As described previously, improvement of the the carrier and optimization of the growth of microorganisms should be approached first in order that survival and activity of microorganisms in the field is assured [8.23–8.25]. Peat has been used as a carrier material standard for most commercial microbial inoculant [8.26]. The use of peat is not recommended in some countries because excessive peat mining will disrupt ecosystem sustainability, therefore other potential materials are necessary [8.27]. Compost is a potential alternative carrier material as the substitute of peat, because it is abundantly available, renewable, and environmentally friendly. Compost-based carrier has a better ability to support the survival of Azotobacter sp. compared to peat as presented in Fig. 8.3.
83 FIG. 8.3. Compost and peat based-Azotobacter sp. during 90 days storage period
Liquid cultures of Azotobacter sp. (10% v/w) are inoculated into carriers in all kinds of materials that have been sterilized by gamma irradiation at a dose of 40 kGy. After storage for 90 days at about 28 ºC, there is a decrease in the population of Azotobacter sp. in peat from 8.0 x 109 to 5.0 x108 cfu (colony forming units)/g. In all carrier-based compost, Azotobacter sp.
population remains high, being around 109 - 1010 cfu/g. These results indicated that the compost-based carrier materials are suitable for use as an alternative instead of peat in the formulation of inoculant of Azotobacter sp.
8.3.2.2. Sterilization of Compost-Based Carrier
Sterilization process of carriers is conducted to prevent the competition between microbial targets and other microorganisms in a nutrient-rich environment25. Cleaning of indigenous microorganisms in the compost-based carrier can be done by steam sterilization or gamma irradiation as shown in Table 8.1. In the steam sterilization treatment by autoclave at 121 ºC for 30 minutes and gamma irradiation at a dose of 10 kGy, sterile compost-based carrier was not generated. However, sterilization by autoclave at 121 ºC for 60 minutes and gamma irradiation at a dose of 20 kGy and 30 kGy produced sterile compost-based carrier (<101 cfu/g).
According to McNamara and co-worker (2003) [8.28], the majority of soil microorganisms including Actinomycetes and fungi can be destroyed by gamma irradiation at a dose of 20 kGy.
Therefore, the sterilization of compost-based carrier (< 101 cfu/g) can be done with an autoclave at 121 ºC for 60 minutes or gamma irradiation at dose of 20 and 30 kGy.
After 90 days of storage period, the population of Azotobacter sp. in the carrier material sterilized by autoclave decreased from 1.2x109 to 8.6x108cfu/g. Meanwhile, in the carrier with sterilization by gamma irradiation at doses of 20 and 30 kGy could maintain the Azotobacter sp. population remains at 109cfu/g. These results indicated that irradiation sterilization at doses of 20–30 kGy is more appropriate for the survival of Azotobacter sp. It was also reported that the gamma irradiation sterilization process does not produce toxic substances to some microbes. In further observation on irradiation dose, it was known that a dose of 25 kGy could produce compost-based carrier with the most appropriate sterility and quality for microbial inoculants. Gamma irradiation at a dose of 25 kGy can clean total aerobic bacteria (5.7x108 cfu/g) and fungi (1.4x107 cfu/g) to an undetectable (10 cfu/g). The gamma irradiation sterilization of compost-based carrier can sustain population of Azotobacter sp., Bacillus circulans, and Bacillus stearothermophilus with high concentrations during the 180 days of storage period. After the storage period, the population of Azotobacter sp., B. circulans, and B.
stearothermophilus were 3.2x1011, 1.7x1011, and 1.9cfu/g, respectively. These results indicated
6 7 8 9 10 11
0 30 60 90
Azotobactersp., Log 10 cfu/g
Storage period (day)
Peat (C:N:P = 720:36:1) Compost A (C:N:P = 304:19:1) Compost B (C:N:P = 380:19:1) Compost C (C:N:P = 300:15:1)
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that carrier-based compost sterilized by gamma irradiation at a dose of 25 kGy has a potential for producing microbial inoculants for plant growth enhancement.
TABLE 8.1. THE EFFECT OF STERILIZATION TREATMENT AGAINST TOTAL CONTENT OF AEROBIC BACTERIA AND FUNGI IN COMPOST-BASED CARRIER
No Sterilization treatment
Microorganism, cfu/g aerobic bacteria
total
fungi total
1 Control (without sterilization) 5.9x108 6.5x106 2 Autoclave, 121 ºC, 30 min 9.0x103 1.3x103
3 Autoclave, 121 ºC, 60 min nd nd
4 Gamma irradiation, 10 kGy 6.4x104 5.0x103
5 Gamma irradiation, 20 kGy nd nd
6 Gamma irradiation, 30 kGy nd nd
Note: cfu = colony forming unit; nd = not detected at 101 cfu/g.
8.3.2.3. Low-Dose Gamma Irradiation Effect on Microbial Inoculant
Study on bioremediation of hydrocarbon contaminants and heavy metals (Pb2+, Cd2+) in the liquid medium showed that low-dose gamma irradiation treatment can improve the performance of microbial inoculants. Microbial inoculants T.viridie, T. harzianum and A. Niger are irradiated at doses of 0 (control), 125, 250 and 500 Gy, then the inoculants added into a liquid medium containing hydrocarbon contaminants (27 ppm) and heavy metals [Pb2+ (27.1 ppm) and Cd2+ (18.8 ppm)], subsequently incubated at room temperature of 28ºC with agitation at 150 rpm for 9 days. Compared to control, it showed that the optimum degradation of total petroleum hydrocarbon (TPH) was obtained at inoculation treatment by irradiated T.viridie, T.
harzianum and A. Niger at a dose of 250 Gy. In this treatment, the ability of T.viridie, T.
harzianum and A. Niger to degrade TPH decreased by 15%, 21%, and 19%, respectively (Fig.
8.4). Gamma irradiation at a dose of 250 Gy also significantly enhances the ability of T. viridie, T. harzianum, and A. Niger in reducing contamination of Pb2+ and Cd2+ in a liquid medium.
The ability of these fungi in reducing Pb2+ increased by 22% (T. viridie), 22% (T. harzianum), and 20% (A. Niger) compared to control as shown in Fig. 8.5, while the ability of these inoculants in reducing Cd2+ also increased by 16% (T. viridie), 14% (T. harzianum), and 12%
(A. Niger) compared to control (Fig. 8.6). These results indicated that gamma irradiation at a dose of 250 Gy is the optimum dose for increasing their ability of T.viridie, T. harzianum, and A. Niger in reducing TPH, Pb2+, and Cd2+ in a liquid medium. The same results also reported by Chakravarty B. and Sen S [8.29] and Afify A.E.M.R. et al. [8.30] that low-dose gamma irradiation has effect on the acceleration of enzyme activity by microbes. According to Afify A.E.M.R. et al. (2012) [8.30], gamma irradiation at a dose of 250 Gy affects the increase in dry weight of mycelia of T. harzinum and T. viridie at 23% and 16%, respectively.
85
8.3.2.4. Application of Irradiated Fungal Inoculant Consortium on Remediation of Degraded Lands in South Tangerang-Banten Province
Bio-compost made from manure is composted and enriched by microbial consortium inoculants which have function as decomposition of organic substances, biological control, N-fixation, and phosphate solubilizing. A total of 200 g of functional microbial consortium-based compost activated in 4 liter solution of 5% molasses, and then used for producing 80 kg of bio-compost.
Before applied to corn plants (Zea mays L., P21variety) and soybean (Glycine max L., Pearl I variety), firstly bio-compost is incubated at 28 ºC for 5 days. Bio-compost applied on degraded land of ex-sand mining in South Tangerang-Banten Province with the characteristics as shown in Table 8.2.
The study was conducted using a randomized block design (RBD) with 4 treatments and 4 replications in each plot = 10 x 20 m2. The dose of NPK, manure, and bio-compost are 200 kg/ha (100%), 2.5 tons/ha and 500 kg/ha, respectively, with the composition of the treatment as shown in Table 8.3. The addition of NPK at dose from 50 to 100% gave the potential increase in biomass of corn crop, biomass of soybean, and dried soybean seeds. Nevertheless, this treatment did not provide the optimal results such as the addition of bio-compost. The addition of manure (from cow dung) as much as 2.5 tons/ha only gave a significant effect on the increase the productivity of soybean biomass. These result suggested to be related to the low nutrient content in the manure, so the plants are not getting enough nutrients. The use of
bio-35 45 55 65 75
0 125 250 500
Degradation of TPH (%)
Absorbed dose (Gy)
T. viridie T.harzianum A. niger
40 60 80 100
0 125 250 500
Reduction of Pb2+(%)
Absorbed dose (Gy) T. viridie T.harzianum A. niger
40 60 80 100
0 125 250 500
Reduction of Cd2+(%)
Absorbed dose (Gy)
T. viridie T.harzianum A. niger FIG. 8.4. Effect of low-dose gamma
irradiation on the ability of fungi to degrade TPH
FIG. 8.5. Effect of low-dose gamma irradiation on the ability of fungi in to reduce Pb2+
FIG. 8.6. Effect of low-dose gamma irradiation on theability of fungi in to reduce Cd2+
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compost as much as 500 kg/ha is more capable to optimize the yield productivity of corn and soybeans compared to other treatments. Addition of bio-compost could increase the productivity of dried biomass of corn crop about 22% (from 4.62 to 5.64 ton/ha) and dried soybeans biomass about 53% (from 3.16 to 4.82 tons/ha). The use of bio-compost could also increase the productivity of dried corn seeds about 52% (from 3.24 to 4.94 tons/ha) and dried soybean seeds of about 77% (from 1.59 to 2.81 tons/ha).
TABLE 8.2. THE CHARACTERISTIC OF DEGRADED LANDS
TABLE 8.3. COMPOSITION OF TREATMENT
No Parameter value criteria No Treatment Composition
1 pH (1:5 H2O) 6.01 Slightly acid 1 A 50% NPK
2 C organic (%) 0.72 very low 2 B 100% NPK
3 Total N (%) 0.05 very low 3 C 50% NPK + manure
4 C/N ratio 16.62 high 4 D 50% NPK + bio-compost
5 P2O5 (ppm) 4 very low
8.3.2.5. Remediation Application on Petroleum Oil Sludge-Contaminated Dry Land in Rejuvenation of Teak Tree (Tectona grandis) in Cepu-Central Java
Dry land contaminated by petroleum oil sludge in traditional oil extraction sites (Cepu) has mild alkaline condition (pH 7.6 - 8.5), very high organic content (C > 5%), low N (0.1 - 0.2), very high C/N ratio (> 25), and very low P2O5 (P < 4 ppm). This condition gradually requires remediation for improvement of soil quality parameters, degradation of hydrocarbon and its potential toxicity, in order to recover the productive functions of land. The initial stages of the remediation carried out by the method of composting at the target site (in-situ composting) and the second stage through the cultivation of non-food crops, namely elephant grass (Pennisetum pupureum). In-situ composting was done on a plot of 150 m2 with 300 g of hydrocarbon degrading functional microbial inoculants, 60 kg of sawdust and 3.6 kg of NPK fertilizer. The treated soil was divided into six subplots @ 20 m2 with a height of 20 cm, then covered with plastic and composted for 60 days. After composting, stem of elephant grass was planted in subplots with the treatment of A = control, B = treated with plant growth enhancer microbial inoculants. A total of 50 g microbial inoculants activated in 5 L of a solution of 5% molasses for 24 h, then diluted with water to 25 L and spread on the surface of plot B. All of subplots were also given 300 g of NPK fertilizer/plot. Maintenance of elephant grass (Pennisetum pupureum) was performed for 90 days. It was shown that application of functional microbial inoculant 50g/20m2 (=25kg/ha) significantly affect plant growth enhancement of elephant grass.
After in-situ composting for 60 and 120 days without treatment, the content of TPH (total petroleum hydrocarbons) reduced by 58% and 77%, respectively. Moreover, the reduction of TPH could be achieved in 82% when treatment in-situ composting followed by stimulated with microbial inoculants consortia. These results indicated that the of microbial inoculant consortia
87 suitable for remediation petroleum oil sludge-contaminated dry land using in-situ post-composting.
Improvement of plant growth in petroleum oil sludge-contaminated land after in-situ post-composting was suggested by detoxification of hydrocarbon decomposition products by rhizosphere microbial isolates. Rhizosphere microbial isolates in the functional microbial inoculant also suggested capable to N-fixation and dissolved phosphate for the growth of grass plants, even though the height of elephant grass plant did not show significant differences, but other parameters such as the weight of the biomass, N, and P uptake showed a significant increase. The weight of dried biomass increased from 47 to 101g, N uptake increased from 415 to 914mg/plant and P uptake increased from 77 to 178 mg/plant. These results suggested that the microbial consortia containing inoculant bacteria isolates (KDB2, KLB5, BM5, KLBN1) and fungal isolates (KLF6, RK1) suitable for plant growth of elephant grass in petroleum oil sludge-contaminated land after in-situ post-composting.
All evaluations showed that consortia of microbial inoculant containing functional hydrocarbon degrading microbial isolates (BMC2, BMC4, BMC6, FMC2, FMC6) from hydrocarbon-contaminated soil has the potential to reduce TPH (total petroleum hydrocarbons), whereas consortia of microbial inoculant containing functional of plant growth enhancers which contain bacterial isolates KDB2, KLB5, BM5, KLBN1 and fungal isolates (KLF6, RK1) have the potential to increase the growth of corn crop (Zea mays L. varieties P21) on dry land with very low available of P (P<4 ppm).
8.3.3. Improvement of natural polymer properties by radiation grafting of adsorbent