially increasing microbial activity. The use of such stimulants serves as a promising means for increasing

biogas production. Coffee spent ground, the residue obtained during the processing of raw coffee powder to prepare “instant coffee” is another residue obtained from the coffee industry which has led to environmental pollution. This residue contains caffeine, tannins and polyphenols which due to their toxic nature were not used beneficially. They might have a beneficial role when combined for anaerobic digestion along with the food wastes. Our future work focuses on this at a pilot scale (one tonne horizontal plug flow reactor is being constructed with the help of Goa Energy Development Agency) along with the study of microbial diversity in anaerobic digestors using molecular tools.

AcknowledgmentsThe authors thank University Grants Commission, Government of India for funding this project (Ref No. SR/33-139/2007). The authors also thank Dr. Rahul Mohan National centre for Antarctic and Ocean Research for SEM images and Dr. Paschke, Helmholtz centre for Environmental Research, Leipzig Germany for surface tension measurements.

REFERENCES

APHA (1998). Standard Methods for the Examination of Water and Wastewater, 18th edn., American Public Health Association, Washington, DC, USA.

Bouallagui H., Ben Cheikh R., Marovani L., Hamdi M. (2003). Mesophilic biogas production from fruit and vegetable waste in a tubular digester. Bioresource Technology, 86: 85-89.

Crowe J.D., Olsson S. (2001). Introduction of laccase activity in Rhizoctonia solani by antagonistic Pseudomonas fluores-cens strains and a range of chemical treatments. Applied Environmental Microbiology, 67: 2080-2094.

Guo Y.Q., Liv J.X., Lu Y., Zhu W.Y., Denman S. E., McSweeney C.S. (2008). Effect of tea saponins on methanogenesis, microbial community structure and expression of mcr A gene, in cultures of rumen microorganisms. Letters in Applied Microbiology, 47: 421-426.

FIG. 6 - SEM of anaerobic digested food waste.

648 V. PRABHUDESSAI et al.

Hiroki M., Kihiro Kimura A., Shinsuke K., Munetaka I. (2004). Updraging of the anaerobic digestion by addition of bioac-tive reagent (saponin). Japanese Journal of Water Treatment Biology, 40: 37-44.

Kim S.H., Lim E.J., Lee S.O., Lee J.D., Lee T.H. (2000). Purification and characterization of biosurfactants from Nocardia spp. L-417. Biotechnol. Appl. Biochem., 31: 249-253.

Kivaisi A.K., Mtila M. (1998). Production of biogas from water hyacinth (Eicchornia crassipes) in a two stage bioreactor. World Journal of Microbiology and Biotechnology, 14: 125-131.

Lastella G., Testa C., Cornacchia G., Notornicola N., Voltasio F., Sharma V.K., (2002). Anaerobic digestion of semi solid organic waste: biogas production and its purification. Energy Conversion and Management, 43 : 63-75.

Ogunseitan O.A. (2002). Caffeine inducible enzyme activ-ity in Pseudomonas putida ATCC 700097. World Journal of Microbiology and Biotechnology, 18: 423-428.

Pandey A., Soccol C.R., Nigam P., Brand D., Mohan R., Roussos S. (2000). Biotechnological potential of coffee pulp and cof-fee husk for bioprocesses. Biochemical Engineering Journal, 6: 153-162.

Pijanowska A., Kaczorek E., Chrzanowski L., Olszanowski A. (2007). Cell hydrophobicity of Pseudomonas spp. and Bacillus spp. bacteria and hydrocarbon biodegradation in the presence of Quillaya saponin. World Journal of Microbiology and Biotechnology, 23: 677-682.

Raposo F., Rubia de la M.A., Borja R., Alaiz M. (2008). Assesment of a modified and optimized method for determining chemical oxygen demand of solid substrates and solutions with high suspended solid content. Talanta, 76: 448-453.

Rollins J.A., Dickman M.B. (1998). Increase in endogenous and exogenous cycle AMP levels inhibits sclerotial develop-ment in Sclerotinia sclerotium. Applied and Environmental Microbiology, 64: 2539-2544.

Singh S., Kumar S., Jain M.C., Kumar D. (2001). Increased biogas production using microbial stimulants. Bioresource Technology, 78: 313-316.

Sliwinski B.J., Soliva C.R., Machmuller A., Kreuzer M. (2002). Efficacy of plant extracts rich in secondary constituents to modify rumen fermentation. Anim. Feed Sci. Technol., 101: 101-114.

Wallace R.J., Arthaud L., Newbold C.J. (1994). Influence of Yucca shidigera extract on ruminal ammonia concentrations and ruminal microorganisms. Appl. Environ. Microbiol., 60: 1762-1767.

Wina E., Muetzel S., Hoffmann E., Makkar H.P.S., Becker K. (2005). Saponins containing methanol extract of Sapindus rarak affect microbial fermentation, microbial activity and microbial community structure in vitro. Anim. Feed Sci. Technol., 121: 159-174.

Wu J.H., Liu W.I., Tseng I.C., Cheng S.S. (2001). Characterization of microbial consortia in a terephthalate degrading anaerobic granular sludge system. Microbiology, 147: 373-382.

Yang Y., Tsukahara K., Sawayama S. (2008). Biodegradation and methane production from glycerol containing synthetic wastes with fixed-bed bioreactor under mesophilic and thermophilic anaerobic conditions. Process Biochemistry, 43: 362-367.

Zhang R., El-Mashad H.M., Hartman K., Wang F., Gaungquig L., Chris C., Gamble P. (2007). Characterization of food waste as feedstock for anaerobic digestion. Bioresource Technology, 98: 929-935.