Journal of Environmental Management 132 (2014) 230e236

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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Algal sludge from Taihu Lake can be utilized to create novel

PGPR-containing bio-organic fertilizers
Miao Zhang 1, Rong Li 1, Liangliang Cao, Juanjuan Shi, Hongjun Liu, Yan Huang,
Qirong Shen*
Key Laboratory of Plant Nutrition and Fertilization in Low-Middle Reaches of the Yangtze River, Ministry of Agriculture, Jiangsu Key Lab for Solid Organic
Waste Utilization, Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, 210095 Nanjing,
Jiangsu Province, PR China

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 31 July 2013
Received in revised form
15 October 2013
Accepted 28 October 2013
Available online 8 December 2013

Large amounts of reoated algal sludge from Taihu Lake result in secondary environmental pollution due
to annual reoatation. This study investigated the possibility to produce bio-organic fertilizer (BIO) using
algal sludge as a solid-state fermentation (SSF) medium. Results showed that addition of algal sludge
contributed to efcient SFF by a plant growth-promoting rhizobacteria (PGPR) strain SQR9 and improved
the nutrient contents in the novel BIO. The optimum water content and initial inoculation size were 45%
and 5%, respectively. After 6 days of SSF, the biomass of strain SQR9 was increased to a cell density of
more than 5
107 CFU g1. Microcystins were rapidly degraded, and a high germination index value was
observed. Plant growth experiments showed that the produced BIO efciently promoted plant growth.
Additional testing showed that the novel SSF process was also suitable for other PGPR strains. This study
provides a novel way of high-value utilization of algal sludge from Taihu Lake by producing low-cost but
high-quality BIOs.
2013 Elsevier Ltd. All rights reserved.

Keywords:
Reoated algal sludge
Solid-state fermentation
Bio-organic fertilizer
Plant growth-promoting rhizob

1. Introduction
Plant growth-promoting rhizobacteria (PGPR) are associated
with plant roots and exert benecial effects on plant development
(Kloepper et al., 1980). They competitively colonize plant roots,
acting not only as biofertilizer but also as antagonists (biopesticides)
of recognized root pathogens, including some bacteria, fungi pathogens and harmful nematodes (Chen et al., 2007). However, biological effect of the PGPR to be applied to soils depends to a greatest
extend on their surviving or reproduction abilities during the period
of application of PGPR and root system construction because the
PGPR need at any time a suitable nutrition either from organic fertilizers or from root exudates (Cao et al., 2011; Chen et al., 2011; ElHassan and Gowen, 2006; Ling et al., 2011; Wang et al., 2013; Zhao
et al., 2011). Several reports have shown that a special bio-organic
fertilizer (BIO) combining PGPR with mature composts could
enhance the activity of PGPR (Cao et al., 2011; Chen et al., 2011; Ling
et al., 2011; Wang et al., 2013; Zhao et al., 2011). Compost not only

* Corresponding author. College of Resources and Environmental Sciences,

Nanjing Agricultural University, 210095 Nanjing, Jiangsu Province, PR China. Tel./
fax: 86 (0)2584396291.
E-mail address: shenqirong@njau.edu.cn (Q. Shen).
1
Miao Zhang and Rong Li contributed equally to this paper.
0301-4797/$ e see front matter 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jenvman.2013.10.031

plays an important role as a suitable substrate, but it also serves as a

growth-promoting medium in SSF (Raviv et al., 2005). The application of these BIOs is becoming more popular in China since more and
more farmers are recognizing the effect of BIOs not only in promoting plant root growth but also in protecting plant roots from soilborn pathogens as well.
Environmentally friendly BIOs could eventually replace some of
the chemical fertilizers and pesticides that are currently overused
in agriculture (ElKramany et al., 2007). Accordingly, research has
focused on the production of efcient and favorable BIO via solidstate fermentation (SSF), which integrates PGPR strains with
compost (Chen and Sun, 2007). Our previous studies have investigated the use of mature compost, oil rapeseed cake and special
microbial species to produce high-quality products via SSF (Cao
et al., 2011; Chen et al., 2011; Ling et al., 2011; Vessey, 2003;
Wang et al., 2013; Zhao et al., 2011). An addition of signicant
quantity of oil rapeseed cakes, usually 40% dry weight (DW), could
lead to a much better growth of microbial species added during the
BIO preparation by providing more nutrients than 100% of matured
composts; however, the production costs signicantly increased by
a simple comparison of matured compost (80 US$ ton1) to oil
rapeseed cakes (420 US$ ton1), thereby limiting large-scale
extension of BIOs. Therefore, new SSF media should be developed
to greatly reduce production costs while supplying essential

M. Zhang et al. / Journal of Environmental Management 132 (2014) 230e236

nutrition for the special microbial species and to further promote

efcacious plant growth.
Eutrophication of Taihu Lake, the third largest freshwater lake in
China, has resulted in annual blooming of cyanobacteria, posing a
major threat to the water supply of lakeside cities (Yan et al., 2012).
Reoatation of algal sludge after blooming was found to be the
most efcient approach for reducing eutrophication (nitrogen and
phosphorus content) in Taihu Lake. Reoated algal sludge is rich in
organic nutrients, especially with protein content as high as 61.76%
of the total solids (Zhong et al., 2012). Several hundred thousand
tons of algal sludge were collected annually in Taihu Lake but
disposed any where, which again resulted in seriously secondary
environmental pollution (Yan et al., 2012). Furthermore, a family of
chemically stable cyclic hepatotoxins called microcystins (MCs)
which are produced by cyanobacteria (Bourne et al., 2006) is toxic
to most of plants, animals and humans. This limited the utilization
of algal sludge. The predominant toxic MCs that have been found in
Taihu Lake algae are microcystin-LR (MC-LR) and microcystin-RR
(MC-RR) (Shen et al., 2003). Therefore, if these toxic substances
are degraded, the algal sludge from Taihu Lake can be used as
protein candidates for the SSF of PGPR, which can signicantly
decrease the production costs of BIOs.
The PGPR strain Bacillus amyloliquefaciens SQR9 was previously
isolated from rhizosphere soil of a healthy cucumber plant in a eld,
where most of the cucumber plants were severely attacked by
Fusarium oxysporum, a typical soil-born pathogen. An expensive
BIO produced by SFF of this strain with organic substrates containing oil rapeseed cakes of 40% DW had an excellent biocontrol
and plant-growth promotion activities in pot experiments (Cao
et al., 2011) and eld demonstrations. Considering its high protein content and other nutrients, the collected algal sludge was
rstly investigated for the development of a new and inexpensive
BIO in the present study, to evaluate whether the collected algal
sludge can be used in the SSF process with the SQR9, to analyze
whether MC-RR and MC-LR can be degraded by the new SSF process, to investigate the efcacy of the algal sludge in combination
with other functional microbes, and to evaluate the effects of the
novel BIO on plant growth.
2. Materials and methods
2.1. Raw materials and bacterial strains
Algal sludge was collected on-site at Taihu Lake and stored in a
pool for a few months before use. The sludge had moisture, carbon,
nitrogen, phosphate, protein, and ash contents of 85e88%, 24.26%,
4.48%, 0.73%, 45%, and 22.44%, respectively, and a free amino acid
concentration of 10.89 mg g1. Mature cattle manure compost was
supplied by Lianye Bio-technology Co., Ltd., Jiangsu, China, and had
moisture, total carbon, total nitrogen, phosphorus and potassium
contents of 25.23%, 13.48%, 1.24%, 0.91% and 1.18%, respectively.
The
bacterial
strains
B.
amyloliquefaciens
SQR9,
B. amyloliquefaciens NJN-6, Paenibacillus polymyxa SQR21-gfp and
Trichoderma harzianum T037 were previously isolated and identied by the Jiangsu Key Lab for Solid Organic Waste Utilization,
Jiangsu, China (Cao et al., 2011; Chen et al., 2011; Ling et al., 2012;
Wang et al., 2013).
2.2. SSF preparation
The SQR9 cells maintained on a 2.5% LB agar plate (Cao et al.,
2011) were pre-cultured in LB medium at 170 rpm and 30  C for
36 h, harvested by centrifugation at 6000 rpm for 5 min, washed
three times with sterilized water, and suspended in the same volume of water.

231

To determine the best additive concentration of algal sludge for

SSF using strain SQR9, cattle manure compost containing ve
different concentrations of algal sludge (1%, 3%, 5%, 7% and 9% DW),
and a control without algal sludge, were arranged in small, coneshaped windrows (0.15 m high by 0.3 m diameter base, approximately 3 kg DW each). The initial inoculation size of strain SQR9
was approximately 106 CFU g1 DW, and the moisture content of
the mixtures was maintained at 40e45%. The mixtures were
maintained at room temperature (20e30  C) for 6 days and
manually turned on a daily basis to promote the growth of the
inoculated PGPR strain. At day 2, 4 and 6 after turning of the piles,
ve sub-samples were taken from symmetrical locations around
the heap and combined to form a composite sample. Triplicate
composite samples were collected and stored at 4  C immediately
prior to analysis. The effects of the following conditions on the SSF
process were examined: the initial moisture content values of the
mixtures (adjusted to 40%, 45%, 50% and 60%); the initial inoculation amounts (set at 0.5%, 1.0%, 5.0%, 10% and 15%); and different
common composts (chicken manure compost, Chinese medicine
residue compost, and pig manure compost, named as J-BIO, Z-BIO,
and P-BIO, respectively).
To evaluate the growth of the other PGPR strains or fungi during
the SSF process, B. amyloliquefaciens NJN-6 (BIO6), P. polymyxa SQR21gfp (BIO21) and T. harzianum T037 (BIO37) were examined. The controls without algal sludge were inoculated with the same amount of
these microbes, and named as BOF6, BOF21 and BOF37, respectively.
A blending ratio of 9.5: 0.5 (cattle manure compost: algal sludge
DW) was chosen for the expanded SSF process. Mixture piles of
20 kg DW (0.7 m high by 0.75 m diameter base) were prepared for a
6-day period SSF with (BIOn) or without (BOFn) algal sludge and
had the optimum moisture content and initial inoculation size.
Cattle manure compost without algal sludge (20 kg DW) and the
bacterial inoculation, named as OFn, was used as a control. Before
each daily turning of the piles, the temperature was measured at a
depth of 0.3 m into the pile. Samples were collected daily as previously described.
The numbers of B. amyloliquefaciens SQR9 and NJN-6 were
determined according to Wang et al. (2013). The number of SQR21gfp cells in fertilizer samples was estimated by a standard dilution
plating technique on LB agar plates containing 50 mg L1 kanamycin. Colony forming units (CFU) with green uorescence were
detected by UV lamps after incubation for 36 h at 30  C. The
number of T. harzianum T037 was determined according to Singh
et al. (2007).

2.3. Physicochemical properties of fertilizers

Fresh fertilizer samples combined with deionized water (1:10
w/w) were oscillated in a rotary shaker for 24 h to obtain aqueous
compost sample extracts. The suspensions were centrifuged at
10000 rpm for 10 min and ltered through 0.45 mm membrane
lters. The pH, electrical conductivity (EC) and total carbon (TC) and
nitrogen (TN) were determined by a pH meter, an EC meter, and an
element analyzer, respectively (Castaldi et al., 2008). Fresh samples
were oven dried at 105  C for 24 h to determine the moisture
content of the compost components. Total phosphorus was determined by spectrophotometer and total potassium was determined
by ame photometry (Bansal and Kapoor, 2000). Total free amino
acids and short peptides (TAPS) were measured according to
Airhart et al. (1974) with some modications. Briey, fresh sample
(5 g) and 0.1 M HCl were added to an erlenmeyer ask, and the
mixture was oscillated on a rotary shaker at 150 rpm and room
temperature (approximately 25  C) for 1 h. Then, the suspension
was centrifuged at 6000 rpm for 10 min and the concentration of

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M. Zhang et al. / Journal of Environmental Management 132 (2014) 230e236

TAPS in the supernatant was determined by the ninhydrin colorimetric method.

2.4. Analysis of MC-LR and MC-RR

28.69 g kg1, and available N, P, and K concentrations of 79.41,

25.77, and 59.44 mg kg1, respectively. Each treatment was
composed of three blocks with each block containing ten pots. After
transferring the seedlings, the agronomic characteristics and biomasses within a specic time period were measured.

Samples were taken at the beginning and end of the SFF process
to determine the MC-LR and MC-RR concentrations. MC-LR and
MC-RR were extracted and puried from the samples according to
Lawton and Edwards (2001) and analyzed by high performance
liquid chromatography (HPLC) with UV detection at 238 nm. The
separation column used for HPLC (internal diameter, 1.6 mm;
length, 25 cm) was a C18 column lled with 5 mm particles. The
mobile phase was methanol: water (55:45, v:v) containing 0.05%
triuoroacetic acid, and the ow rate was 0.6 ml min1. The column
temperature was 40  C.

The pot experiments were repeated twice, and similar trends

were observed. Therefore, only data from the April to June 2012 pot
experiment are presented herein. The differences among the
treatments were assessed using a one-way ANOVA analysis, and
the calculated means were subjected to Duncans multiple range
test at P  0.05. SPSS, version 17.0, was used for statistical analysis
(SPSS Inc., Chicago, IL).

2.5. Tomato seed germination test

3. Results and discussion

Seed germination and root length tests were performed on the

water extracts by mechanically shaking the fresh samples for 2 h at
a solid: water ratio of 1:10 (w/v) (Zucconi et al., 1981). Five ml of
ltrate was added to the bottom of a plate covered with lter paper.
Twenty tomato seeds were evenly distributed on the lter paper
and incubated in the dark at 25  C for 3 days. Each pile sample was
analyzed in triplicate. The treatments were evaluated by counting
the number of germinated seeds and measuring the length of the
root radical. Seeds with hypocotyls of less than 3 mm were
considered unsuccessfully germinated.
The germination index was computed as follows: (GI)
(%) [Seed germination (%)
root length of treatment
100%]/
[Seed germination (%)
root length of control].

3.1. SSF and strain SQR9

2.6. Pot experiment

Pot experiments were performed to evaluate the effects of the
BIO produced in this study on plant growth in a greenhouse located
at the Jiangsu Key Lab for Solid Organic Waste Utilization, Yixing,
China, from April to June and August to October 2012. The pot experiments were designed as follows: the control, the pot soil was
supplemented with 2% (w/w DW) cattle manure compost and
planted with eggplant (OFp) and cucumber (OFc); treatment 1, the
pot soil was supplemented with 2% (w/w DW) BOF and planted
with eggplant (BOFp) and cucumber (BOFc); treatment 2, the pot
soil was supplemented with 2% (w/w DW) BIO and planted with
eggplant (BIOp) and cucumber (BIOc). The BOF and BIO were prepared by the enlarged SSF and contained strain SQR9 concentrations of 0.93
107 and 5.83
107 CFU g1 (DW), respectively. The
soil for the pot experiment was collected from uncultivated grasslands and had a pH of 6.34, an organic matter concentration of

2.7. Statistical analysis

Although culture-independent methods have received attention

in recent years, selective plate counting was used to monitor the
numbers of different bacterial and fungal strains in this study.
Culture-independent methods could overestimate the number of
dead microbes because the DNA is extracted from both living and
dead microbes (He et al., 2008; Mijangos et al., 2009); thus, the
classical technique of counting colony-forming units on agar plates
was more suitable for this study.
Fig. 1a shows the effects of different additive-concentrations of
algal sludge on the cell density of strain SQR9 over time. The
changes in cell density for the treatments added with 1%, 5% and 7%
algal sludge followed the same trend, increasing to the maximum
density in the rst 4 days, then slightly decreasing toward the end
of the SSF process, while the cell density for the treatments with 3%
and 9% algal sludge was relatively stable after reaching the
maximum density in the rst 4 days. No obvious change was
observed in the control. At the end of the SSF process, the
maximum cell density (5.79
107 CFU g1 DW) was obtained in the
treatment with 5% algal sludge, followed by treatments with 3%, 1%,
9% and 7% algal sludge, while the minimum cell density
(1.11
107 CFU g1 DW) was shown in the control. The results
showed that 5% algal sludge (DW) is the best additiveconcentration for the novel SSF process.
To obtain the optimum SSF process, the effects of different water
contents and initial inoculation amounts on the SSF efciency of
strain SQR9 are shown in Fig. 1b and c. The cell density increased
with an increase in the inoculation amount when the amount
added was less than 5.0%, being the optimum inoculation amount.
No signicant difference was observed between the inoculation

Fig. 1. The effects of different additive concentrations of algal sludge on cell density for PGPR strain SQR9 during SSF (a), and the effects of initial inoculation sizes (b) and different
moisture contents (c) on the cell density of PGPR SQR9 after SSF. Bars with different letters indicate signicant differences among the eight treatments, as dened by Duncans test
(P < 0.05).

M. Zhang et al. / Journal of Environmental Management 132 (2014) 230e236

amounts of 10% and 15%. For moisture content, no signicant difference was observed between the treatments with the moisture
content values of 40% and 45%, both of which were more suitable
than the other treatments.
Chicken manure compost, pig manure compost, and Chinese
medicine residue compost were selected to evaluate whether the
addition of blue algae could normally promote bacterial growth
during the SSF process. The changes in cell density for the treatments with algal sludge added followed the same trend. A rapid
increase in biomass concentration was observed within the rst 4
days (more than 5
107 CFU g1 DW on the fourth day); however,
growth slowed down during the later stages of the SSF (Fig. S1aec),
which was most likely due to limited nutrients and other factors
associated with the batch growth of microorganisms (Zhu et al.,
2012). Relatively low and stable bacterial concentrations were
observed for the controls.
To reduce the eutrophication, several hundred thousand tons of
blue algae have been reoated annually from Taihu Lake (Yan et al.,
2012), thus, economical methods are desired for reclaiming this
waste resource. SSF has received renewed interests due to recent
developments in the eld of microbial biotechnology. The solid
phase in SSF provides a rich and complex source of nutrients that
may or may not be sufcient to the overall nutritional requirements
of the particular microorganism that is cultivated on that substrate.
Medium supplementation is necessary in non-traditional SSF, as it
induces biomass formation (Singh et al., 2009). In previous researches that investigated the conversion of waste to biofertilizer,
carbon sources, such as starch (Ogbo, 2010) and corn our (Zhu
et al., 2012), were added as nutrition to promote the growth of
microbres during fermentation. In the present study, the possibility
of utilizing algal sludge in SSF with PGPR strain SQR9 for the production of BIO was investigated. Algal sludge represents a potential
source of protein, polysaccharides, N and P (Mulbry et al., 2005),
which may support the growth of functional bacteria. Our study
showed that the addition of algal sludge to four different types of
compost normally promoted B. amyloliquefaciens SQR9 growth. The
optimum amount of algal sludge and bacteria to add into the
compost was both 5%. However, when the initial water content was
more than 50%, the number of the functional bacterium decreased.
Moisture content plays an important role in the growth of bacteria
and the optimum moisture content for a particular type of SSF and
its microbe-substrate system should be determined (Singh et al.,
2009). Weight ratios of water to substrate in SSF are usually between 1:1 and 1:10 (Reid, 1989). The results from the present study
showed that the optimum moisture content for the SSF investigated here is 45%.
3.2. Effects of the novel SSF process on the growth of other microbes
The effects of the novel SSF process on the growth of other
microbes are shown in Fig. 2. Two PGPR bacterial strains,
B. amyloliquefaciens NJN-6 and P. polymyxa SQR21 previously

233

isolated in our laboratory (Ling et al., 2011; Wang et al., 2013) were
selected to estimate the effects of the novel SSF process on the
growth of other PGPR strains. Selective plate counting was used to
monitor the number of B. amyloliquefaciens NJN-6; however,
because it is difcult to determine the number of wild P. polymyxa
SQR21, P. polymyxa SQR21-gfp was used in this study. Compared to
the control, the novel SSF process facilitated signicant growth and
biomass production of these two strains (Fig. 2b and c). A rapid
increase in biomass concentration within the rst 4 days was
observed; however, the growth slowed down during the later
stages of fermentation, which was most likely limited by the
available nutrients. We were also interested in whether the novel
SSF process facilitated fungi growth. T. harzianum T037, previously
isolated in our laboratory and effectively suppresses Fusarium wilt
disease of the cucumber, was selected; however, it failed as part of
the novel SSF process, as the cell density of this fungal strain
continually uctuated till the end of the SFF process (Fig. 2a). This
uctuation may be due to the unique characteristics of solid-state
cultivation, which provides a higher concentration of moisture
and is not favorable for mycelial organisms (Singh et al., 2009).
3.3. Variations in the physicochemical properties of the expanded
SSF process
The variations in the different physicochemical properties for
different treatments of the expanded SSF process are shown in
Fig. 3. Temperature readings for all of the treatments followed the
same trend, increasing to the maximum value in the rst 3 days,
and then slightly decreasing toward the end of the SSF process
(Fig. 3a). Temperature was used as an index to predict the microbial
activity in the fertilizer (Rainisalo et al., 2011). The BIOn treatment,
with algal sludge, showed the highest temperature; this was due to
the addition of organic matter and ambient nutrients from the algal
sludge, which encouraged microbial decomposition. During hightemperature composting, the high temperature is related to the
properties and the ratios of raw materials in the compost (Nigam
and Pandey, 2009). In this study, algal sludge was mixed with
mature compost which was thoroughly decomposed by hightemperature composting, and the mixture was turned over daily,
thus resulting in normal temperature SSF so that the functional
microbe could be successfully solid-state fermented. The EC of the
BIOn treatment decreased to its minimum value within the rst 2
days, then increased to 3.35 toward the end of the SSF process
(Fig. 3b). OFn and BOFn treatments showed similar patterns of
change in EC (Fig. 3b), which reects the salinity level in the raw
material. A stable pH value was shown for the OFn and BOFn
treatments. The pH value of the BIOn treatment slightly decreased
throughout the SSF procedure (Fig. 3c). This decrease may be due to
the decomposition of organic matter and the production of organic
and inorganic acids by the microorganisms (Mathur, 1991). After
the SSF process, no signicant differences in phosphorus, potassium and total carbon content were observed for the three

Fig. 2. Effects of the novel SSF process on the variations in cell density for Trichoderma harzianum T037 (a), PGPR NJN-6 (b) and SQR21 (c).

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M. Zhang et al. / Journal of Environmental Management 132 (2014) 230e236

Fig. 3. Changes in temperature (a), EC (b), pH (c) and TCAP (d) during the SSF process for BOFn (mature cattle manure compost, 100%), BIOn (cattle manure compost and blue algae,
95:5, w/w), and OFn treatments (mature cattle manure compost, 100%). All treatments were inoculated with PGPR strain SQR9 (5%, v/w DW).

fertilizers. Compared with the OF and BOF treatments, the total

nitrogen value in the BIO treatment was signicantly higher, due to
the addition of algal sludge (Table 1).
3.4. Variations in TAPS content
The concentrations of TAPS in the different treatments over time
are shown in Fig. 3d. The concentration of TAPS for the BIOn
treatment increased to its maximum value (4.4 g kg1 DW) on the
third day and subsequently decreased slightly to 2.3 g kg1 DW
toward the end of the SSF process; this concentration was signicantly higher than that of the OFn (0.15 g kg1 DW) control and
BOFn (0.39 g kg1 DW) treatment. The variation in the concentrations of the TAPS for the BIOn treatment is due to the protein from
the algal sludge, which was hydrolyzed to TAPS by microbes during
the SSF process. Therefore, the results showed that the addition of
algal sludge not only promoted SSF for the functional microbe, but
it also improved the TAPS content in the resulting BIO. Several reports have demonstrated that amino acids are important for plant
growth. Nsholm et al. (1998) found that some amino acids, such as
tryptophan and methionine, are precursors of ethylene and IAA and
Table 1
Selected physicochemical properties for OF, BOF and BIO, which were produced by
the following treatments: BOFn (mature cattle manure compost, 100%), BIOn (cattle
manure compost and blue algae, 95:5, w/w), and OFn (mature cattle manure
compost, 100%), respectively. All treatments were inoculated with the PGPR strain
SQR9 (5%, v/w DW).

OF
BOF
BIO

Total
carbon (%)

Total
nitrogen (%)

P2O5 (%)

K2O (%)

GI value (%)

12.93  0.08a
12.11  0.19a
13.75  0.32a

0.99  0.02b
0.91  0.08b
1.28  0.03a

1.97  0.04a
1.82  0.16a
2.06  0.03a

1.42  0.04a
1.39  0.06a
1.36  0.01a

90  14b
1002b
1187a

All values are the mean of three replicates. Numbers followed by  represent the
standard errors.
Values with the different letter after the number in the same column are signicantly different at P < 0.05 according to Duncans test.

that L-tryptophan can enhance nitrogen absorption. Kinnersley

et al. (1998) reported that the exogenous addition of amino acids
could increase crop yields to a certain extent. In this study, more
TAPS were observed in the BIO, indicating that this fertilizer may be
more effective in promoting plant growth.
3.5. GI values and MC-LR and MC-RR concentrations of the
produced fertilizer
The GI value of the fertilizer produced by BIOn treatment was
signicantly higher than that of those produced by other treatments. All of the fertilizers reached GI values above 50%, indicating
that the application of these fertilizers would not injure the plants
(Table 1) (Zucconi et al., 1981).
The concentrations of MC-LR and MC-RR in the raw materials
and the produced fertilizer were determined in this study. MC-LR
and of MC-RR were degraded from initial concentrations of 0.22
and 5.24 mg g1 DW, respectively, down to undetectable level
(0.1 mg g1 DW). Microcystins are a group of monocyclic heptapeptides that are hardly degraded by conventional methods due to
their thermostability and lack of volatility after boiling (Kujbida
et al., 2006). In this study, rapid biodegradation rates of MC-LR
and MC-RR, two main toxic components belonging to the microcystins in Taihu Lake (Shen et al., 2003), were observed through the
use of novel SSF technology. This may be due to the activity of
microbes during the SSF process. Previous reports have already
demonstrated that a signicant amount of microbes could degrade
microcystins in the environment (Chen et al., 2008).
3.6. Effects of the BIO on eggplant and cucumber growth
Thirty days after being applied to the cucumber plants, the
fertilizer produced from the BIOn treatment resulted in greater
plant height, stem diameter and above- and below-ground fresh
weights than the fertilizers produced from the BOFn treatment and
OFn control. By the end of the pot experiments, the BIO application

M. Zhang et al. / Journal of Environmental Management 132 (2014) 230e236

signicantly increased the plant height, stem diameter, and the

above- and below-ground fresh and dry weights of the plant by
7.77%, 3.28%, 53.18%, 31.41%, 9.76% and 21.88%, respectively, when
compared with BOFc treatment, and by 10.84%, 15.41%, 82.39%,
67.43%, 29.10% and 119.01%, respectively, when compared with OFc
control. No signicant differences in the SPAD and soluble protein
contents of the cucumbers were observed during the pot experiments; nevertheless, the soluble protein content of the BIOc
treatment was the highest. These results show that the BIO application effectively promoted cucumber growth. Compared with the
OFc control, the BOFc treatment signicantly increased the plant
height, stem diameter and the above-ground dry weight thirty days
after being added to the pots and at the end of the experiment, and
no signicant differences in any other indices were observed during
the pot experiment; nevertheless, the index-values of the BOFc
treatment were all higher than that of the OFc control (Fig. S2).
From thirty days after being added to the pots until the end of
the pot experiments, the fertilizer produced from the BIOn treatment had signicantly increased the plant height, stem diameter,
and the above-ground dry and fresh weights of the eggplants, when
compared with the other two fertilizers. At the end of the pot experiments, the BIOp treatment increased the plant height, stem
diameter, soluble protein content, SPAD, and the above- and belowground dry and fresh weights of the eggplants by 25.24%, 33.91%,
64.66%, 57.54%, 81.56%, and 62.92%, respectively, when compared
with the BOFp treatment, and by 35.78%, 26.15%, 68.51%, 68.89%,
93.31%, and 74.12%, respectively, when compared with the OFp
control. No signicant differences in the indices of the eggplant
were observed for the fertilizers produced from the BOFp treatment
and OFp control during the pot experiments; nevertheless, the
index-values for the fertilizer produced from the BOFp treatment
were all higher than that of the OFp control (Fig. S3).
Based on the results of the pot experiments, the fertilizer produced from the BOF treatment signicantly promoted plant growth
when compared with the OF control. This result may be due to the
functional PGPR (strain SQR9) in the BOF. PGPR are free-living
bacteria that have benecial effects on plants: they enhance
emergence, colonize roots, and stimulate growth (Idriss et al.,
2002). B. amyloliquefaciens SQR 9 is a Gram-positive, plant-associated bacterium, which can colonize the cucumber root rapidly and
efciently after rhizosphere inoculation, thus stimulating plant
growth (Cao et al., 2011). In the present study, results also
conrmed that the PGPR strain B. amyloliquefaciens SQR 9 could
promote plant growth in a greenhouse experiment. The highest
yield in biomass was found in treatments where the BIO was
applied; this is possibly due to the large amounts of PGPR strains
and biologically active substances, such as amino acid and peptides,
which stimulated the growth of the plant. The results were similar
to those obtained from other studies in our lab, where the application of BIO to the soil gave the best results (Cao et al., 2011; Zhao
et al., 2011). The greenhouse experiments showed great potential
for the use of the novel BIO as a soil additive to promote plant
growth. In order to determine whether the use of the produced BIO
could avoid a new eutrophication of lake and water reservoirs,
physical characteristics of the soil samples collected at the end of
the pot experiment were analyzed. As shown in table S1, except Ec,
which was signicant lower in the soil collected from BIOp treatment than that in BOFp treatment and OFp control, no signicant
differences in the physical characteristics detected among samples
supplied by different fertilizers with or without algal sludge for the
two plants were observed, indicating that the application of the
produced BIO is satisfactory for the water resource. The combined
results showed that utilization of collected algal sludge in the SSF
process as a medium increased the number of strain SQR9, promoted the plant growth and thus attain economic viability. This

235

study provides a novel resource recovery method to reclaim algal

sludge from Taihu Lake.
4. Conclusion
In this study, reoated algal sludge was successfully used to
produce novel BIO that contains more nutrients than commonly
used compost and a large amounts of PGPR strains. The GI value of
the BIO was satisfactory using the GI criterion. Pot experiments
demonstrated that the BIO produced by the novel SSF process
effectively promoted plant growth and is satisfactory for the water
resource. The novel SSF process could also be performed using
other PGPR strains. Therefore, this study provides a novel way of
high-value utilization of algal sludge from Taihu Lake by producing
low-cost but high-quality BIOs.
The novel SSF technology created via the new medium, reoated
algal sludge which was free of charge, could effectively reduce the
BIO costs and avoid the secondary environmental pollution if
extensively applied in factory production, indicating that more and
more farmers could be capable of utilizing the novel low-cost but
high-quality BIOs to improve the crop yield and quality, thus largescale extension of BIOs may be achieved. However, the efciency of
the novel process in factory and the direct effects of the novel
products in eld experiment should be further studied.
Acknowledgements
This research was supported by the National Natural Science
Foundation of China (41101231), the Chinese Ministry of Science
and Technology (2013AA102802), the Priority Academic Program
Development of the Jiangsu Higher Education Institutions (PAPD),
the 111 project (B12009), the Key Technology R&D Program of
Jiangsu, China (BE2012377), the Jiangsu Postdoctoral Science
Foundation (1102079C), the Specialized Research Fund for the
Doctoral Program of Higher Education (20110097120001), the
China Postdoctoral Science Foundation (2011M501248) and
(2012T50479), and the Youth Foundation of Nanjing Agricultural
University (KJ2011007).
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jenvman.2013.10.031.
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